Plant Breeding Reviews, Volume 30

PLANT BREEDING REVIEWS Volume 30 Plant Breeding Reviews, Volume 30. Edited by Jules Janick Copyright © 2008 John Wiley...

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PLANT BREEDING REVIEWS Volume 30

Plant Breeding Reviews, Volume 30. Edited by Jules Janick Copyright © 2008 John Wiley & Sons, Inc.

Plant Breeding Reviews is sponsored by: American Society for Horticultural Science Crop Science Society of America Society of American Foresters National Council of Commercial Plant Breeders International Society for Horticultural Science

Editorial Board, Volume 30 I. L. Goldman C.H. Michler Rodomiro Ortiz

PLANT BREEDING REVIEWS Volume 30

edited by

Jules Janick Purdue University

John Wiley & Sons, Inc.

1 This book is printed on acid-free paper. *

Copyright # 2008 by John Wiley & Sons, Inc. All rights reserved Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada Wiley Bicentennial Logo: Richard J. Pacifico No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 646-8600, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at www.wiley.com/go/permissions. Limit of Liability/Disclaimer of Warranty: While the publisher and the author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor the author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information about our other products and services, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. For more information about Wiley products, visit our web site at www.wiley.com. Library of Congress Cataloging-in-Publication Data: ISBN-13: 978-0-470-17152-3 ISBN-10: 0-470-17152-9 ISSN: 0730-2207 Printed in the United States of America 10 9 8 7 6 5 4 3 2 1

Contents Contributors

viii

1. Dedication: Francesco Salamini Plant Geneticist and Plant Breeder

1

Dorothea Bartels I. II. III. IV. V. VI.

Biographical Sketch Scientific Achievements The Man Acknowledgments Literature Cited Publications of Francesco Salamini

2. Epigenetics and Plant Breeding

2 5 12 14 14 15

49

Athanasios S. Tsaftaris, Alexios N. Polidoros, Aliki Kapazoglou, Eleni Tani, and Nives M. Kovacˇevic´ I. Introduction II. Molecular Epigenetic Mechanisms III. Plant Biological Phenomena Involving Epigenetic Mechanisms IV. Epigenetic Mechanisms and Plant Development V. Implications in Plant Breeding VI. Outlook VII. Acknowledgments VIII. Literature Cited

3. Enhancing Crop Gene Pools with Beneficial Traits Using Wild Relatives

51 57 85 104 119 143 145 145

179

Sangam L. Dwivedi, Hari D. Upadhyaya, H. Thomas Stalker, Matthew W. Blair, David J. Bertioli, Stephan Nielen, and Rodomiro Ortiz I. Introduction II. Genetic Resources from Wild Relatives III. Barriers and Approaches to Interspecific Gene Transfer

181 183 184 v

vi

CONTENTS

IV. Beneficial Traits from Wild Relatives Contributing to Crop Gene Pools V. Biotechnological Approaches to Enhance Utilization of Wild Relatives in Crop Improvement VI. Outcomes of Wild Relatives Use in Genetic Enhancement of Crops VII. Future Outlook VIII. Acknowledgments IX. Literature Cited

4. Allelopathic Crop Development: Molecular and Traditional Plant Breeding Approaches

188 197 211 215 215 216

231

Cećile Bertin, Leslie A. Weston, and Harlene Kaur I. Introduction II. Common Breeding Techniques for Selection of Weed-Suppressive Crops III. Use of Transgenes to Produce Allelopathic Crops IV. Use of Whole-Genome Techniques to Understand Allelopathic Phenomena V. Conclusion VI. Literature Cited

5. Molecular Genetics and Breeding for Fatty Acid Manipulation in Soybean

232 234 239 249 253 254

259

Andrea J. Cardinal I. Introduction II. Glycerolipid Biosynthesis in Plants III. Breeding for Improved Oil Quality, Mapping, and Molecular Basis for Altered Fatty Acid Composition IV. Genetic Relationships Among Loci, Pleiotropy, and Environmental Effects on Fatty Acid Composition V. Research Needs VI. Acknowledgments VII. Literature Cited

6. Improving Breeding Efficiency for Early Maturity in Peanut

260 261

265

282 286 288 288

295

S. N. Nigam and R. Aruna I. Introduction II. Botany

296 297

CONTENTS

III. IV. V. VI.

vii

Genetics and Breeding Cultural Manipulations to Shorten Crop Duration Summary Literature Cited

7. Ploidy Manipulation for Breeding Seedless Triploid Citrus

302 314 315 316

323

Patrick Ollitrault, Dominique Dambier, François Luro, and Yann Froelicher I. Introduction II. Natural Polyploidy in Citrus III. Ploidy Manipulation for Triploid Seedless Cultivar Breeding IV. Conclusion V. Literature Cited

8. Breeding Southern Highbush Blueberries

324 326 332 344 345

353

Paul Lyrene I. Introduction II. Biology of the Highbush Blueberry III. Genetic Resources for Breeding Southern Highbush Blueberry IV. Origins of Highbush Blueberry Breeding V. Methods VI. Breeding Objectives VII. Recommending Cultivars for Commercial Plantings VIII. Outlook and Issues IX. Literature Cited

9. Coffee Germplasm Resources, Genomics and Breeding

354 356 359 369 373 380 402 403 406

415

Fernando E. Vega, Andreas W. Ebert, and Ray Ming I. II. III. IV. V.

Introduction Germplasm Resources Genomics Breeding Literature Cited

416 418 426 430 441

Subject Index

449

Cumulative Subject Index

451

Cumulative Contributor Index

475

Contributors

R. Aruna, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India, [email protected] Dorothea Bartels, University of Bonn, Kirschallee 1, 53115 Bonn, Germany, [email protected] Cećile Bertin, McGill University, Department of Plant Science, Raymond Building, 21111 Lakeshore Road, Ste. Anne de Bellevue, Quebec H9X 3V9, Canada, [email protected] David J. Bertioli, Universidade Catolica de Brasilia, Pos Graduacao Campus II, SGAN 916, Brasilia, DF CEP 70.790-160, Brazil, [email protected] Matthew W. Blair, Centro Internacional de Agricultura Tropical (CIAT), A.A. 6713 Cali, Colombia, [email protected] Andrea J. Cardinal, Department of Crop Science, Box 7620, North Carolina State, University, Raleigh, North Carolina 27695-7620, USA, andrea_cardinal@ ncsu.edu Dominique Dambier, CIRAD, UPR, Ame´lioration des Plantes a` Multiplication Ve´ge´tative, TA50/PS4, Boulevard de la Lironde, 34398 Montpellier, France, [email protected] Sangam L. Dwivedi, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India, s.dwivedi@ cgiar.org Andreas W. Ebert, Plant Genetic Resources and Biotechnology Unit, Department of Agriculture and Agroforestry, Tropical Agricultural Research and Higher Education Center (CATIE), Turrialba, Costa Rica, [email protected] Yann Froelicher, CIRAD, UPR, Ame´lioration des Plantes a` Multiplication Ve´ge´tative, San Giuliano, 20230 San Nicolao, France, yann.froelicher@ cirad.fr Aliki Kapazoglou, Institute of Agrobiotechnology, CERTH, 57001, Thermi, Greece, [email protected] Harlene Kaur, Max Planck Institute for Chemical Ecology, Molecular Biology Program, 07745 Jena, Germany, [email protected] Nives M. Kovac˘evic´, Department of Agronomy, University of Wisconsin-Madison, 1575 Linden Drive, Madison, WI 53706, USA, [email protected] François Luro, INRA, GECA, San Giuliano, 20230 San Nicolao, France, luro @corse.inra.fr Paul Lyrene, Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611, USA, [email protected] viii

CONTRIBUTORS

ix

Ray Ming, Department of Plant Biology, 288 ERML, MC-051, 1201 W. Gregory Drive, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA, [email protected] Stephan Nielen, PBI, EMBRAPA Recursos Genéticos e Biotecnologıá, Parque Estaçaõ Ecologica Norte, Final Av W5 Norte, Brası´lia-DF 70770-900, Brazil, [email protected] S. N. Nigam, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India, [email protected] Rodomiro Ortiz, Director, Resource Mobilization, CIMMYT, Apdo. Postal 6-641, Mexico, 06600, D.F., Mexico, [email protected] Patrick Ollitrault, CIRAD, UPR, Ame´lioration des Plantes a` Multiplication Ve´ge´tative, TA50/PS4, Boulevard de la Lironde, 34398 Montpellier, France, [email protected] Alexios N. Polidoros, Institute of Agrobiotechnology, CERTH, 57001, Thermi, Greece, [email protected] H. Thomas Stalker, North Carolina State University, PO Box 7620, Raleigh, North Carolina 27695, USA, [email protected] Eleni Tani, Institute of Agrobiotechnology, CERTH, 57001, Thermi, Greece, [email protected] Athanasios S. Tsaftaris, Department of Genetics and Plant Breeding, AUTh, 54124, Thessaloniki, and Institute of Agrobiotechnology, CERTH, Thermi 57001, Greece, [email protected] Hari D. Upadhyaya, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru PO, 502324, A.P., India, H.Upadhyaya@ cgiar.org Fernando E. Vega, Insect Biocontrol Laboratory, U. S. Department of Agriculture, Agricultural Research Service, Bldg. 011A, BARC-W, Beltsville, Maryland 20705, USA, [email protected] Leslie A. Weston, Department of Horticulture, Cornell University, Ithaca, New York 14853, USA, [email protected]

Francesco Salamini

1 Dedication: Francesco Salamini Plant Geneticist and Plant Breeder Dorothea Bartels University of Bonn Kirschallee 1, 53115 Bonn, Germany

I. BIOGRAPHICAL SKETCH A. From Childhood to Science B. Postgraduate Studies C. Return to Italy II. SCIENTIFIC ACHIEVEMENTS A. Maize Genetics 1. Wax Biosynthesis 2. Seed Proteins B. Origin of Agriculture and Domestication of Modern Crop Plants C. Plant Breeding in Germany D. Potato: Genetics, Biotechnology, and Genomics E. Sugarbeet F. Science Politics III. THE MAN A. Advisor and Editor B. The Collector C. Impact IV. ACKNOWLEDGMENTS V. LITERATURE CITED VI. PUBLICATIONS OF FRANCESCO SALAMINI

I first got to know Francesco Salamini in 1986, when he offered me a position as a group leader in his Department of Plant Breeding and Yield Physiology at the Max Planck Institute (MPI) for Plant Breeding in Cologne, Germany. Dr. Salamini had been appointed as director of the MPI and head of the department of plant breeding one year before. The board of directors of MPI was now complete, together with Professors Jeff Schell, Heinz Saedler, and Klaus Hahlbrock. These four scientists


1

2

D. BARTELS

with different but complementary expertise formed a crew of captains who steered the MPI as an internationally highly prestigious research institution through calm and rough waters. The position at the MPI was certainly a challenge to Francesco Salamini, as he could not create a new department according to his ideas but he had to take over the existing department and gradually shape it to his vision with the opportunity to make only a very few new appointments. When Dr. Salamini joined the MPI, he began a gradual process of getting the department into shape. His task was to build a bridge between the emerging field of molecular biology and traditional plant breeding. This required that he be an expert in both molecular biology and plant breeding methods. As a research director in Germany, he also had to become acquainted with the German scientific culture and familiar with the German scientific community. At the time he arrived at the MPI, most of the internationally prestigious plant science research institutes had decided to mainly focus research on Arabidopsis thaliana or other model plants and to use these systems to address fundamental questions in research. However, according to the long term research programs of the MPI Dr. Salamini was supposed to stay close to agriculturally relevant plants in his research projects. These plants ranged from potato, barley, maize, sugarbeet, progenitors of ancient hexaploid wheat, to exotic desiccation-tolerant plants, the so-called resurrection plants. The choice of the plant was decided by the scientific questions asked, which, in turn, were driven by agricultural problems.

I. BIOGRAPHICAL SKETCH A. From Childhood to Science Francesco Salamini was born in 1939 in Castelnuovo Bocca D’Adda, situated in the Plain of Lombardy south of Milan, Italy. He spent his childhood in a rural community where he grew up on a farm. He helped his father and became involved in various tasks around the farm, such as hoeing and irrigating the fields. Maize, lucern, and clover were the main crops grown. The intensive farming and the rural environment influenced Francesco’s upbringing. He was interested in hunting, fishing, bird watching, and cultivating plants. In 1959, Francesco began to study agricultural sciences at the Catholic University in Piacenza (Italy), where he graduated magna cum laude with honors in February 1963. During these studies he became inter-

1. DEDICATION: FRANCESCO SALAMINI

3

ested in plant genetics with maize the main subject of his studies. His teacher and mentor was Professor Angelo Bianchi, at that time the leading maize geneticist in Italy. Francesco’s close interaction with Professor Bianchi on the scientific and personal level was extremely fruitful and has continued ever since. Professor Bianchi strongly influenced Francesco’s scientific education and direction and inspired him to become an enthusiastic and dedicated maize geneticist. Francesco’s first scientific publication was a collaboration between mentor and student (Bianchi and Salamini 1962). In graduate studies, which Francesco continued in Piacenza, he studied the glossy genes in maize, which are involved in the biosynthesis of epicuticular waxes. The waxes determine the appearance of the leaf surface and are responsible for physiological characters, such as transpiration protection. His graduate studies laid the foundation for further biochemical and molecular studies on the glossy genes and their function in wax synthesis. The genetic analysis of glossy genes in maize provided the basis for intensive biochemical analysis of the synthesis of epicuticular waxes, which he later carried out with his friend and colleague, Giorgio Bianchi, an organic chemist at the University of Pavia. Francesco resumed this early work on wax biosynthesis about 30 years later for a molecular genetic analysis (Tacke et al. 1995). During his studies at Piacenza University, Francesco married Marisa, who actively supported his career wherever he went. B. Postgraduate Studies After graduating from Piacenza, Francesco obtained a National Research Council fellowship and continued his investigations on maize mutants for two years, as did Carlo Lorenzoni, Gian Piero Soressi, and Antonio Ghidoni, all of whom were to obtain professorships in plant genetics. Francesco benefited from these interactions and maintained close personal and scientific relationships with these individuals over decades. Intensive and lively discussions of maize genetics are easy to imagine in this group of scientists. In 1966 Francesco Salamini had his first scientific appointment as a research scientist at the Institute of Plant Breeding in Bologna, one of the 23 institutes of the Italian Ministry of Agriculture. This position introduced new aspects to his maize research, and he initiated very important studies on seed quality traits. Maize seed quality is of enormous importance for human nutrition and livestock feed. Throughout his work, Francesco has focused on the subject of maize seed quality closely coupled with a genetic approach to understanding parameters that

4

D. BARTELS

determine seed quality. Recognizing that new expertise was needed to complement the genetic approach, he decided to join the laboratory of Oliver Nelson at the department of botany and plant pathology at Purdue University in West Lafayette, Indiana, for one year (1969– 1970). The research there enabled Francesco to apply a combination of enzymology protein biochemistry and plant physiology to problems of maize seed quality. This was a very rewarding and scientifically influential time for Francesco Salamini. Knowledge obtained during this time influenced his thinking for many years. Dr. Nelson had discovered that the maize endosperm mutants opaque-2 and floury-2 improved protein quality of maize seeds, an important nutritional aspect, and introduced Francesco Salamini to biochemical genetics. Later Dr. Salamini was one of the researchers who discovered the molecular basis for the opaque-2 mutation (Lohmer et al. 1991). C. Return to Italy Upon his return to Italy in 1970, Francesco Salamini diversified his research and started a program on genetics and breeding of common bean at the Research Institute of Vegetable Crops, Section of Montanaso Lombardo near Milan. The project focused on Italian bean cultivars, and studies were carried out to analyze the effects of chemical mutagenesis on dormant bean seeds. Mutation frequencies and formation of chimeras were investigated (Motto et al. 1975). Questions of seed dormancy are still today of high relevance. Interestingly, Marten Koornneef, Francesco’s successor as director of the MPI in Cologne, is analyzing dormancy in seeds of Arabidopsis thaliana. Besides basic genetic studies, the bean breeding programs were begun to introduce genes for virus resistance into Italian germplasm. This approach was successful, and several breeding lines were released, some of which were cultivated. Francesco Salamini’s ability to conduct several research projects in parallel became apparent at this time, and it is therefore not surprising that he continued his genetic studies in maize. He also lectured at the University of Piacenza. His dedication to pass his knowledge on to young people was rewarded 1971 by a professorship in plant genetics at the University of Piacenza, a position he kept until 1975. Francesco’s love for maize genetics fell on fertile soil when he became research director of the maize section of the Institute of Cereal Crops in Bergamo, Italy, a position which he kept from 1975 to 1985. Bergamo has been the center for his family life, and all of the three children have been educated there. On the scientific side, the directorship of the maize institute allowed him to generate extremely valuable, unique, and large


5

maize genetic stocks and breeding lines. Francesco has always been a passionate maize geneticist, and he has been the driving force to a vast number of genetic crosses in the field and to the subsequent evaluation of the crosses. It was a particular experience for scientific staff or collaborators to be a member of the maize genetic team in Bergamo. A citation from one of his close colleagues, Dr. Mario Motto, may describe the atmosphere and Francesco’s dedication to practical maize genetics. ‘‘Frequently during those years one could see him in the Bergamo cornfields, wearing one of those hats from some seed companies, walking in the nursery looking for mutants or to discuss plant-breeding philosophy. While at the end of the pollination day the crew members might advocate a break to the air-conditioned shed, in attempts to survive the heat and humidity of the field, he invariably preferred to stay in the fields and talked with the occasional ‘poor guy’ some intricate crossing schemes to develop new inbred lines or to figure out the parental lines of some hybrid combinations. Although most of the times the hypothesis, as usual in sciences, were incorrect, he tried to convince the occasional listener about his skill in discovering the right materials for maize improvement.’’ In his career, Francesco Salamini has had a good sense for adopting new technical approaches and to go to places where it is done best. In 1976, as plant tissue culture methods became accepted tools for obtaining genetic variations, he decided to spend a short time in the laboratory of Dr. Harold Smith at the Brookhaven National Laboratory Upton, NY, USA, in order to become acquainted with tissue culture techniques. Despite being a very active maize geneticist in Bergamo, Francesco always sought new challenges. This he found in Germany as a director of the Max Planck Institute of Plant Breeding in Cologne. He kept this position from 1985 until 2004, when he turned 65 and officially retired from his position as director. Reaching 65 did not signal a stop to Francesco’s active scientific life, it only meant that it was time to move back to Italy, where he became a professor of genetic technologies at the University of Milan and a director at the CISI-Centro Interdisciplinare Studi Biomolecolari affilicarioni industrali. According to his wife and other Italian colleagues, Francesco Salamini has never been so busy.

II. SCIENTIFIC ACHIEVEMENTS Yield increase of crop plants has been the most important factor supporting agricultural progress. Yield depends on the capability of plants to accumulate storage products, but it is also influenced by the

6

D. BARTELS

architecture of the plant and by the ability to withstand diseases. These parameters guided the nature of the research projects that were developed by Francesco Salamini. The genetic basis of developmental traits relevant to crop production and the genetic diagnosis of crop trait variability have been the two major research directions while Dr. Salamini was the director of the department of plant breeding and yield physiology at the MPI. He developed research projects using diverse crop and model plants. In 1999 the MPI issued an information brochure entitled ‘‘Genomics at the Max-Planck-Institut.’’ Out of the seven projects described in the brochure, five were initiated by Francesco Salamini: sugarbeet, potato, maize, barley, and Einkorn wheat. Dr. Salamini has always had inside knowledge of international scientific movements and goals. Therefore, when the first plant genome project in Germany ZIGIA (Arabidopsis functional genomics) was set by his colleague Heinz Saedler, his dedicated involvement was not surprising. In addition to these research projects, Dr. Salamini started research on potato cytogenetics, photosynthesis as elementary process of high-energy utilization in Arabidopsis thaliana, virus resistance in potato as well as in tropical plant species, and desiccation tolerance in resurrection plants, which are unique higher plants expressing desiccation tolerance in vegetative tissues. It is beyond the scope of this article to summarize all Dr. Salamini’s research activities and achievements equally; therefore, I have chosen to describe some highlights. Several of the projects have originated and developed under the guidance of Dr. Salamini, but individual researchers, including myself, have been given the freedom to continue the projects as independent research projects. In other words, Francesco Salamini has sown the seeds for proliferating successful research projects. Due to his inspiration and continuous intellectual and financial support, I have studied desiccation tolerance in resurrection plants (Bartels and Salamini 2001), and I am continuing these studies at the University of Bonn. Francesco has contributed to my research with ideas and with help to solve problems, when necessary. When we obtained new results, he was usually the first to hear about it, because he was genuinely interested and could share my enthusiasm. I am very grateful to him for this opportunity. The project on photosynthesis, a basic plant process determining yield and fitness, conducted by Dario Leister, has followed a similar course. Dario started as a doctoral student and recently became a professor of botany at the University of Munich, where he continues the photosynthesis research initiated by Francesco Salamini with Professor Carlo Soave. A genetic approach was used to identify tools for optimization of the photosynthesis process (as described in Biehl et al. 2005).


7

A. Maize Genetics Francesco Salamini has studied maize genetics for nearly 40 years. Emphasis has been on two main research complexes: the biosynthesis of epicuticular waxes and the development of maize kernels and protein quality with regard to nutritional parameters. 1. Wax Biosynthesis. The genetics of wax biosynthesis was the first maize genetics research project undertaken by Francesco Salamini. Initially mutants were isolated that interfere with the wax biosynthesis. The identification of mutants was relatively easy, as they often cause visual phonotypes like glossy or blunt leaf surfaces. Several classes of glossy (Gl) mutants were distinguished: those that interrupt the elongation-decarboxylation reactions at specific sites, those that block the reduction of the C32 aldehyde to alcohol, and those that regulate the level of wax production without affecting the lengths of the fatty acid chains (Avato et al. 1984). In order to understand which biochemical steps were affected in the wax biosynthesis by the glossy mutations, several genes were tagged by inserting the En/Spm transposable element in the gene of interest through genetic crosses in the experimental maize plots in Bergamo. Tagging of the genes led to the molecular isolation of the glossy1 (Gl1) and glossy 2 (Gl2) genes, which encode a b-ketoacyl reductase and a putative acetyl transferase, respectively (Tacke et al. 1995; Sturaro et al. 2005). 2. Seed Proteins. Zeins are the main storage proteins of the maize kernel. Zeins are deposited in the endosperm, and their amino acid compositions determine nutritional quality. Zeins are subdivided in several classes, and they are encoded by a large gene family. Understanding the control of zein synthesis and other maize proteins was a large research effort with several important, fruitful collaborations. One of Dr. Salamini’s first collaborators was Carlo Soave, a very knowledgeable plant biochemist from Milan. This collaboration led to the genetic mapping and characterization of structural zein genes, and it initiated work on zein synthesis regulation, a project that was later also tackled molecularly at the MPI in Cologne. Several mutations affecting zein accumulation were isolated, such as opaque2 (o2), opaque 7 (o7), floury 2 (fl2), and others (Soave and Salamini 1984). The nature of the regulatory genes was of high interest, and an intensive genetic approach to clone the genes was carried out at the institute in Bergamo. The strategy was to use the transposable elements Bg, which stands for ‘‘Bergamo,’’ and the Ac element from Barbara McClintock to tag the regulatory genes. In a strong

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competition with American scientists, the o2 mutation was identified and molecularly cloned using transposon tagging. It was shown that the o2 gene encodes a transcription factor belonging to the bZIP class family. The work on the o2 gene was much appreciated scientifically and led to further studies, including studies on the interaction of o2 and o6 in zein deposition, the transcriptional function of o2, and the regulation of o2 translation (Motto et al. 1997). These projects were carried out by close collaboration between the maize group at the MPI, Cologne, led by Dr. Richard Thompson, and the maize group in Bergamo led by Dr. Motto. The genetic maize fields in Bergamo were a starting point for closer connections between Francesco Salamini and Cologne, where Professors Peter Starlinger and Heinz Saedler became interested in maize transposable elements. The first connection was made during a visit of Peter Starlinger, a professor in genetics from the University of Cologne. Besides bacterial transposable elements, he started to study the maize elements Ac/Ds, which led him to visit Dr. Salamini in Bergamo. The two became colleagues and close friends. The mutations in the o2 gene had some practical effects on Italian maize breeding lines. In lines carrying mutations in the o2 gene, the level of the zein storage proteins is reduced and other seed proteins are more prevalent. This results in higher levels of tryptophan and lysine and thus in improved nutritional quality of the maize kernel flour. As a consequence, the institute of Bergamo released several o2 inbred lines and lines with o2-based synthetic gene pools. A curious observation was made in a breeding program attempting to transfer the opaque phenotype into elite maize inbred lines. A somatic instability at the opaque-2 locus was discovered, which sparked off consultations with Professor Peter Peterson, a pioneer of maize genetics using transposable elements (Peterson 2002). Together Professor Peterson and Dr. Salamini studied the significance and behavior of transposable elements in maize and the power to generate genetic variability (Peterson and Salamini 1986). Dr. Salamini discovered a particular transposable element system in maize, the Bg (= Bergamo) system. It was shown that 10 to 12 copies of Bg homologous sequences are present in the maize genome. The insertions of these elements were responsible for the instability often observed at the o2 locus (Salamini 1980, 1981). The Bg elements were characterized and isolated in Bergamo (Motto et al. 1989). B. Origin of Agriculture and Domestication of Modern Crop Plants The research project on Triticum monococcum (wild einkorn) was set up to understand species domestication and the establishment of


9

agriculture. The diploid einkorn is considered to be one of the primary domesticates and thus one of the ancestors of our modern bread wheat, Triticum aestivum. More than 200 wild einkorn lines were collected from primary and secondary habitats for a fingerprint analysis. Molecular markers of about 200 lines have been used as a diagnostic tool to show that einkorn wheat originated from the Fertile Crescent region in the Karacadag Mountains, southeast Turkey (Heun et al. 1997). This discovery was reported in Germany’s newspapers with the headline ‘‘Unser Weizen ist ein Tu¨rke’’ (literally: our wheat is a Turkish man). Comparative work with barley demonstrated the monophyletic nature of barley domestication; it identified the wild populations from IsraelJordan to be most similar to the cultivated varieties. Barley migrated from the Near East to South Asia, and Nepal and the Himalayas are considered the center of barley domestication. Various genetic crosses, mutagenesis programs, and segregating populations have been made and propagated with the objective of understanding the genetic basis of the domestication process. Traits like seed size, baking quality of flour, and fragile rachis (important for stability during mechanical harvesting) have been studied. This work is still in progress, and today Francesco Salamini is harvesting the results of a huge genetic program. This project provides insights into the evolution of traits and characters of cereals, but it also teaches a lesson in sociocultural history. ‘‘A better understanding of the genetic differences between wild grasses and domesticated crops adds important facets to the continuing debate on the origin of western agriculture and the societies to which it gave rise,’’ Francesco has stated. C. Plant Breeding in Germany The German plant breeders had high expectations when Francesco Salamini came to the country and took his position as head of the department for plant breeding at the MPI. After an initial hesitation and skepticism, a fruitful dialogue developed between Dr. Salamini and the major German breeders, in particular representatives of the Kleinwanzlebener Saatzucht AG (KWS) and potato breeders. This dialogue led to an understanding of their different positions and ‘‘cultures’’ and resulted in common projects mainly introducing the concept of markerassisted breeding to companies. The first project, which was developed between KWS and Francesco Salamini, was the European EUREKA project trying to identify quantitative trait loci for yield and dry mass in maize. After trust and confidence had been established, more collaborative projects were developed. Two major projects have to be

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mentioned here: potato breeding, conducted by Dr. Christiane Gebhardt, and molecular markers in sugarbeet, a project conducted by Dr. Katharina Schneider and others (see below for details). Francesco Salamini introduced the concept of marker-assisted breeding at the start of his career at the MPI. A series of marker techniques were carried out: initially restriction fragment length polymorphism (RFLP), followed by amplified length polymorphism (AFLP), and more recently single-nucleotide polymorphism (SNP). Using various genetic markers, a basic genetic network was developed for potato and later sugarbeet, two difficult species for genetic analysis. The markers allow for plant traits to be followed in breeding programs and for the eventual isolation of genes of interest. The department at MPI developed the marker technique, and many scientists, particularly from Italy, came to learn it. Breeders in Germany recognized that Dr. Salamini’s strength was combining traditional and molecular breeding approaches. Francesco was fascinated by the possibilities of genome analysis and the application of this knowledge to improve agricultural plants, and he was able to transmit his enthusiasm to the practical plant breeders. His ideas have influenced many research projects, and he has always been interested in practical applications of knowledge. Because of his international experience and his deep understanding of plant breeding, Dr. Salamini had a huge impact on the approaches and discussions in the German plant breeding scene while he was Max Planck director. D. Potato: Genetics, Biotechnology, and Genomics The move to Germany induced Dr. Salamini to develop new research projects around the potato, a crop very relevant to German agriculture. Research and breeding of potatoes had a tradition at the MPI. A field station in Scharnhorst in northern Germany was dedicated to multiply potato lines for breeding purposes. This potato breeding station was in Dr. Salamini’s department, but was closed later, when laboratory-based techniques became more relevant. Potato was the crop plant that was used as a model to demonstrate that the development of molecular markers can in part overcome the lack of genetic approaches. Constructions of a physical and genetic map of the potato were linked to other potato research projects: potato cytogenetics, the generation of efficient anther plant-producing clones, the establishment of a potato population transformed with the maize transposable element Ac to obtain mutations by insertions, problems associated with potatoes stored at low temperatures, engineering virus-resistant potatoes by genetic transformation,


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the exploration of self-incompatibility, and a genetic system preventing self-fertilization. Just listing all the potato research projects and correlating them with their respective publications shows the multitude of approaches and reflects the advances achieved in potato research by the activities of Dr. Salamini and his department. Major outcomes of these projects were a physical and genetic map of potato, fingerprinting techniques to identify varieties and species, the generation of a potato leaf roll virus-resistant potato line, identification of genes relevant to potato carbohydrate metabolism and yield, as well as the identification and most recently the isolation of genes involved in disease resistance. The focus was on the diseases caused by cyst-forming nematodes, by the late-blight pathogen Phytophthora infestans, and by the potato leaf roll virus (PLRV). Late blight caused by P. infestans destroyed the potato crop in Ireland in the 1840s and resulted in mass starvation and emigration. Natural resistance in wild potato lines was explored to learn more about the genetic background of the disease. E. Sugarbeet A collaborative project was set up between Dr. Salamini’s department and the breeding company KWS Saat AG to analyze the organization of the sugarbeet genome with respect to such agronomically important traits as disease resistance and sugar yield. KWS provided and propagated the plant material for the analysis, while the MPI developed the molecular markers. The results were analyzed and discussed together (Hunger et al. 2003). Therefore, this project improved information transfer between the research community and industry. The focus of the project was the cloning of a gene that confers resistance to the virusinduced disease Rhizomania. In a second approach, molecular markers were identified for sugar metabolism. F. Science Politics Due to the leadership positions Francesco Salamini held in Germany and in Italy, he had to assume responsibility in science politics in order to assure funding for plant science in Europe and to direct its achievements to beneficial uses for the public. Dr. Salamini has been active on different levels. He had great input into various European activities to promote plant sciences, such as the Advanced Molecular Initiative in Community Agriculture (AMICA) initiative in connection with European Union projects and participation in the European Plant Sciences Organization (EPSO). He also helped to get modern plant biotechnology,

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including genetically modified plants, accepted by the general public. This has been a long process, which still is ongoing, but Francesco Salamini has contributed immensely with discussions and convincing statements on these controversial issues. His approach has always been directed toward applying genetically modified organisms for a sustainable and more ecologically oriented agriculture. An example can be found as ‘‘A Case Presentation’’ given at the InterAcademy Panel on International Issues (http://domino148a.nae.edu/iap/iapga.nsf/Events/ 16D39D5C7A1342DD85256D9F00488928?opendocument).

III. THE MAN A. Advisor and Editor Francesco Salamini has been influential in transmitting novel research contributions from the scientific community through journals. He served as editor for a number of journals: Theoretical and Applied Genetics, Journal of Genetics & Breeding, Journal of Plant Breeding, Maydica, Molecular Breeding, Genetical Research, Atti Lincei, Plant Biology, Plant Biotechnology Journal, and Caryologia. Dr. Salamini takes his editorial responsibilities very seriously, and he often reshapes and restructures manuscripts to help authors present their research adequately. Dr. Salamini is always ready to share his expertise with other scientists and to use his experience and broad knowledge to help individuals and organizations to promote science. As a result, he has been a member of many national and international advisory boards and scientific councils, such as the Board of Trustees CIMMYT (Mexico), the EMBO fellowship committee, advisory board of the German Plant Breeders (GFP), advisory board of International Centre for Agricultural Research CGIAR, the Plant Genome Initiative, GABI (Germany), Genome Programme Genoplante, Paris (France), and Scientific Counsel for Biotechnologies in Agriculture, Lombardy Region (Italy). Most recently he has served as the president of the Scientific Committee, Parco Tecnologico Padano Foundation, Lodi (Italy). Dr. Salamini was often asked to solve difficult or complicated problems within the Max Planck Institute and elsewhere. As an advisor, Francesco is a moderate, reliable, and fair colleague whose advice is actively sought, highly regarded, and respected in the scientific community. He does not try to turn the situation to his advantage, but always provides advice in the interest of promoting and advancing science,


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although this may have not always been apparent at first sight. Many of his approaches may have been unconventional and have led to unexpected discoveries. His original, unconventional ideas and his focus on fairness in science were the basis for a close friendship with his colleague Professor Jeff Schell. B. The Collector Francesco Salamini has long been an avid hunter and collector. As a child he collected wild animals and plants. Later on he extended these collections to large scientific collections of maize and barley mutants, Triticum monococcum lines (wild einkorn), and even publications in scientific journals. (To date, he has published more than 573 manuscripts with numerous collaborators, beginning in 1962.) His collections are eclectic, from flowers from the alpine mountains to stamps displaying mushrooms. His mushroom-collecting trips are legendary: After his mushroom forays in the Eifel hills, west of Cologne, he often returns with an enormous hoard of Boletus edulis, which he shares with friends and colleagues. C. Impact Francesco Salamini’s research and scientific achievements have been officially recognized and honored by several prestigious awards. He was elected to the Italian National Academy of Science (1990) and the Academia Europea, London, UK (1992). In 1995 he received the Invernizzi Foundation-Prize for his career. He has been a member of the European Molecular Biology Organization (EMBO) since 1992. In addition, his activities have been recognized by his membership in the Accademia dei Georgofili, Florence (Italy) and the Agriculture National Academy, Bologna (Italy). Plant science and plant breeding have undergone dramatic changes in the last 40 years as a result of the ability of bioinformatics to handle large data sets and through the advance of new technologies of molecular biology, such as polymerase chain reactions, complete genome sequences, and others. Dr. Salamini has always had a vision of how to use these new technologies for plant breeding. He has an excellent sense for new opportunities in science through the application of technological developments. This meant that sometimes he was not understood by scientific colleagues, plant breeders, or decision-making politicians because he was ahead of his time. He was not prepared to make compromises when he wanted to achieve a scientific goal, which often

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seemed to be far away. His persistence and patience coupled with intelligent and energetic leadership formed the basis for scientific success. ‘‘Life scientists are evolving from hunters to farmers’’ is a quotation from Dr. Frank Gannon, the previous executive director of EMBO, when he reflected on changes in life sciences. However, this statement characterizes very well the scientific life of Francesco Salamini. He started with ‘‘hunting’’ for mutants, for breeding lines, for isolated genes, for genetic and molecular markers, for a wide selection of plant species to be researched, for project grants, for scientific publications, and so on. Dr. Salamini’s career has spanned more than 40 years of active research in plant sciences. It is clear that he, together with his many scientific friends, collaborators, and students, has been able to put single achievements in a broad biological context, an effort that has greatly advanced and changed research areas relevant to using plants for the benefit of humankind. Scientists who have been trained, educated, and stimulated by Francesco Salamini today hold leading positions in institutions, universities, and breeding companies all over Europe. Dr. Salamini should be proud of his scientific family in the plant sciences. It is with both pride and humility that I dedicate Volume 30 of Plant Breeding Reviews to a great scientist, a committed plant breeder, and an inspiring mentor.

IV. ACKNOWLEDGMENTS Much information about Francesco Salamini’s early years in Italy was obtained from a dedication written in a special issue of Maydica by A. Bianchi and M. Motto 2002. I would like to thank Dr. J.F. Seitzer for making available his manuscript of a speech on ‘‘Forschung and Zu¨chtung—Symbiose,’’ which he presented on the occasion of Dr. Salamini’s departure from the MPI, Cologne. I would like to apologize to colleagues who collaborated with Dr. Salamini but whose work was not adequately described due to space restrictions.

V. LITERATURE CITED Avato, P., G. Avato, E. Gentinetta, and F. Salamini. 1984. Effect of trichloroacetic acid on wax composition of normal and mutant maize (Zea mays L.). J. Expt Bot. 35(151):245–251.


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Bartels, D., and F. Salamini. 2001. Desiccation tolerance in the resurrection plant Craterostigma plantagineum: a contribution to the study of drought tolerance at the molecular level. Plant Physiol. 127:1346–1353. Bianchi, A., and F. Salamini. 1962. Ultrastruttura della superficie fogliare di mutanti di mais e di frumento duro. Genetica Agraria 15:1–13. Bianchi, A., and M. Motto. 2002. Francesco Salamini: Pathways in plant genetics. Maydica 47:141–146. Biehl, A, E. Richly, C. Noutsos, F. Salamini, and D. Leister. 2005. Analysis of 101 nuclear transcriptomes reveals 23 distinct regulons and their relationship to metabolism, chromosomal gene distribution and co-ordination of nuclear and plastid gene expression. Gene 344:33–41. Heun, M., R. Scha¨fer-Pregl. R. Klawan, R. Castagna, M. Accerbi, B. Borghi, F. Salamini. 1997. Site of Einkorn wheat domestication identified by DNA fingerprinting. Science 278:1312–1314. Lohmer, S., M. Maddaloni, M. Motto, N. Di Fonzo, H. Hartings, F. Salamini, and R.D. Thompson. 1991. The maize regulatory locus Opaque-2 encodes a DNA-binding protein which activates the transcription of the b-32 gene. EMBO J. 10:617–624. Motto, M., G.P. Soressi, and F. Salamini. 1975. Mutation frequencies and chimeric formation in Phaseolus vulgaris after ems treatment of dormant seeds. Radiation Bot. 15:291–299. Motto, M., R.D. Thompson, and F. Salamini, 1997. Genetic regulation of carbohydrate and protein accumulation in seeds. pp. 479–522. In: B. Larkins and L.K. Vasil (eds.), Cellular and molecular biology of plant seed development. Kluwer Academic Publishers, Dordrecht, Netherlands. Motto, M., R.D. Thompson, N. Di Fonzo, G. Ponziani, C. Soave, M. Maddaloni, H. Hartings, and F. Salamini. 1989. Transpositional ability of the Bg-rbg system of maize transposable elements. Maydica 34:107–122. Peterson, P.A. 2002. Early beginnings of mobile element studies: Controlling elements vs. gene inserts, pre-molecular concepts. Maydica 47:147–167. Peterson, P.A., and F. Salamini. 1986. A search for active mobile elements in the Iowa StiffStalk synthetic maize population and some derivatives. Maydica 31:163–172. Salamini, F. 1981a. Controlling elements at the Opaque-2 locus of maize: their involvement in the origin of spontaneous mutation. Cold Spring Harbor Symp. Quant. Biol. 45:467–476. Soave, C., and F. Salamini. 1984. Organization and regulation of zein genes in maize endosperm. Phi. Trans. R. Soc. London B. 304:341–347. Sturaro, M., H. Hartings, E. Schmelzer, R. Velasco, F. Salamini, and M. Motto. 2005. Cloning and characterization of GLOSSY1, a maize gene involved in cuticle membrane and sax production. Plant Physiol. 138:478–489. Tacke, E., C. Korfhage, D. Michel, M. Maddaloni, M. Motto, S. Lanzini, F. Salamini, and H.P. Do¨ring. 1995. Transposon tagging of the maize GLOSSY2 locus with the transposable element En/Spm. Plant J. 8:907–917.

VI. PUBLICATIONS OF FRANCESCO SALAMINI Bianchi, A., and F. Salamini. 1962. Ultrastruttura della superficie fogliare di mutanti di mais e di frumento duro. Genetica Agraria 15:1–13. Salamini, F.. 1963. La superficie fogliare del mais. Maydica 8:67–72.

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Bianchi, A., and F. Salamini. 1963. Detection of linkage in maize by means of balanced lethal systems. Maydica 8:52–58. Bianchi, A., and F. Salamini. 1964. Ultrastruttura di superfici fogliari di mutanti radioindotti in Triticum durum. Genetica Agraria 18:183–194. Lorenzoni, C., M. Pozzi, and F. Salamini. 1964. Frequenze di mutanti in popolazioni di mais italiana. Genetica Agraria 19:146–158. Bianchi, A., B. Borghi, C. Lorenzoni, M. Pozzi, and F. Salamini. 1964. Mendelian characters in Italian maize. Maize Gen. Coop. Newsletter 38:89–91. Lorenzoni, C., and F. Salamini 1965. Cases of close linkages between endosperm and seedling traits. Maize Gen. Coop. Newsletter 39:113–114. Borghi, B., C. Lorenzoni, M. Pozzi, and F. Salamini. 1965. Reversion frequency of alleles of the gl1 locus and of some of their compounds. Maize Gen. Coop. Newsletter 39:111– 112. Salamini, F., and B. Borghi, 1966. Analisi genetica di mutanti glossy di mais. II. Frequenze di reversione al locus gl1. Genetica Agraria 20:239–248. Borghi, B., and F. Salamini. 1966. A three point test for an endosperm trait in chromosome 7. Maize Gen. Coop. Newsletter 40:77–78. Borghi, B., and F. Salamini 1966. Analisi genetica di mutanti glossy di mais. I. Frequenze per i diversi loci. Maydica 11:45–57. Borghi, B., and F. Salamini 1966. Analisi genetica di mutanti glossy di mais. I. Frequenze per i diversi loci. Genetica Agraria 20:237–238. Salamini, F. 1967. Ricombinazione intracistronica nel mais: il locus Su1. Atti Ass. Genet. Ital. 12:417–425. Salamini, F. 1967. Reversion frequency of alleles of the su1 locus and of some their compounds. Maize Gen. Coop. Newsletter 41:97–100. Salamini, F. 1967. An unstable locus affecting aleurone and anther color. Maize Gen. Coop. Newsletter 41:100–101. Salamini, F., and T. Ekpenyong, 1967. Weight of opaque (o2) and normal kernels on the same ear. Maize Gen. Coop. Newsletter 41:101–102. Bianchi, A., and F. Salamini. 1967. Eredita` e miglioramento genetico del mais. Sementi Elette 13:256–313. Bianchi, A., F. Salamini, and A. Ghidoni, 1967. Genetical and cytological data on Marocco, maize. Maize Gen. Coop. Newsletter 41:107–108. Buiatti, M., P. Rotili, and F. Salamini. 1967. Problemi del miglioramento vegetale: produzione cerealicolo-foraggera in funzione della zootecnia. pp. 236–254. In: Crisi dell’Agricoltura e Ricerca. Ist. Gramsci-CESPE, Bologna. Piva, G., F. Salamini, and E. Santi, 1967. Caratteristiche proteiche e valore alimentare del mais omozigote per l’allele ‘‘opaque-2’’ isolato da una popolazione italiana. Alimentazione Animale 11:1–11. Salamini, F. 1968. Variabilita` mendeliana nelle varieta` italiane di mais. Maydica 13: 1–45. Bianchi, A., and F. Salamini. 1968. Further data on an unstable factor affecting anther and aleurone color. Maize Gen. Coop. Newsletter 42:91. Bianchi, A., and F. Salamini. 1968. Il controllo genetico della sintesi delle proteine. In: Fattori genetici per il miglioramento qualitativo del mais. Scienza dell’Alimentazione 1:26–34. Bianchi, A., and F. Salamini. 1968. Il miglioramento genetico del mais. pp. 17–32. In: La Maiscoltura. Ist. di Tecn. e Prop. Agr., Roma, Ekpenyong, T., and F. Salamini. 1968. Biochemical and breeding aspects of opaque-2. Maize Gen. Coop. Newsletter 42:89–91.


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Salamini, F., and G. Baldi, 1969. Nitrogen, protein and amino acid content of opaque-2 and normal kernels in F2 maize with different genetic backgrounds. Genetica Agraria 23(1– 4):111–114. Salamini, F., G. Baldi, and A. Bianchi, 1969. Azoto totale, frazioni proteiche e aminoacidi in diverse F2 di mais segreganti seme normale e opaco-2. Maydica 14:3–22. Bianchi, A., and F. Salamini. 1969. Il mais. Quaderno II. Trattato di Genetica Agraria speciale. Bianchi, A., F. Salamini, and R. Parlavecchio. 1969. On the origin of controlling elements in Maize. Genetica Agraria 22:335–344 Bianchi, A., F. Salamini, and F. Restaino. 1969. Concomitant occurrence of different controlling elements. Maize Gen. Coop. Newsletter 43:91. Salamini, F., and C. Lorenzoni, 1970. Genetical analysis of glossy mutants of maize. III. Intracistron recombination and high negative interference at the gl1 locus. Mol. Gen. Genet. 105:225–232. Salamini, F., B. Borghi, and C. Lorenzoni. 1970. The effect of the opaque-2 gene on yield in maize. Euphytica 19:531–538. Lorenzoni, C., and F. Salamini. 1970. Analisi di mutanti glossy in mais. III. Possibili meccanismi di reversione. Atti Ass. Genet. Ital. 15:184–185. Lorenzoni, C., and F. Salamini. 1970. Further data on the cp2(collapsed-2) location. Maize Gen. Coop. Newsletter 44:93. Lorenzoni, C., and F. Salamini. 1970. A rt (rootless) mutant detectable at an early seedling stage. Maize Gen. Coop. Newsletter 44: 93–44. Bianchi, A., F. Salamini, and R. Parlavecchio. 1970. On the origin of regulatory elements in maize. Atti Ass. Genet. Ital. 15:335–344. Tsai, C.Y., F. Salamini, and O.E. Nelson, 1970. Enzymes of carbohydrate metabolism in the developing endosperm of maize. Plant Physiol. 46:299–306. Salamini, F. 1971. Mais: evoluzione e miglioramento genetico. Gazzettino Allevatore 4:3–4. Salamini, F., and L. Uncini, 1971. Frequenze di esincrocio nel fagiolo di spagna (Phaseoluscoccineus L., Phaseolus multiflorus, Willd). Ann. Ist. Sper. Ort., 2: 9–14. Salamini, F., G.P. Soressi, and C. Lorenzoni, 1971. Induzione di contemporaneita` di maturazione in cultivar di pomodoro a sviluppo determinato mediante trattamento con acido 2-cloro-etilfosfonico. Rivista Agronomia 5:49–53. Bianchi, A., C. Lorenzoni, and F. Salamini. 1971. Acquisizioni e prospettive nel miglioramento del mais. In: Convegno Naz. Aspetti Problemi Maiscoltura Italiana, Pacini, Pisa. Lorenzoni, C., M. Pozzi, and F. Salamini. 1971. Opaque endosperm mutants in Italian varieties. Maize Gen. Coop. Newsletter 45:89. Lorenzoni, C., G. Baldi, T. Maggiore, and F. Salamini. 1971. Spighette biflore sulla infiorescenza femminile del mais. Maydica 16:65–82. Lorenzoni, C., T. Maggiore, M. Scandura, V. Servello, and F. Salamini. 1971. Effetti del genotipo e dell’ambiente sulla qualita` del mais opaco-2. Maydica 16:101–116. Salamini, F., C.Y. Tsai, and O.E. Nelson, 1972. Multiple forms of glucosephosphate isomerase in maize. Plant Physiol. 50:256–261. Salamini, F., B. Borghi, C. Lorenzoni, T. Maggiore, and B.M. Mariani, 1972. Ibridi opaco-2 in italia settentrionale. II. Attitudine combinatoria in linee convertite. Maydica 17:120– 133. Lorenzoni, C., and F. Salamini. 1972. Genes for spikelet bearing two fertile pistillate flowers. Maize Gen. Coop. Newsletter 46:98–99. Baldi, G., M. Buiatti, and F. Salamini. 1972. Introduzione al miglioramento genetico qualitativo. Quaderno X. Trattato di Genetica Agraria Speciale. Edagricole, Bologna.

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Maggiore, T., C. Lorenzoni, G.P. Soressi, B.M. Mariani, and F. Salamini. 1972. Ibridi opaco-2 in Italia settentrionale. I. Prove di confronto 1971. Maydica 17:67–94. Salamini, F. 1973. Variabilita` genetica per quantita` e qualita` delle proteine delle leguminose con particolare riferimento al genere ‘‘Phaseolus’’. Convegno Istituto Ital-Lat. Americano, Roma, IILA:299–317. Baldi, G., and F. Salamini. 1973. Variability of essential amino acid content in seeds of 22 Phaseolus species. Theor. Appl. Genet. 43:75–78. Gentinetta, E., and F. Salamini. 1973. Phosphoglucose isomerase I and II in developing and germinating rice endosperm. Il Riso 22(4):303–311. Baldi, G., C. Lorenzoni, T. Maggiore, and F. Salamini. 1973. Fenotipico della cariosside e contenuto in aminoacidi nelle proteine endospermiche della linea pura di mais Lo32o2. Maydica 18:7176. Gentinetta, E., C. Lorenzoni, M. Odoardi, and F. Salamini. 1973. Collapsed endosperm-1 (cp1), una nuova mutazione del cromosoma 7 del mais interessante il metabolismo dei carboidrati. Maydica 18:2–15. Odoardi, M., E. Gentinetta, G.P. Soressi, and F. Salamini. 1973. Metabolismo del saccarosio seme di fagiolo in diversi stadi dello sviluppo. Ann. Ist. Sper. per l’Orticoltura 4:57–68. Odoardi, M., E. Gentinetta, G.P. Soressi, and F. Salamini. 1973. Forme multiple della fosfoesoisemerasi nel seme di fagiolo durante lo sviluppo. Ann. Ist. Sper. per l’Orticoltura 4:69–71. Bianchi, A., B. Borghi, C. Lorenzoni, F. Salamini, and G.P. Soressi, 1973. Acquisizioni di genetica vegetale e insegnamento. Atti Ass. Genet. Ital. 18:79–90. Lorenzoni, C., F. Coppolino, T. Maggiore, B.M. Mariani, and F. Salamini. 1973. Prove di attitudine combinatoria con linee precoci di recente costituzione. Maydica 18:112–131. Maggiore, T., C. Lorenzoni, B.M. Mariani, M. Odoardi, and F. Salamini. 1973. Ibridi opaco2 in Italia settentrionale. IV. Prova di confronto tra tipi scelti nel 1971. Maydica 18:40– 44. Lorenzoni, C., N. Di Fonzo, T. Maggiore, B.M. Mariani, and F. Salamini. 1973. Ibridi opaco-2 in Italia settentrionale. III. Ricerca di genotipi che riducono l’umidita` della granella alla raccolta. Maydica 18:33–39. Lorenzoni, C., T. Maggiore, B.M. Mariani, A.M. Stanca, and F. Salamini. 1973. Ibridi opaco-2 in Italia settentrionale. V. Prove di attitudine combinatoria 1972. Maydica 18:86–111. Maggiore, T., B. Borghi, C. Lorenzoni, B.M. Mariani, F. Salamini, and G.P. Soressi, 1973. Determinated hybrid tomatoes. I Comparison between hybrids and vaieties and definition of a standard fruit-type for mechanical harvesting. Genetica Agraria 37:235–280. Soressi, G.P., B. Borghi, C. Lorenzoni, T. Maggiore, B.M. Mariani, and F. Salamini. 1973. Ibridi di pomodoro determinati. II. Valutazione dell’attitudine combinatoria di 106 varieta`. Sementi Elette 19(4):3–35. Salamini, F., 1974. Fagiolo comune. Monografie di genetica Agraria 2:141–154. Salamini, F. 1974. Principi e metodi per una moderna tecnica di miglioramento delle sementi. Monografie di Genetica Agraria 2:19–32. Soressi, G.P., E. Gentinetta, M. Odoardi, and F. Salamini. 1974. Leaf peroxidase activity in tomato mutants affecting plant morphology. Biochem. Genetics 12:181–198. Maggiore, T., C. Lorenzoni, B.M. Mariani, M. Omacini, and F. Salamini. 1974. Ibridi opaco-2 in Italia settentrionale. Maydica 19:28–46. Maggiore, T., C. Lorenzoni, M. Amigoni, F. Coppolino, and F. Salamini. 1974. Ibridi opaco-2 in Italia settentrionale. VII. Prove di confronto fra i migliori ibridi 1973-1974. Annali Ist. Sper. Cereal. 5:91–195.


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Lorenzoni, C., T. Maggiore, M. Amiconi, M. Motto, and F. Salamini. 1974. Ibridi opaco-2 in Italia settentrionale. VIII. Influenza della densita` di coltivazione sul comportamento di ibridi a elevata produttivita`. Annali Ist. Sper. Cereal. 5:185–190. Salamini, F., T. Maggiore, C. Lorenzoni, and G.P. Soressi. 1975. Ibridi di pomodoro. III. Attitudine combinatoria di 86 varieta` da industria. Sementi Elette 21:3–23. Salamini, F., E. Fornasari, E. Gentinetta, T. Maggiore, and C. Lorenzoni, 1975. Efficacy of the DBC test in the identification of maize inbreds with high-quality proteins. Maydica 20:185–195. Bianchi, G., and F. Salamini. 1975. Glossy mutants of maize. IV. Chemical composition of normal epicuticular waxes. Maydica 20:1–3. Lorenzoni, C., F. Salamini. 1975. Glossy mutants of maize. V. Morphology of the epicuticular waxes. Maydica 20:5–19. Bianchi, G., P. Avato, and F. Salamini, 1975. Glossy mutants of maize. VI. Chemical constituents of glossy-2 epicuticular waxes. Maydica 20:165–173. Motto, M., G.P. Soressi, and F. Salamini, 1975. Mutation frequencies and chimeric formation in Phaseolus vulgaris after ems treatment of dormant seeds. Radiation Botany 15:291–299. Maggiore, T., C. Lorenzoni, F. Coppolino, B.M. Mariani, and F. Salamini, 1975. Ibridi di opaco-2 in Italia settentrionale. X. Prove di attitudine combinatoria 1975. Ann. Ist. Sper. Cerealicoltura 6:93–101. Soave, C., F. Pioli, A. Viotti, F. Salamini, and P.G. Righetti, 1975. Synthesis and heterogeneity of endosperms proteins in normal and opaque-2 maize. Maydica 20:83–94. Gentinetta, E., T. Maggiore, F. Salamini, C. Lorenzoni, F. Pioli, and C. Soave. 1975. Protein studies in 46 opaque-2 strains with modified endosperm texture. Maydica 20:145–164. Salamini, F. 1976. Mais: ricerche genetiche e fisiologia della produzione conducono questa pianta verso nuove mete. L’Informatore Agrario 32:21917–21920. Salamini, F., and A. Allavena. 1976. Fagiolo e fagiolino per serra: indicazioni e prospettive. Colture protette 5:53-54. Maggiore, T., M. Motto, F. Salamini, and G.P. Soressi, 1976. Attitudine combinatoria per l’adattabilita` alla coltura protetta in pomodoro (L. esculentum Mill.). Sementi Elette 22:9–16. Odoardi, M., S. Palmieri, G. Giacomozzi, G.P. Soressi, and F. Salamini. 1976. Enzimi del metabolismo dei carboidrati durante lo sviluppo del seme in pisello liscio e rugoso. Rivista di Agronomia 10(4):233–239. Soave, C., P.G. Righetti, C. Lorenzoni, E. Gentinetta, and F. Salamini. 1976. Expressivity of the opaque-2 gene at the level of zein molecular components. Maydica 21:61–75. Maggiore, T., and F. Salamini. 1977. Maiscoltura e foraggicoltura: contrapposizione o integrazione? Problemi della coltivazione del mais. pp. 13–28. In: E. A. Fiera di Padova. ed., Dimostrazione di macchine, impianti, attrezzature e sistemi per la raccolta, la lavorazione e la conservazione dei foraggi e del mais. Fiera di Padova, Padova. Bianchi, G., P. Avato, and F. Salamini. 1977. Glossy mutants of maize. VII. Chemistry of glossy 1, glossy 3, and glossy 7 epicuticular waxes. Maydica 22:9–17. Di Fonzo, N., E. Fornasari, and. F. Salamini. 1977. SDS-protein subunits in normal, opaque-2 and floury-2 maize endosperms. Maydica 22:77–88. Di Fonzo, N., E. Fornasari, E. Gentinetta, F. Salamini, and C. Soave. 1977. Proteins and carbohydrate accumulation in normal, opaque-2 and floury maizes. pp. 199–212. In: B.J. Miffling and M. Zoeschke (eds.), Carbohydrate and protein synthesis. Seminar CEE, Giessen, Germany. Lorenzoni, C., T. Maggiore, F. Salamini, et al. 1977. Mais: scelte varietali. L’Informatore Agrario 8:25639–25680.

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Giannazza, E., V. Viglienghi, P.G. Righetti, F. Salamini, and C. Soave. 1977. Amino acid composition of zein molecular components. Phytochemistry 15:315–317. Maggiore, T., S. Edallo, M. Perenzin, E. Gentinetta, and F. Salamini. 1977. Effetti della concimazione azotata sulla qualita` proteica del mais normale, opaco-2 e farinoso-2. Rivista di Agronomia 11:158–166. Righetti, P.G., E. Giannazza, F. Salamini, E. Galante, A. Viotti, and C. Soave. 1977. Zein; macromolecular properties, biosynthesis and genetic regulation. Electrofocusing and isotachophoresis, pp. 200–211. W. de Gruyter and Co., Berlin. Soave, C., A. Viotti, F. Salamini, E. Gentinetta, E. Giannazza, and P.G. Righetti. 1977. Techniques for the separation of seeds proteins of maize. pp 61-68 In: B.J. Miflin, P.R. Shewry (eds.), Techniques for the separation of barley and maize proteins. D.G. Agriculture, C.E.C., EUR 5687e., Bruxelles . Maggiore, T., M. Bertolini, C. Lorenzoni, B.M. Mariani, M. Perenzin, and F. Salamini. 1977. Prove di ibridi di mais sperimentali da granella tardivi, medi e precoci. Annali Ist. Sper. Cerealicoltura, Roma, 8 Suppl. no. 1. Lorenzoni, C., P. Alberi, A. Viotti, C. Soave, N. Di Fonzo, E. Gentinetta, T. Maggiore, and F. Salamini. 1977. Biochemical characterisation of high and low protein strains of maize. pp. 173–197.In: B.J. Miffling and M. Zoeschke (Eds).Carbohydrate and protein synthesis. Seminar CEE, Giessen, Germany. Salamini, F., A. Fadda, A. Allavena, M. Motto, and G.P. Soressi, 1978. Nuove varieta` di fagiolo resistente al mosaico comune (BCMV) costituite in Italia. L’Informatore Agrario 34:3390–3393. Bianchi, A., and F. Salamini. 1978. Modelli morfo-fisiologici per seconda coltura e trinciati. Quaderni di Agricoltura Ricerca, Suppl. no. 1:42–63. Bianchi, G., P. Avato, and F. Salamini. 1978. Glossy mutants of maize. VIII. Accumulation of fatty aldehydes in surface waxes of gl5 maize seedlings. Biochemical Genetics 16:1015–1021. Motto, M., G.P. Soressi, and F. Salamini. 1978. Seed size inheritance in a cross between wild and cultivated common beans (Phaseolus vulgaris L.). Genetica 49:31–36. Nucca, R., C. Soave, M. Motto, and F. Salamini. 1978. Taxonomic significance of the zein isoelectric focusing pattern. Maydica 23:239–249. Palmieri, S., M. Odoardi, G.P. Soressi, and F. Salamini. 1978. Indoleacetic acid oxidase activity in two high-peroxidase tomato mutants. Physiol. Plant. 42:85–90. Soave, C., N. Suman, A. Viotti, and F. Salamini. 1978. Linkage relationships between regulatory and structural gene loci involved in zein synthesis in maize. Theor. Appl. Genet. 52:263–267. Motto, M., C. Lorenzoni, E. Gentinetta, T. Maggiore, and F. Salamini. 1978. Expected genetic gain for protein related traits and allocation of resources in a modified opaque-2 synthetic. Maydica 23:35–44. Soave, C., S. Dossena, C. Lorenzoni, N. Di Fonzo, and F. Salamini. 1978. Expressivity of the floury-2 allele at the level of zein molecular components. Maydica 23:145– 152. Martiniello, P., C. Lorenzoni, A.M. Stanca, T. Maggiore, E. Gentinetta, and F. Salamini. 1978. Seed quality differences between normal, floury-2 and opaque-2 maize inbreds. Euphytica 27:411–417. Maggiore, T., C. Lorenzoni, F. Salamini, et al. 1978. Scelte varietali. L’Informatore Agrario 34:623–685. Salamini, F. 1979. Il mais a ‘‘foglie erette’’. Civilta` Verde 33, Suppl. no 1: 6–13. Salamini, F. 1979. Fisiologia e produttivita`. In: Maiscoltura degli anni 80. Italia Agricola 116:262–270.


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Salamini, F., A. Fadda, and G.P. Soressi, 1979. Inge P73: varieta` rampicante di fagiolo tipo borlotto resistente al mosaico comune (BCMV). Ann. Ist. Sper. Orticoltura, 8:1–7. Salamini, F., N. Di Fonzo, E. Gentinetta, and C. Soave, 1979. A dominant mutation interfering with protein accumulation in maize seeds. Seed protein improvement in cereals and grain legumes. IAEA- SM 230/38: 97–108, Vienna. Salamini, F., A. Fadda, A. Allavena, and G.P. Soressi. 1979. Mary P2: Varieta` nana di fagiolo da granella a seme screziato resistente al mosaico comune (BCMV). Ann. Ist. Sper. Orticoltura, 8(121):1–6. Gentinetta, E., and F. Salamini. 1979. Free sugar fraction of the amylose-related mutants of maize. Biochemical Genetics 17:405–414. Soave, C., and F. Salamini. 1979. High-lysine genes in maize: theoretical and applied aspects. Genetica Agraria, Mon. 4:107–140. Bianchi, G., P. Avato, and F. Salamini. 1979. Glossy mutants of maize. IX. Chemistry of glossy-4, glossy-15 and glossy-18 surface waxes. Heredity 42:391–395. Bianchi, G., P. Avato, and F. Salamini. 1979. The absence of fatty acid aldehydes in gl1 gl2 waxes. Maize Gen. Coop. Newsletter 53:103. Di Fonzo, N., E. Gentinetta, and F. Salamini. 1979. Action of the opaque-2 mutation on the accumulation of storage products in maize endosperm. Plant Sci. Lett. 14:345– 354. Gentinetta, E., M. Zambello, and F. Salamini. 1979. Free sugars in developing maize grain. Cereal Chem. 56(2):81–83. Motto, M., G.P. Soressi, and F. Salamini. 1979. Growth analysis in a reduced leaf mutant of common bean (Phaseolus vulgaris L.). Euphytica 28:593–600. Motto, M., F. Salamini, R. Reggiani, and C. Soave, 1979. Evaluation of genetic purity in hybrid corn (Zea mays L.) seed production through zein isoelectrophoretic patterns. Maydica 24:223–233. Motto, M., A. Fadda, G.P. Soressi, and F. Salamini. 1979. Lisa P71: varieta` nana di fagiolo da granella a seme bianco resistente al virus del mosaico comune (BCMV). Ann. Ist. Sper. Orticoltura 8(123):1–6. Soave, C., A. Viotti, N. Di Fonzo, and F. Salamini. 1979. Maize prolamin: Synthesis and genetic regulation. pp. 165–174. In: Seed protein improvement in cereals and grain legumes. IAEA-SM 230/39, Vienna. Maggiore, T., C. Lorenzoni, M. Motto, M. Perenzin, and F. Salamini. 1979. Produzione di sostanza secca totale e di granella in ibridi di mais tardivi. Rivista di Agronomia 13:431– 435. Maggiore, T., M. Bertolini, C. Fogher, C. Lorenzoni, P. Manmana, A. Vederio, and F. Salamini. 1979. Prove di ibridi di mais sperimentali da granella tardivi, medi e precoci. Anni 1978–1979. Ann. Ist. Sper. Cerealicoltura 10, Suppl. n8 1:1–7. Lorenzoni, C., C. Fogher, M. Bertolini, N. Di Fonzo, E. Gentinetta, T. Maggiore, and F. Salamini. 1979. Prove di attitudine combinatoria in linee di opaco-2. Prove di attitudine combinatoria di linee farinoso-2. Anni 1978–1979. Ann. Ist. Sper. Cerealicoltura, Vol. 10, Suppl. 3. Maggiore, T., C. Lorenzoni, F. Salamini, et al. 1979. Prove comparative 1978. L’Informatore Agrario 35:4717–4753. Salamini, F.1980. Genetic instability at the Opaque-2 locus of maize. Mol. Gen. Genet. 179:497–507. Gavazzi, G., and F. Salamini. 1980. ‘‘Quaderni di Biologia.’’: mutagenesi e miglioramento delle piante coltivate. Piccin, Padova, Roma, Milano. . Di Fonzo, N., F. Coppolino, and F. Salamini. 1980. The main haemolymph protein of Ostrinia nubilalis HB during larval development. Redia 63:81–85.

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Soave, C., A. Viotti, N. Di Fonzo, and F. Salamini. 1980. Recent evidence concerning the genetic regulation of zein synthesis. pp. 219–-226. In: C.J. Leaver (ed.), Genome organization and expression in plants. Plenum, New York. Manzocchi, L.A., M.G. Daminati, E. Gentinetta, and F. Salamini. 1980. Viable defective endosperm mutants in maize. I. Kernel weight, protein fractions and zein subunits in mature endosperms. Maydica 25:105–116. Di Fonzo, N., E. Fornasari, F. Salamini, R. Reggiani, and C. Soave. 1980. Interaction of maize mutants floury-2 and opaque-7 with opaque-2 in the synthesis of endosperm proteins. J. Hered. 71:397–402. Lorenzoni, C., C. Fogher, M. Bertolini, N. Di Fonzo, E. Gentinetta, T. Maggiore, and F. Salamini. 1980. On the relative merit of opaque-2, floury-2, and opaque-2floury-2 in breeding maize for quality. Maydica 25:33–39. Maggiore, T., E. Gentinetta, M. Motto, M. Perenzin, F. Salamini, and C. Lorenzoni, 1980. Variability for protein and fibre content in three diallels of maize for silage production. pp. 135–153. In: W. G. Pollmer and R. H. Phipps (eds.), Improvement of quality traits of maize for grain and silage use. Martinus Nijhoff, The Hague, the Netherlands. Motto, M., N. Di Fonzo, M. Perenzin, E. Gentinetta, T. Maggiore, F. Salamini, and C. Lorenzoni. 1980. Genetic control of protein percentage in a diallel involving highprotein inbreds. pp. 117–134. In: W.G. Pollmer and R.H. Phipps (eds.), Improvement quality traits of maize for grain silage use. Martinus Nijhoff, The Hague, the Netherlands. Lorenzoni, C., M. Bertolini, E. Gentinetta, C. Fogher, N. Di Fonzo, T. Maggiore, and F. Salamini. 1980. Miglioramento qualitativo del mais: confronto fra genotipi opaco-2, farinoso-2 e opaco-2 farinoso-2. Annali UCSC Piacenza 20:75–100. Maggiore, T., C. Lorenzoni, F. Salamini et al. 1980. Prove di ibridi commerciali per la produzione di granella. L’Informatore Agrario 36:9243–9270. Salamini, F. 1981. Quale seme di mais? Un dibattito utile. Terra e Vita 22:23–25. Salamini, F. 1981. Prove di confronto tra ibridi di mais da granella. Agricoltura Ricerca 4:19–34. Salamini, F. 1981. Report of consultancy POL/77/07 (maize breeding). Warsaw, 11–20 November. Salamini, F. 1981. Controlling elements at the Opaque-2 locus of maize: their involvement in the origin of spontaneous mutation. Cold Spring Harbor Symp. Quant. Biol. 45:467– 476. Salamini, F. 1981. Genetic basis of physiological traits related to maize productivity. Rivista di Agronomia 15:3–19. Soave, C., and F. Salamini. 1981. Il seme del mais. pp. 20–26. In: Mais. Monografia Tecnica CIBA-GEIGY. Bianchi, A., F. Salamini, F. Coppolino, and G. Mariani, 1981. Four-years summary (1977– 1980) of results obtained. Probleme de Prectoria Plantelor 9:97–100. Edallo, S., C. Zucchinali, M. Perenzin, and F. Salamini. 1981. Chromosomal variation and frequency of spontaneous mutation associated with in vitro culture and plant regeneration in maize. Maydica 26:39–56. Fogher, C., C. Lorenzoni, E. Gentinetta, and F. Salamini. 1981. The surface of amyloseextender starch granules. Maydica 26:57–62. Soave, C., R. Reggiani, N. Di Fonzo, and F. Salamini. 1981. Clustering of genes for 20 kd zein subunits in the short arm of maize chromosome 7. Genetics 97:363–377. Soave, C., L. Tardani, N. Di Fonzo, and F. Salamini. 1981. Zein level in maize endosperm depends on a protein under control of the opaque-2 and opaque-6 loci. Cell 27:403–410.


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Vederio, A., and F. Salamini. 1983. Al mais la notte piace fresca. Il Giornale di Agricoltura 93:36–41. Marotta, R., M. Bremenkamp, and F. Salamini. 1983. A simple method for filter hybridization of labeled restriction fragments excised from gels. Anal. Biochem. 130:27–31. Motto, M., M. Perenzin, F. Salamini, and C. Soave. 1983. Maize chromosome 7 revisited. Maydica 28:25–39. Galante, E., A. Vitale, L. Manzocchi, C. Soave, and F. Salamini. 1983. Genetic control of a membrane component and zein deposition in maize endosperm. Mol. Gen. Genet. 192:316–321. Bertolini, M., N. Di Fonzo, E. Gentinetta, M. Motto, A. Vederio, F. Salamini, C. Fogher, C. Lorenzoni, T. Maggiore, and A. Bianchi, 1983. Lo876o2, linea pura di mais ‘‘high lysine’’ sviluppata in Italia. Sementi Elettere 29:21–25. Salamini, F. 1984. Il concetto di trasposizione genica e la sua rilevanza negli studi applicativi. Genetica Agraria 6:31–48. Salamini, F., and E. Lupotto, 1984. La biotecnologia nel miglioramento genetico delle produzioni vegetali. L’Italia Agricola 121:127–143. Salamini, F., N. Di Fonzo, E. Gentinetta, R. Reggiani, and C. Soave, 1984. Trasporto degli assimilati e produttivita` in mais. Agricoltura Ricerca 6:5–11. Devreux, M., and F. Salamini. 1984. Self-incompatibility and transposable elements. Plant Cell Incompatibility Newsletter 16:2–3. Soave, C., and F. Salamini. 1984. Organization and regulation of zein genes in maize endosperm. Phi. Trans. R. Soc. London B. 304:341–347. Soave, C., and F. Salamini. 1984. The role of structural and regulatory genes in the development of maize endosperm. Develop. Gen. 5:1–25. Avato, P., G. Bianchi, and F. Salamini. 1984. Genetic control of epicuticular lipids in maize (Zea mays L.). pp. 503–506. In: P.A. Starlinger and W. Eichenberger (eds.), Function and metabolism of plant lipids. Elsevier Science Publ., Amsterdam, the Netherlands. Bianchi, G., P. Avato, and F. Salamini. 1984. Surface waxes from grain, leaves and husks of maize (Zea mays L.). Cereal Chem. 61:45–47. Avato, P., G. Avato, E. Gentinetta, and F. Salamini. 1984. Effect of trichloroacetic acid on wax composition of normal and mutant maize (Zea mays L.). J. Experimental Botany 35:245–251. Torti, G., L. Lombardi, L.A. Manzocchi, and F. Salamini. 1984. Indole-3-acetic acid content in viable defective endosperm mutants of maize. Maydica 29:335–343. Montanelli, C., N. Di Fonzo, R. Marotta, M. Motto, C. Soave, and F. Salamini. 1984. Occurrence and behaviour of the components of the o2-m(r)-Bg system of maize controlling elements. Mol. Gen. Genet. 197:209–218. Hartings, H., Bonanomi, C. Soave, N. Di Fonzo, and F. Salamini. 1984. Mapping of genes for minor zein SDS subunits and revision of zein genes nomenclature. Genetica Agraria 38:447–464. Zangara, L., N.G. Di Bartolomeo, F. Salamini, and M. Bertolini, 1984. Caratterizzazione morfo-fisiologica degli ibridi di mais iscritti al registro Nazionale nel 1984. pp. 3–23. In: Repertorio nazionale delle Novita` Vegetali iscritte 1983/194. Ed. Ente Fiera Vicenza. Do¨ring, H.P., M. Freeling, M.A. Johns, P. Kunze, A. Merckelbach, F. Salamini, and P. Starlinger. 1984. A Ds-mutation of the Adh1 gene in Zea mays L. Mol. Gen. Genet. 193:199–204. Bianchi, A., C. Lorenzoni, T. Maggiore, M. Bertolini, F. Coppolino, N. Di Fonzo, E. Gentinetta, M. Motto, A. Vederiio, and F. Salamini. 1984. Linee di mais selezionate per gli ambienti italiani. Agricoltura Ricerca 6:5–12.


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Ardigo`, A., F. Salamini, et al. 1984. Ibridi commerciali per la produzione di granella. L’Informatore Agrario 40:49–65. Salamini, F. 1985. Aspetti molecolari della genetica delle piante: il concetto di trasposizione genica e la sua rilevanza negli studi applicativi. In: Atti Acc. Agric. Sci. Lett. di Verona. 36:105–118. Salamini, F. 1985. Protocolli di metodologie sperimentali di Genetica Agraria. CNR, IPRA Quaderno no. 1. Salamini, F., M. Bremenkamp, N. Di Fonzo, L. Manzocchi, R. Marotta, M. Motto, and C. Soave. 1985. Genetic regulation of zein deposition in maize. pp. 543–553. In: L. van Vloten Doting, S.P.G. Groot, and C.T. Hall (eds.), Molecular form and function of the plant genome. Plenum, New York. Salamini, F., M. Brembilla, M. Bremenkamp, N. Di Fonzo, L. Manzocchi, R. Marotta, C. Montanelli, M. Motto, G. Ponziani, and C. Soave. 1985. Alleles of the opaque-2 locus in maize and molecular approaches to zein regulation. pp. 715–721. In: M. Freeling (ed.), Plant genetics. Alan R. Liss, Inc., New York. Brandolini A., and F. Salamini (Eds). 1985. Breeding strategies for maize production improvement in the tropics. Proc. Inter. Expert Cons. Firenze and Bergamo, FAO and Ist. Agr. Oltremare. Pub. 100. Trapani, N., and F. Salamini. 1985. Germination capacity of endosperm mutants of maize under osmotic stress conditions. Maydica 30:121–124. Avato, P., G. Bianchi, and F. Salamini. 1985. Absence of long chain aldehydes in the wax of the glossy-11 mutant of maize. Phytochemistry 24:1995–1997. Marotta, R., M. Motto, and F. Salamini. 1985. Mutagenesi da trasposizione in mais. Quad. n. 1. Protocolli di Metodologie Sperimentali di Genetica Agraria. pp. 19–20. CNR, IPRA Roma. Bianchi, A., G. Bianchi, P. Avato, and F. Salamini. 1985. Biosynthetic pathways of epicuticular wax of maize as assessed by mutation, light, plant age and inhibitor studies. Maydica 30:179–198. Bianchi, A., and F. Salamini. 1985. Metodologia sperimentale per il confronto tra ibridi di mais. L’Informatore Agrario 41:57–59. Gentinetta, E., N. Di Fonzo, G. Ponziani, F. Salamini, and C. Soave, 1985. Comparison of nitrogen assimilation enzymes in single and double high lysine mutants of maize. Genetica Agraria 39:193–200. Reggiani, R., C. Soave, N. Di Fonzo, E. Gentinetta, and F. Salamini. 1985. Factors affecting starch and protein content in developing endosperms of high and low protein strains of maize. Genetica Agraria 39:221–232. Zangara, L., N.G. Di Bartolomeo, F. Salamini, and M. Bertolini, 1985. Caratterizzazione morfo-fisiologica degli ibridi di mais iscritti al Registro Nazionale Varieta` nel 1985. pp. 3–52. In: Repertorio nazionale ibridi di mais iscritti nel 1985. Ed. Ente Fiera di Vicenza. Ardigo`, A., L. Bocchi, F. Salamini, et al. 1985. Ibridi commerciali per la produzione di granella. L’Informatore Agrario 41:63–84. Salamini, F. 1986. Photosensitivity in maize: evaluation, genetics and breeding for insensitivity. pp. 143–157. In: A. Brandolini and F. Salamini (eds.), Breeding strategies for maize improvement in the tropics.Proc. Inter. Expert Cons. Firenze and Bergamo, FAO and Ist. Agr. Oltremare. Pub. 100. Salamini, F., C. Lorenzoni, and C. Soave. 1986. The use of advanced methodologies in plant quality improvement—two selected cases. Congr. Deut. Gesell. fu¨r Qualita¨tsforschung (Pflanzliche Nahrungsmittel) DGQ XXI., pp. 95–116.Vortratagung. Geisenheim, Rheingau.

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Peterson, P.A., and F. Salamini. 1986. A search for active mobile elements in the Iowa StiffStalk Synthetic maize population and some derivatives. Maydica 31:163–172. Torti, G., L. Manzocchi, and F. Salamini. 1986. Free and bound indoleacetic acid is low in the endosperm of the maize mutant defective endosperm-B18. Theor. Appl. Genet. 72:602–605. Allavena, A., A. Fadda, G.P. Soressi, and F. Salamini. 1986. ’Grazia’ e ’Patrizia’ bush bean. HortScience 21:1081–1082. Di Fonzo, N., L. Manzocchi, F. Salamini, and C. Soave, 1986. Purification and properties of an endospermic protein of maize associated with the Opaque-2 and Opaque-6 genes. Planta 167:587–594. Motto, M., R. Marotta, N. Di Fonzo, C. Soave, and F. Salamini. 1986. Ds-induced alleles at the opaque-2 locus of maize. Genetics 112:121–133. Pozzi, G.L., E. Gentinetta, F. Salamini, and M. Motto, 1986. Genotypic variation for cold tolerance among twelve maize (Zea mays L.) publications. Plant Breed. 97:2–12. Marotta, R., G. Ponziani, M. Motto, H. Hartings, A. Gierl, N. Di Fonzo, C. Soave, A. Bianchi, and F. Salamini. 1986. Genetic instability at the Shrunken ans waxy loci in the o2m(r)Bg strain of maize. Maydica 31:131–151. Gentinetta, E., D. Ceppi, C. Lepori, G. Perico, M. Motto, and F. Salamini. 1986. A major gene for delayed senescence in maize. Pattern of photosynthases accumulation and inheritance. Plant Breed. 97:193–203. Bochicchio, A., C. Vazzana, A. Raschi, and F. Salamini. 1986. Effetto dello stadio di maturazione e dell’eta` del seme sulla germinazione e primo accrescimento a 208C e in condizioni di cold test, per ibrido di mais di larga diffusione in Italia. Rivista di Agronomia 20:395–405. Salamini, F. 1987. Wheat in Europe: economical aspects, marketing problems, protection of consumers and safeguard of the environment. pp. 282–286. In: B. Borghi (ed.), Hard wheat: Agronomic, technological, biochemical and genetic aspects. ECSC-EEC-EAEC, Bruxelles, Luxembourg. Marocco, A., and F. Salamini. 1987. Strategie di clonaggio dei geni delle piante. Agricultura Ricerca 77:75–84. Uhrig, H., and F. Salamini. 1987. Dihaploid plant production for 4 genotypes of potato by the use of efficient anther plants producing tetraploid strains (4 EAPP-clones). Proposal of a breeding methodology. Plant Breed. 98:228–235. Avato, P., G. Bianchi, and F. Salamini. 1987. Ontogenetic variations in the chemical composition of maize surface lipids. pp. 549–551. In: P.K. Stumpf, J. B. Mudd, and W.D. Nes (eds.), The metabolism, structure, and function of plant lipids.Plenum, New York. Motto, M., N. Di Fonzo, C. Soave, and F. Salamini. 1987. Chromosomal and genetic instabilities originating from revertant derivatives of the o2m(r) mutable allele of maize. Maydica 32:249–272. Avato, P., G. Bianchi, A. Nayak, F. Salamini, and E. Gentinetta, 1987. Epicuticular waxes of maize as affected by the interaction of mutant gl8 with gl3, gl4 and gl5. Lipids 22:11–16. Reddy, A.R., L. British, F. Salamini, H. Saedler, and W. Rhode. 1987. The A1 (Anthocyanin-1) locus in Zea mays encodes dihydroquercetin reductase. Plant Sci. 52:7–13. Rohde, W., E. Barzen, A. Marocco, Zs. Schwarz-Sommer, H. Saedler, and F. Salamini. 1987. Isolation of genes that could serve as traps for transposable elements in Hordeum vulgare. Barley Gen. 5:533–541. Salamini, F. 1988. Risikoabscha¨tzung und Richtlinien bei gentechnologischen Experimenten in der Pflanzenzu¨chtung. Zusammenfassende Stellungnahme des Direktoriums des MPI fu¨r Zu¨chtungsforschung. pp. 1–6. Ko¨ln.


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Salamini, F., C. Lorenzoni, and C. Soave. 1988. The use of advanced methodologies in plant quality improvement—two selected cases. pp. 117–128. Hodowla Roslin Aklimatyzacja I Nasiennictwo, Tom 32 Zeszyt 1/2, presented at XI. Eucarpia Congress, Warsaw. Bartels, D., and F. Salamini. 1988. Model systems to approach abiotic stresses in plants. In: Proc. of 3rd Intern. Symp. Durum wheat. The future of cereals for feeding and development of biotechnical research, pp. 243–244. ISC, Foggia. Ceccarelli, S., and F. Salamini. 1988. Miglioramento per produtivita`. pp. 495–511. In: G.T. Scarascia-Mugnozza (ed.), Miglioramento genetico vegetale. Patron, Bologna. Soave, C., and F. Salamini. 1988. Genetica biochimica e fisiologica della produzione. pp. 474–494. In: G.T. Scarascia-Mugnozza (ed.), Miglioramento genetico vegetale, Ed. Patron, Bologna. Bartels, D., M. Singh, and F. Salamini. 1988. Onset of desiccation tolerance during development of the barley embryo. Planta 175:485–492. Di Fonzo, N., H. Hartings, M. Brembilla, M. Motto, C. Soave, E. Navarro, J. Palau, W. Rhode, and F. Salamini. 1988. The b-32 protein from maize endosperm, an albumin regulated by the O2 locus: nucleic acid (cDNA) and amino acid sequences. Mol. Gen. Genet. 212:481–487. Knapp, S., H. Uhrig, and F. Salamini. 1988. Genetic transformation of diploid Solanum tuberosum by direct DNA transfer and mediated by Agrobacterium tumefaciens. pp. 355–356. In: K.J.Puite et al. (eds.), Progress 7th Int.. Protoplast Symp. Wageningen, the Netherlands, 6–11 December 1987. K.J.), Kluwer Acad. Publ., Boston. Rohde, W., D. Becker, and F. Salamini. 1988. Structural analysis of the waxy locus from Hordeum vulgare. Nucleic Acid Res. 16:7185–7186. Singh, A.K., H. Uhrig, and F. Salamini. 1988. Implications of chromosome pairing in a monohaploid and its colchidoploid of Solanum tuberosum (¼12). Genome 30:347–351. Bochicchio, A., C. Vazzana, A. Raschi, D. Bartels, and F. Salamini. 1988. Effect of desiccation on isolated embryos of maize. Onset of desiccation tolerance during development. Agronomie 8:29–36. Knapp, S., G. Coupland, H. Uhrig, F. Salamini, and P. Starlinger, 1988. Transposon of the maize transposable element Ac in Solanum tuberosum. Mol. Gen. Genet. 213:285–290. Di Fonzo, N., H. Hartings, M. Maddaloni, L. Manzocchi, G. Ponziani, J. Palau, F. Salamini, C. Soave, A. Spada, R. Thompson, and M. Motto 1988. Maize seed storage proteins: Genetic structure and regulation. In: Proc. 3rd Int. Symp. Durum Wheat. The future of cereals for human feeding and development of biotechnological research. pp.165–180. ISC, Foggia. Di Fonzo, N., H. Hartings, M. Maddaloni, L. Manzocchi, G. Ponziani, J. Palau, F. Salamini, C. Soave, A. Spada, R. Thompson, and M. Motto 1988. Maize seed storage proteins: Genetic structure and regulation. pp. 165–180. In: G. Wittmer (ed.), The future of cereals for human feeding and development of biotechnological research. Foggia, Italy. Motto, M., M. Maddaloni, G. Ponziani, M. Brembilla, R. Marotta, N. Di Fonzo, C. Soave, R. Thompson, and F. Salamini. 1988. Molecular cloning of the o2-m5 allele of Zea mays using transposon marking. Mol. Gen. Genet. 212:488–494. Salamini, F. 1989. Biotecnologie agrarie alle soglie del 2000: Implicazioni ecologiche e sociali. Verso una ondata innovativa in agricoltura. Univ. degli Studi di Pavia, January, pp. 101–118. Salamini, F., D. Bartels, and C. Gebhardt. 1989. Plant biotechnologies for developing countries. Proc. Int. Symp. CTA and FAO, Luxembourg, 26–30 June. E. Baylis, The Trinity Press, UK, pp. 59–76.

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Salamini, F., and Lo Piparo, G. 1989. Progetto di ricerca MAF per le tecnologie avanzate. Agricoltura 199:5. Salamini, F. 1989. The interaction between conventional plant breeding and molecular based approaches in the improvement of cultivated crops. pp. 138–146. In: U. Leone, G. Rialdi, and R. Vanore (eds.), Advanced technologies for increased agricultural production. CNR-USG, Genova, Italy. Salamini, F. 1989. Biotecnologie agrarie alle soglie del 2000: Implicazioni ecologiche e sociali. Verso una ondata innovativa in agricoltura. pp. 101–118. Atti Inaug. Anno Acc. 1988–89. Universita`, Pavia. Lorenzetti, F., and F. Salamini. 1989. Biotecnologie e innovazione in agricoltura. Riv. di Agron. 23:337–371. Rohde, W., and F. Salamini. 1989. Approcci moleculari per il miglioramento della resistenza ai virus. pp. 24–29. Atti Con. Tech. Biol. Inn. Fitoiatria, SIN-AFI, Bologna. Bianchi, A., C. Lorenzoni, and F. Salamini. 1989. Genetica dei cereali. Edizioni Agricole, Bologna. Cattivelli, L., Lorenzetti, F., and F. Salamini. 1989. Biotecnologia e moglioramento genetico delle piante. Agricoltura 201/202:66–79. Marocco, A., Spena, A., and F. Salamini. 1989. Organizazzione genomica. pp. 107–168. In: A. Bianchi, C. Lorenzoni, and F. Salamini (eds.), Genetica dei cereali.Edizioni Agricole, Bologna. Rohde, W., A. Marocco, and F. Salamini. 1989. Genetic engineering in crop improvement. pp. 27–69. In: P.K. Gupta and T. Tsuchiya (eds.). Chromosome engineering in plants: Genetics, breeding, evolution, Part A. Elsevier Sci. Publ., Amsterdam, the Netherlands. Singh, A.K., F. Salamini, and H. Uhrig 1989. Chromosome pairing in 14 F1 hybrids among 11 diploid potato species. J. Genet. Breed. 43:1–5. Verderio, A., T. Maggiore, M. Bertolini, and F. Salamini. 1989. Mais. pp. 527–560. In: A. Bianchi, C. Lorenzoni, and F. Salamini (eds.), Genetica dei cereali.Edizioni Agricole, Bologna, Tacke, E., S. Sarkar, F. Salamini, and W. Rohde. 1989. Cloning of the gene for the capsid protein leafroll virus. Arch. Virol. 105:153–163. Wefels, E., H. Sommer, F. Salamini, and W. Rohde, 1989. Cloning of the potato virus Y genes encoding the capsid protein CP and the nuclear inclusion protein NIb. Arch. Virol. 107:123–134. Marocco, A., M. Wissenbach, D. Becker, J. Paz-Ares, H. Saedler, F. Salamini, and W. Rohde. 1989. Multiple genes are transcribed in Hordeum vulgare and Zea mays that carry the DNA binding domain of the myb oncoproteins. Mol. Gen. Genet. 216:183– 187. Gebhardt, C., C. Blomendahl, U. Schachtschabel, T. Debener, F. Salamini, and E. Ritter, 1989. Identification of 2n breeding lines and 4n varieties of potato (Solanum tuberosum, ssp. tuberosum) with RFLP-fingerprints. Theor. Appl. Genet. 78:16–22. Gebhardt, C., E. Ritter, T. Debener, U. Schachtschabel, B. Walkemeier, H. Uhrig, and F. Salamini. 1989. RFLP analysis and linkage mapping in Solanum tuberosum. Theor. Appl. Genet. 78:65–75. Maddaloni, M., N. Di Fonzo, H. Hartings, N. Lazzaroni, F. Salamini, R.D. Thompson, and M. Motto. 1989. The sequence of the zein regulatory gene opaque-2 (O2) of Zea mays. Nucl. Acids Res. 17:7532. Hartings, H., M. Maddaloni, N. Lazzaroni, N. Di Fonzo, M. Motto, F. Salamini, and R. Thompson, 1989. The O2 gene which regulates zein deposition in maize endosperm encodes a protein with structural homologies to transcriptional activators. EMBO J. 8:2795–2801.


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Kirch, H.-H., H. Uhrig, F. Lottspeich, F. Salamini, and R.D. Thompson, 1989. Characterization of proteins associated with self-incompatibility in Solanum tuberosum. Theor. Appl. Genet. 78:581–588. Motto, M., R.D. Thompson, N. Di Fonzo, G. Ponziani, C. Soave, M. Maddaloni, H. Hartings, and F. Salamini. 1989. Transpositional ability of the Bg-rbg system of maize transposable elements. Maydica 34:107–122. Bartels, D., C. Gebhardt, S. Knapp, W. Rohde, R. Thompson, H. Uhrig, and F. Salamini. 1989. Combining conventional plant breeding procedures with molecular based approaches. Genome 31:1014–1026. Motto, M., N. Di Fonzo, H. Hartings, M. Maddaloni, F. Salamini, C. Soave, and R.D. Thompson. 1989. Regulatory genes affecting maize storage protein synthesis. Oxford Surveys of Plant Mol. and Cell Biol. 6:87–114. Salamini, F. 1990. Developmental mutants of barley. Fundacion Juan March, Serie Universitaria 251, Approaches to plant development, Madrid, pp. 31–32. Debener, T., F. Salamini, and C. Gebhardt 1990. Phylogeny of wild and cultivated Solanum species based on nuclear restriction fragment length polymorphisms (RFLPs). Theor. Appl. Genet. 79:360–368. Ritter, E., C. Gebhardt, and F. Salamini. 1990. Estimation of recombination frequencies and construction of RFLP linkage maps in plants from crosses between heterozygous parents. Genetics 125:645–654. Wise, R.P., W. Rohde, F. Salamini. 1990. Nucleotide sequence of the Bronze-1 homologous gene from Hordeum vulgare. Plant Mol. Biol. 14:277–280. Piatkowski, D., K. Schneider, F. Salamini, and D. Bartels 1990. Characterization of five ABA-responsive cDNA clones isolated from the desiccation-tolerant plant Craterostigma plantagineum and their relationship to other water-stress genes. Plant Physiol. 94:1682–1688. Rohde, W., Rosch, K., Kro¨ger, K., and F. Salamini. 1990. Nucleotide sequence of a Hordeum vulgare gene encoding a glycine-rich protein with homology to vertebrate cytokeratins. Plant Mol. Biol. 14:1057–1059. Rohde, W., D. Becker, B. Kull, and F. Salamini. 1990. Structural and functional analysis of two waxy gene promoters from potato. J. Gen. Breed. 44:1–6. Tacke, E., D. Pru¨fer, F. Salamini, and W. Rohde 1990. Characterization of a potato leafroll luteovirus subgenomic RNA: Differential expression of viral proteins by internal translation initiation and UAG suppression. J. Gen. Virol 71:2265–2272. Tacke, E., D. Pru¨fer, E. Wefels, F. Salamini, and W. Rohde 1990. Construction of potato lines transgenic for genes of potato leafroll virus. Potato Research 33:150. Barone, A., E. Ritter, U. Schachtschabel, T. Debener, F. Salamini, and C. Gebhardt. 1990. Localization by restriction fragment length polymorphism mapping in potato of a major dominant gene conferring resistance to the potato cyst nematode Globodera rostochiensis. Mol. Gen. Genet. 224:177–182. Bartels, D., K. Schneider, G. Terstappen, D. Piatkowski, and F. Salamini. 1990. Molecular cloning of abscisic acid-modulated genes which are induced during desiccation of the resurrection plant Craterostigma plantagineum. Planta 181:27–34. Concilio, L., H. Uhrig, C. Gebhardt, R. Thompson, W. Rohde, and F. Salamini. 1990. The use of biotechnology in plant breeding with particular reference to potato tuber quality. pp. 46–72.Proc. EAPR Congr. Edinburgh, UK. Hartings, H., N. Lazzaroni, A. Marsan, A. Aragay, R. Thompson, F. Salamini, N. Di Fonzo, J. Palau, and M. Motto 1990. The b-32 protein from maize endosperm: Characterization of genomic sequences encoding two alternative central domains. Plant Mol. Biol. 14:1031–1040.

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Maddaloni, M., G. Bossinger, N. Di Fonzo, M. Motto, F. Salamini, and A. Bianchi 1990. Unstable alleles of the glossy-1 locus of maize show a light-dependent variation in the pattern of somatic reversion. Maydica 35:409–420. Maddaloni, M., N. Di Fonzo, H. Hartings, S. Lohmer F. Salamini, R. Thompson, and M. Motto. 1990. Regulatory genes affecting maize seed storage protein synthesis. Atti Associazione Genetica Italiana 36:117–118. Motto, M., N. Di Fonzo, H. Hartings, M. Maddaloni, L. Pirovano, S. Lohmer, F. Salamini, and R. Thompson 1990. Genetic and molecular based approaches for improving grain protein and grain yield in maize. Int. Advanced Course Maize Breeding, Production, Processing and Marketing in Mediterranean Countries, Maize 90, Belgrade. pp. 141– 156. Lo Pinto, M., and F. Salamini. 1991. Evoluzione dei sistemi agricoli e interventi di ricerca ai fini di una agricultura possibile per il duemila. pp. 93–110. In: Scienza e tecnologia per lo sviluppo nella pace. Atti aggiornamento culturale Universita` Cattolica Sacro Cuore. Vita e Pensiero, Milano.. Debener, T., F. Salamini, and C. Gebhardt 1991. The use of RFLPs (Restriction Fragment Length Polymorphisms) detects germplasm introgressions from wild species into potato (Solanum tuberosum ssp. tuberosum) breeding lines. Plant Breed. 106:173–181. Rohde, W., S. Doerr, F. Salamini, and D. Becker 1991. Structure of a chalcone synthase from Hordeum vulgare. Plant Mol. Biol. 16:1103–1106. Lohmer, S., M. Maddaloni, M. Motto, N. Di Fonzo, H. Hartings, and F. Salamini, and R.D. Thompson 1991. The maize regulatory locus Opaque-2 encodes a DNA-binding protein which activates the transcription of the b-32 gene. EMBO J. 10:617–624. Fladung, M., G. Bossinger, G.W. Roeb, and F. Salamini. 1991. Correlated alterations in leaf and flower morphology and rate of leaf photosynthesis in a midribless (mbl) mutant of Panicum maximum Jacq. Planta 184:356–361. Thompson, R.D., H. Kaufmann, H.-H., Kirch, and F. Salamini. 1991. Self-incompatibility in flowering plants. pp. 367–385. In: J. Harding, F. Singh, and J.N.M. Mol (eds.), Genetics and breeding of ornamental species.Kluwer Academic Publishers, Boston Ritter, E., T. Debener, A. Barone, F. Salamini, and C. Gebhardt. 1991. RFLP mapping on potato chromsomes of two genes controlling extreme resistance to potato virus X (PVX). Mol. Gen. Genet. 227:81–85. Bianchi, G., A. Gamba, C. Murelli, F. Salamini, and D. Bartels. 1991. Novel carbohydrate metabolism in the resurrection plant Craterostigma plantagineum. Plant J. 1:355–359. Hinze, K., R.D. Thompson, E. Ritter, F. Salamini, and P. Schulze-Lefert 1991. Restriction fragment length polymorphism-mediated targeting of the ml-o resistance locus in barley (Hordeum vulgare). Proc. Natl. Acad. Sci. (USA) 88:3691–3695. Bartels, D., Engelhardt K., R. Roncarati, K. Schneider, Rotter, M., and F. Salamini. 1991. An ABA and GA modulated gene expression in the barley embryo encodes an aldose reductase related protein. EMBO J. 10:1037–1043. Bertolini, M., Bosio, M., Bressan, M., F. Coppolino, N. Di Fonzo, E. Gentinetta, F. Introzzi, E. Lupotto, T. Maggiore, M. Perenzin, M. Snidaro, P. Valoti, A. Vederiio, A. Bianchi, C. Soave, Lorezoni, C., M. Motto, and F. Salamini. 1991. Breeding activity of the maize station of Bergamo: synthetic gene pools and inbreds released in the period 1975–1989. Maydica 36:87–106. Hartings, H., C. Spilmont, N. Lazzaroni, V. Rossi, F. Salamini, R.D. Thompson, and M. Motto 1991. Molecular analysis of the Bg-rbg transposable element system of Zea mays L. Mol. Gen. Genet. 227:91–96. Hartings, H., V. Rossi, N. Lazzaroni, R.D. Thompson, F. Salamini, and M. Motto 1991. Nucleotide sequence of the Bg transposable element of Zea mays L. Maydica 36:355–359.


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Belletti, A., C. Petrini, A. Minguzzi, V. Landini, C. Piazza, and F. Salamini. 1991. Yield potential and adaptability to Italian conditions of sweet sorghum as biomass crop for energy production. Maydica 36:283–291. Marocco, A., A. Santucci, S. Cerioli, M. Motto, N. Di Fonzo, R.D. Thompson, and F. Salamini. 1991. Three high-lysine mutations control the level of ATP-binding HSP70-like proteins in the maize endosperm. Plant Cell 3:507–515. Gebhardt, C., E. Ritter, A. Barone, T. Debener, B. Walkemeier, U. Schachtschabel, H. Kaufmann, R.D. Thompson, M.W. Bonierbale, M.W. Ganal, S.D. Tanksley, and F. Salamini. 1991. RFLP maps of potato and their alignment with the homoeologous tomato genome. Theor. App. Genet. 83:49–57. Bartels, D., K. Schneider, D. Piatkowski, R. Elster, G. Iturriaga, G. Terstappen, B. Le Tran, and F. Salamini. 1991. Molecular analysis of desiccation tolerance in the resurrection plant Craterostigma plantagineum. VI. NATO Advanced Study. Institute Plant Molecular Biology, eds. R. G. Herrmann and B. Larkins, 663–671. Maddaloni, M., L. Barbieri, S. Lohmer, M. Motto, F. Salamini, and R. Thompson 1991. Characterization of an endosperm-specific developmentally regulated protein synthesis inhibitor from maize seeds. J. Genet. and Breed. 45:377–380. Salamini, F. 1992. Scienza e vita nel momento attuale III. Genetica e miglioramento delle piante coltivate: contributo allo sviluppo di sistemi agricoli eco-compatibili. Istituto Lombardo di Scienze e Lettere, Milano. pp. 171–187. Salamini, F., and M. Motto 1992. Regulatory genes affecting storage protein synthesis in maize endosperm. Agr. Med. 122:61–66. Salamini, F. 1992. Una agricoltura per il 2000: Sistemi agronomici e interventi della ricerca genetica. pp. 43–61. In: Milano da Coltivare. Atti Progetto Ambiente Parco Agricolo Sud, Parco Sud, Milano. Coltivare. Gebhardt, C., and F. Salamini. 1992. Restriction fragment length polymorphism analysis of plant genomes and its application to plant breeding. 135:201–237. In: K.W. Jeon and M. Friedlander (eds.), A survey of cell biology. Int. Rev. Cytol. Academic Press, New York. Meyer, R., F. Salamini, and H. Uhrig 1992. Biotechnology and plant breeding: relevance of cell genetics in potato improvement. pp. 11–21. In: W, Powell, J.R. Hillman, and J.A. Raven, (eds), Opportunities and problems in plant biotechnology. Proc. Royal Society of Edinburgh, April 1991, 99B (3/4). Bossinger, G., W. Rohde, Lundqvist, U., and F. Salamini. 1992. Genetics of barley development: mutant phenotypes and molecular aspects. pp. 231–263. In: P.R. Shewry (ed.), Barley: Genetics, biochemistry, molecular biology and biotechnology. CAB International, Alden Press, Oxford, UK. Bossinger, G., U. Lundqvist, W. Rohde, and F. Salamini. 1992. Genetics of plant development in barley. pp. 989–1022. In: L. Munck (ed.), Barley genetics VI. Proc. of the 6th Int. Barley Gen. Symp. 1991, Helsingborg, Sweden. Munksgaard Int. Publ., Copenhagen, Denmark. Bossinger, G., M. Maddaloni, M. Motto, and F. Salamini. 1992. Formation and cell lineage patterns of the shoot apex of maize. Plant J. 2:311–320. Iturriaga, G., K. Schneider, F. Salamini, and D. Bartels. 1992. Expression of desiccationrelated proteins from the resurrection plant Craterostigma plantagineum in transgenic tobacco. Plant Mol. Biol. 20:555–558. Scheffler, J.A., J. Hesselbach, H. Hemme, and F. Salamini. 1992. Sampling potato genotypes that maintain chip quality under low temperature storage. J. Genet. Breed. 46:103–110. Ko¨hler, S., I. Coraggio, D. Becker, and F. Salamini. 1992. Pattern of expression of meristemspecific cDNA clones of barley (Hordeum vulgare L.). Planta 186:227–235.

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Bartels, D., C. Hanke, K. Schneider, D. Michel, and F. Salamini. 1992. A desiccationrelated Elip-like gene from the resurrection plant Craterostigma plantagineum is regulated by light and ABA. EMBO J. 11:2771–2778. Barzen, E., W. Mechelke, E. Ritter, J.F. Seitzer, and F. Salamini. 1992. RFLP markers for sugar beet breeding: Chromosomal linkage maps and location of major genes for rhizomania resistance, monogermy and hypocotyl colour. Plant J. 2:601– 611. Bianchi, G., A. Gamba, C. Murelli, F. Salamini, and D. Bartels. 1992. Low molecular weight solutes in desiccated and ABA-treated calli and leaves of Craterostigma plantagineum. Phytochemistry 31:1917–1922. Go¨rg, R., U. Schachtschabel, E. Ritter, F. Salamini, and C. Gebhardt 1992. Discrimination among 136 tetraploid potato varieties by fingerprints using highly polymorphic DNA markers. Crop Sci. 32:815–819. Dale, P.L., M. Baga, A.A. Szalay, D. Piatkowski, F. Salamini, and D. Bartels. 1992. Expression of luciferase in transgenic tobacco mediated by a promoter element from desiccation tolerant Craterostigma plantagineum. pp. 65–769. In: C.M. Karssen, L.C. von Loon, and D. Vreugdenhil (eds.), Progress in plant growth regulation. Kluwer Academic Publ., Dordrecht the Netherlands. Leonards-Schippers, C., W. Gieffers, F. Salamini, and C. Gebhardt 1992. The R1 gene conferring race-specific resistance to Phytophthora infestans in potato is located on potato chromosome V. Mol. Gen. Genet. 233:278–283. Tacke, E., B. Kull, D. Pru¨fer, S. Reinold, J. Schmitz, F. Salamini, and W. Rohde 1992. PLRV gene expression in potato. In: Viral pathogenesis and disease resistance. Proc. 5th Int. Symp. Biotechnol. Plant Protection. pp. 353–367. Uhrig, H., C. Gebhardt, E. Tacke, W. Rohde, and F. Salamini. 1992. Recent advances in breeding potatoes for disease resistance. In: Advances in potato crop protection. Neth. J. Plant Path. 98 ( Suppl. 2):193–210. Rohde, W., F. Salamini, R. Ashburner, and J.W. Randles. 1992. An EcoRI repetitive sequence family of the coconut palm Cocos nucifera L. shows sequence homology to copia-like elements. J. Genet. Breed. 46:391–394. Bartels, D., R. Alexander, K. Schneider, R. Elster, R. Velasco, J. Alamillo, G. Bianchi, D. Nelson, and F. Salamini. 1993. Desiccation-related gene products analyzed in a resurrection plant and in barley embryos. pp. 119–127. In: R.J. Close and E.A. Bray (eds.), Plant responses to cellular dehydration during environmental stress. Current Topics in Plant Physiology, Am. Soc. Plant Physiol. Series Vol. 10. Rizzi, E., C. Balconi, P. Ajmone-Marsan, F. Salamini, R. Thompson, and M. Motto. 1992. Growth requirements of the o6 mutant and its relationship with the gene encoding the rip protein b-32 of maize endosperm. Maydica 37:275–281. Salamini, F., and M. Motto. 1993. The role of gene technology in plant breeding. pp. 138– 159. In: M.D. Hayward, N.O Bosemark, and I.Romagosa (eds.), Plant breeding. Principles and prospects. Chapman & Hall, London, Schneider, K., B. Wells, E. Schmelzer, F. Salamini, and D. Bartels. 1993. Desiccation leads to the rapid accumulation of both cytosol and chloroplast proteins in the resurrection plant Craterostigma plantagineum Hochst. Planta 189:120–131. Petrini, C., A. Belletti, and F. Salamini. 1993. Accumulation and distribution of dry matter and soluble carbohydrates in two sweet sorghum cultivars: Influence of sowing date and harvesting time. Eur. J. Agron. 2:185–192. Kaufmann, H., F. Salamini, and R. Thompson. 1991. Sequence variability and gene structure at the self-incompatibility locus of Solanum tuberosum. Mol. Gen. Genet. 226:457–466.


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Garcia-Maroto, F., F. Salamini, and W. Rohde. 1993 Molecular cloning and expression patterns of three alleles of the Deficiens-homologous gene St-Deficiens from Solanum tuberosum. Plant J. 4:771–780. Meyer, R., F. Salamini, and H. Uhrig. 1993. Isolation and characterization of potato diploid clones generating a high frequency of monohaploid or homozygous diploid androgenetic plants. Theor. Appl. Genet. 85:905–912. Wefels, E., F. Salamini, and W. Rohde. 1993. Modification at the N-terminus of the potato virus Y capsid protein CP does not interfere with CP-mediated virus resistance in transgenic potato (Solanum tuberosum L.). J. Genet. Breed. 47:89–93. Wolter, M., K. Hollricher, F. Salamini, and P. Schulze-Lefert 1993. The mlo resistance alleles to powdery mildew infection in barley trigger a developmentally controlled defence mimic phenotype. Mol. Gen. Genet. 239:122–128. Gebhardt, C., D. Mugniery, E. Ritter, F. Salamini, and E. Bonnel. 1993. Identification of RFLP markers closely linked to the H1 gene conferring resistance to Globodera rostochiensis in potato. Theor. Appl. Genet. 85:541–544. Balconi, C., E. Rizzi, M. Motto, F. Salamini, and R. Thompson. 1993. The accumulation of zein polypeptides and zein mRNA in cultured endosperms of maize is modulated by nitrogen supply. Plant J. 3:325–334. Bianchi, G., A. Gamba, R. Limiroli, N. Pozzi, R. Elster, F. Salamini, and D. Bartels. 1993. The unusual sugar composition in leaves of the resurrection plant Myrothamnus flabellifolia. Physiologia Plant. 87:223–226. Lohmer, S., M. Maddaloni, M. Motto, F. Salamini, and R.D. Thompson. 1993. Translation of the mRNA of the maize transcriptional activator Opaque-2 is inhibited by upstream open reading frames present in the leader sequence. Plant Cell 5:65–73. Michel, D., F. Salamini, D. Bartels, P. Dale, M. Baga, and A. Szalay. 1993. Analysis of a desiccation and ABA-responsive promoter isolated from the resurrection plant Craterostigma plantagineum. Plant J. 4:29–40. Gallusci, P., J. Muth, S. Lohmer, F. Salamini, and R. Thompson. 1993. Opaque-2: A plant leucine zipper transcriptional activator. J. Cell Biochem. Abstr. 17A:157. Kaufmann, H., H.-H. Kirch, T. Wemmer, A. Peil, F. Lottspeich, H. Uhrig, F. Salamini, and R.D. Thompson. 1992. Sporophytic and gametophytic self-incompatibility pp. 115– 125. In: M. Cresti and A.Tiezzi (eds.), Sexual plant reproduction. Springer-Verlag, Berlin. Castagna, R., B. Borghi, G. Bossinger, and F. Salamini. 1993. Induction and characterization of Triticum monococcum mutants affecting plant and ear morphology. J. Genet. Breed. 47:127–138. Bartels, D., R. Velasco, K. Schneider, F. Forlani, A. Furini, and F. Salamini. 1993. Resurrection plants a smodel systems to study desiccation tolerance in higher plants. pp. 47–58. In: T. Mabry, H. Nguyen, and R. Dixon (eds.), Applications as prospects of biotechnology for arid and semiarid lands IC2 Institute, Univ.Texas, Austin. ¨ berlacker, F. Vogt, D. Becker, F. Salamini, and W. Rohde. 1993. Myb Wissenbach, M., B. U genes from Hordeum vulgare: Tissue-specific expression of chimeric Myb promoter/ GUS genes in transgenic tobacco. Plant J. 4:411–422. Gianinetti, A., M. Cantoni, C. Lorenzoni, F. Salamini, and A. Marocco. 1993. Altered levels of antioxidant enzymes associated with two mutations in tomato. Physiol. Plant. 89:157–164. Mauri, I., M. Maddaloni, S. Lohmer, M. Motto, F. Salamini, R. Thompson, and E. Martegiani. 1993. Functional expression of the transcriptional activator Opaque-2 of Zea mays in transformed yeast. Mol. Gen. Genet. 241:319–326.

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Gallusci, P., F. Salamini, and R.D. Thompson. 1994. Differences in cell type-specific expression of the gene Opaque-2 in maize and transgenic tobacco. Mol. Gen. Genet. 244:391–400. Gebhardt, C., E. Ritter, and F. Salamini. 1994. RFLP map of the potato. pp. 271–285. In: I.K. Vasil and R.L. Philipps (eds.), DNA-based markers in pants. Vol. I: Kluwer Academic Publ., Dordrecht, The Netherlands. Nelson, D., F. Salamini, and D. Bartels. 1994. Abscisic acid promotes novel DNA-binding activity to a desiccation-related promoter of Craterostigma plantagineum. Plant J. 5:451–458. Van Berkel, J., F. Salamini, and C. Gebhardt. 1994. Transcripts accumulating during cold storage of potato (Solanum tuberosum L.) tubers are sequence related to stress-responsive genes. Plant Physiol. 104:445–452. Velasco, R., F. Salamini, and D. Bartels. 1994. Dehydration and ABA increase mRNA levels and enzyme activity of cytosolic GAPDH in the resurrection plant Craterostigma plantagineum. Plant Mol. Biol. 26:541–546. Alexander, R., J. Alamillo, F. Salamini, and D. Bartels. 1994. A novel embryo-specific barley cDNA clone encodes a protein with homologies to bacterial glucose and ribitol dehydrogenase. Planta 192:519–525. Michel, D., A. Furini, F. Salamini, and D. Bartels. 1994. Structure and regulation of an ABA- and desiccation-responsive gene from the resurrection plant Craterostigma plantagineum. Plant Mol. Biology 24:549–560. Do¨rr, I., S. Miltenyi, F. Salamini, and H. Uhrig. 1994. Selecting somatic hybrid plants using magnetic protoplast sorting. Bio/Technology 12:511–515. Furini, A., C. Koncz, F. Salamini, and D. Bartels. 1994. Agrobacterium-mediated transformation of the desiccation-tolerant plant Craterostigma plantagineum. Plant Cell Reports 14:102–106. Pirovani, L., S. Lanzini, H. Hartings, N. Lazzaroni, V. Rossi, R. Joshi, R.D. Thompson, F. Salamini, and M. Motto. 1994. Structural and functional analysis of an Opaque-2related gene from sorghum. Plant Mol. Biol. 24:515–523. El-Kharbotly, A., C. Leonards-Schippers, D.J. Huigen, E. Jacobsen, A. Pereira, Stiekema, F. Salamini, and C. Gebhardt. 1994. Segregation analysis and RFLP mapping of the R1 and R3 alleles conferring race-specific resistance to Phytophthora infestans in progeny of dihaploid potato parents. Mol. Gen. Genet. 242:749–754. Leonards-Schippers, C., W. Gieffers, R. Scha¨fer-Pregl, E. Ritter, S.J. Knapp, F. Salamini, and C. Gebhardt. 1994. Quantitative resistance to Phytophthora infestans in potato: A case study of QTL mapping in an allogamous plant species. Genetics 137:67–77. Alamillo, J.M., R. Roncarati, P. Heino, R. Velasco, D. Nelson, R. Elster, G. Bernacchia, A. Furini, G. Schwall, F. Salamini, and D. Bartels. 1994. Molecular analysis of desiccation tolerance in barley embryos and in the resurrection plant Craterostigma plantagineum. Agronomie 2:161–167. Bartels, D., P. Heino, D. Nelson, D. Michel, A. Furini, G. Bernacchia, R. Velasco, R. Roncarati, R. Elster, G. Schwall, J. Alamillo, and F. Salamini. 1994. Analysis and regulation of gene expression in the resurrection plant Craterostigma plantagineum. NATO ASI Series Vol. H81 Plant Mol. Biology. Coruzzi, G., Puigdome`nech, P. (eds.) Springer-Verlag, Berlin. pp. 267–275. Cerioli, S., A. Marocco, M. Maddaloni, M. Motto, and F. Salamini. 1994. Early event in maize leaf epidermis formation as revealed by cell lineage studies. Development 120:2113–2120. Castagna, R., G. Maga, M. Perenzin, M. Heun, and F. Salamini. 1994. RFLP-based genetic relationships of Einkorn wheats. Theor. Appl. Genet. 88:818–823.


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Do¨rr, I., H. Uhrig, W. Rohde, E. Tacke, C. Gebhardt, and F. Salamini. 1994. Advanced approaches to the breeding of potato. pp. 85–100. The 2nd Korea-Germany Joint Symposium in Plant Biotechnology. Gyeongsang National University, Korea. Speulman, E., and F. Salamini. 1995. A barley cDNA clone with homology to the DNAbinding domain of the steroid hormone receptors. Plant Sci. 106:91–98. Speulman, E., and F. Salamini. 1995. GA3-regulated cDNAs from Hordeum vulgare leaves. Plant Mol. Biol. 28:915–926. Spena, A., and F. Salamini. 1995. Genetic tagging of cells and cell layers for studies of plant development. Methods Cell Biol. 49:331–354. Furini, A., F. Salamini, and D. Bartels. 1995. T-DNA tagging of a gene inducing desiccation tolerance in Craterostigma plantagineum. pp. 513–518. In: M. Terzi, R. Cella, A. Falavigna (eds.), Current issues in plant molecular and cellular biology. Kluvier Acad. Pub., London. Kull, B., F. Salamini, and W. Rohde. 1995. Genetic engineering of potato starch composition: Inhibition of amylose biosynthesis in tubers from transgenic potato lines by the expression of antisense sequences of the gene for granule-bound stach synthase. J. Genet. Breed. 49:69–76. Niewo¨hner, J., F. Salamini, and C. Gebhardt. 1995. Development of PCR assays diagnostic for RFLP marker alleles closely linked to alleles Gro1 and H1, conferring resistance to the root cyst nematode Globodera rostochiensis in potato. Mol. Breed. 1:65–78. Barzen, E., W. Mechelke, E.E. Ritter Schulte-Kappert, and F. Salamini. 1995. An extended map of the sugar beet genome containing RFLP and RAPD loci. Theor. Appl. Genet. 90:189–193. Gebhardt, C., B. Eberle, C. Leonards-Schippers, B. Walkemeier, and F. Salamini. 1995. Isolation, characterization and RFLP linkage mapping of a DNA repeat family of Solanum spegazzinii by which chromosome ends can be localized on the genetic map of potato. Genet. Res., Camb. 65:1–10. Kull, B., F. Salamini, and W. Rohde. 1995. Genetic engineering of potato starch composition: inhibition of amylose biosynthesis in tubers from transgenic potato lines by the expression of potato starch composition: inhibition of amylose biosynthesis in tubers from transgenic potato lines by the expression of antisense sequences of the gene for granule-bound starch sunthase. J. Genet. Breed. 49:69–76. Petrini, C., A. Belletti, and F. Salamini. 1995. Breeding and growing sweet sorghum for energy. pp. 215–244. In: B.J. Vanstone (ed.), Renewable energy in agriculture and forestry Bioenergy Agreement International Energy Agency, Faculty of Forestry, Univ. Toronto, Ontario M5S 3B3, Canada. Roncarati, R., F. Salamini, and D. Bartels. 1995. An aldose reductase homologous gene from barley: Regulation and function. Plant J. 7:809–822. Montag, K., F. Salamini, and R.D. Thompson. 1995. ZEMa, a member of a novel group of MADS box genes, is alternatively spliced in maize endosperm. Nucl. Acids Res. 23:2168–2177. Michel, D., H. Hartings, S. Lanzini, M. Michel, M. Motto, G.R. Riboldi, F. Salamini, and H.P. Do¨ring. 1995. Insertion mutations at the maize Opaque-2 locus by transposable element families Ac, En/Spm and Bg. Mol. Gen. Genet. 248:287–292. Becker, J., P. Vos, M. Kuiper, F. Salamini, and M. Heun. 1995. Combined mapping of AFLP and RFLP markers in barley. Mol. Gen. Genet. 249:65–73. Kleines, M., R. Elster, J. Ingram, G. Bernacchia, G. Schwall, F. Salamini, and D. Bartels. 1995. Regulation of the carbohydrate metabolism in the resurrection plant Craterostigma plantagineum. In: STRESSNET. Proceedings of the 2nd STRESSNET conference. Salsomaggiore, Italy, 21–23 September.

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Hueros, G., S. Varotto, F. Salamini, and R.D. Thompson. 1995. Molecular characterization of BET1, a gene expressed in the endosperm transfer cells of maize. Plant Cell 7:747– 757. Alamillo, J., C. Almoguera, D. Bartels, and J. Jordano. 1995. Constitutive expression of small heat shock proteins in vegetative tissues of the resurrection plant Craterostigma plantagineum. Plant Mol. Biol. 29:1093–1099. Michel, D., F. Salamini, M. Motto, and H.-P. Do¨ring. 1994. An unstable allele at the maize Opaque2 locus is caused by the insertion of a double Ac element. Mol. Gen. Genet. 243:334–342. Mu¨ller, M., J.R. Muth, P. Gallusci, S. Knudsen, M. Maddaloni, M. Motto, D. Schmitz, M.B. S½rensen, F. Salamini, D. von Wettstein, and R.D. Thompson. 1995. Regulation of storage protein synthesis in cereal seeds: development and nutritional aspects. J. Plant Physiol. 145:606–613. Mu¨ller, K.J., N. Romano, O. Gerstner, F. Garcia-Maroto, C. Pozzi, F. Salamini, and W. Rohde 1995. The barley Hooded mutation caused by a duplication in a homeobox gene intron. Nature 374:727–730. Hartings, H., N. Lazzaroni, V. Rossi, G.R. Riboldi, R.D. Thompson, F. Salamini, and M. Motto. 1995. Molecular analysis of opaque-2 alleles from Zea mays L. reveals the nature of mutational events and the presence of a hypervariable region in the 5’ part of the gene. Genet. Res., Camb. 65:11–19. Bernacchia, G., G. Schwall, F. Lottspeich, F. Salamini, and D. Bartels. 1995. The transketolase gene family of the resurrection plant Craterostigma plantagineum: differential expression during rehydration phase. EMBO J. 14:610–618. Tacke, E., C. Korfhage, D. M. Michel, M. Maddaloni, M. Motto, S. Lanzini, F. Salamini, and H.-P. Do¨ring. 1995. Transposon tagging of the maize Glossy2 locus with the transposable element En/Spm. Plant J. 8:907–917. Meksem, K., D. Leister, F. Salamini, and C. Gebhardt. 1995. A high-resolution map of chromosome V of potato based on RFLP and AFLP markers in the vicinity of the R1 locus. Mol. Gen. Genet. 249:74–81. Ballvora, A., J. Hesselbach, J. Niewo¨hner, F. Salamini, and C. Gebhardt. 1995. Marker enrichment and high-resolution map of the segment of potato chromosome VII harbouring the nematode resistance gene Gro1. Mol. Gen. Genet. 249:82–90. Mu¨ller, K., C. Pozzi, F. Salamini, and W. Rohde. 1995. Homeotic gene expression and plant development. pp. 1597–1604. Proc. Forum for Applied Biotechnology. Med. Fac. Landbouww. Univ. Gent, 60/4a. Castagna, R., B. Borghi, N. Di Fonzo, M. Heun, and F. Salamini. 1995. Yield and related traits of einkorn (T. monococcum ssp.monococcum) in different environments. Eur. J. Agron. 4:371–378. Salamini, F. 1996. Geni, transgeni e agricoltura del futuro. L’Informatore Agrario 46:7–10. Ritter, E., and F. Salamini. 1996. The calculation of recombination frequencies in crosses of allogamous plant species with applications to linkage mapping. Genet. Res. Camb. 67:55–65. Bernacchia, G., F. Salamini, and D. Bartels. 1996. Molecular characterization of the rehydration process in the resurrection plant Craterostigma plantagineum. Plant Physiol. 111:1043–1050. Scha¨fer-Pregl, R., F. Salamini, and C. Gebhardt. 1996. Models for mapping quantitative trait loci (QTL) in progeny of non inbred plants and their behaviour in presence of distorted segregation ratios. Genet. Res. Camb. 67:43–54. Tacke, E., F. Salamini, and W. Rohde. 1996. Genetic engineering of potato for resistance to potato leafroll virus (PLRV) infection. Proc. EAPR Virol. Sect. Meeting. pp. 15–22.


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Simons, G., T. van der Lee, P. Diergaarde, R. van Daelen, J. Groenendijk, A. Frijters, R. Bu¨schges, K. Hollricher, S. To¨psch, P. Schulze-Lefert, F. Salamini, M. Zabeau, and P. Vos. 1997. AFLP-based fine mapping of the Mlo gene to a 30-kb DNA segment of the barley genome. Genomics 44:61–70. Bartels, D., J. Chandler, C. Bockel, W. Frank, M. Kleines, M.J. Rodrigo, J. Phillips, J.-B. Mariaux, A. Furini, and F. Salamini. 1997. Investigating the molecular basis of desiccation tolerance using the resurrection plant Craterostigma plantagineum as an experimental system. Acta Physiologiae Plant. 19:399–403. Salamini, F. 1998. Transgenic plant products in animal and human nutrition. G. Piva (ed.), Proc. 5th Int. Feed Production Conference. Picenza, Italy. pp. 54–70. Salamini, F. 1998. Clonal analysis in plant development. Cellular integration of signalling pathways in plant development. Cell Biol. 104:233–235. Salamini, F. 1998 Mutants of maize. Book review. [M.G. Neuffer, E.A. Coe, S.R. Wessler (eds.)] Plant Sci. 131:107–108. Salamini, F. 1998. Piante transgeniche: dal laboratorio al campo. Quaderni dei I Georgofili 6:71–95. Salamini, F., and K. Stu¨ber. 1998. Pflanzenzucht. In: Lexikon der Bioethik, Band 3, Hrsg. Korff, W., Beck, L., Mikat, P., im Auftrag der Go¨rres-Gesellschaft. Gu¨tersloher Verlagshaus, pp. 802–804. Salamini, F., B. Reiss., C. Koncz, and J. St. Schell. 1998. Pflanzenbiotechnologie, die Zukunft fu¨r Forschung und Anwendung. Bayerischer Monatsspiegel 34:61– 64. Velasco, R., F. Salamini, and D. Bartels. 1998. Gene structure and expression analysis of the drought- and abscisic acid-responsive CDeT11-24 gene family from the resurrection plant Craterostigma plantagineum Hochst. Planta 204:459–471. Frank, W., J. Phillips, F. Salamini, and D. Bartels. 1998. Two dehydration-inducible transcripts from the resurrection plant Craterostigma plantagineum encode interacting homeodomain-leucine zipper proteins. Plant J. 15:413–421. Borghi, B., R. Castagna, M. Corbellini, and F. Salamini. 1998. Le caratteristiche qualitative del frumento monococco. Molini d’Italia. pp. 33–39. Hueros, G., J. Rahfeld, F. Salamini, and R.D. Thompson. 1998. A maize FK506-sensitive immunophilin, mzFKBP-66, is a peptidylproline cis-trans isolerase that interacts with calmodulin and a 36 kDa cytoplasmic protein. Planta 205:121–131. Mariaux, J.B., C. Bockel, F. Salamini, and D. Bartels. 1998. Desiccation- and abscisic acidresponsive genes encoding major intrinsic proteins (MIPs) from the resurrection plant Craterostigma plantagineum. Plant Mol. Biol. 1089–1099. Bockel, C., F. Salamini, and D. Bartels. 1998. Isolation and characterization of genes expressed during early events of the dehydration process in the resurrection plant C. plantagineum. J. Plant Physiol. 152:158–166. Castiglioni, P., C. Pozzi, M. Heun, V.M. Terzi, K.J. Mu¨ller, W. Rohde, and F. Salamini. 1998. An AFLP-based procedure for the efficient mapping of mutants and DNA probes in barley. Genetics 149:2039–2056. Marocco, A., C. Soave, and F. Salamini. 1998. Genomics and genetic engineering. 15th Congr. European Association for Research on Plant Breeding—Eucarpia, 21–25 September, pp. 134–173. Bertolini, M., A. Bianchi, E. Lupotto, F. Salamini, A. Verderio, and M. Motto 1998. Maize. 15th Congress of the European Association for Research on Plant Breeding—Eucarpia, 21–25 September 1998, pp. 209–229. Scha¨fer-Pregl, R., E. Ritter, L. Concilio, J. Hesselbach, L. Lovatti, B. Walkemeier, H. Thelen, F. Salamini, and C. Gebhardt. 1998. Analysis of quantitative trait loci (QTLs) and

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Maitz, M., G. Santandrea, Z. Zhang, S. Lal, L.C. Hannah, F. Salamini, and R.D. Thompson. 2000. rfg1, a mutation reducing grain filling in maize through effects on basal endosperm and pedicel development. Plant J. 23:29–42. Mu¨ller, K., C. Pozzi, J. Mu¨ller, F. Salamini, and W. Rohde. 2000. Molecular analysis of homeotic genes involved in barley development. European J. Physiol. 439, Supplement R14–R15. Varotto, C., P. Pesaresi, D. Maiwald, J. Kurth, F. Salamini, and D. Leister. 2000. Identification of photosynthetic mutants of Arabidopsis by automatic screening for altered effective quantum yield of photosystem 2. Photosynthetica 38:497–504. ˆ nick, M. Tizzano, F. Schiavon, F. Salamini, and D. Varotto, C., P. Pesaresi, P. Jahns, A. LeO Leister. 2000. Single and double knock-outs of the genes for photosystem I subunits PSIG, -K and -H of Arabidopsis thaliana: Effects on PSI composition, photosynthetic electron flow and state transitions. Plant Physiol. 129:616–624. Salamini, F. 2001. Il frumento monococco e l’origine dell’agricoltura. Le Scienze 120:4–9. Salamini, F. 2001. Biotecnologie in sala da pranzo. Magazine Bipielle 1:20–21. Salamini, F. 2001. Piante geneticamente modificate. pp. 109–113. In: P. Hrelia and G.C. Forti (eds.), La tossicologia per la qualita` e la sicurezza alimentare. Pa`tron Editore, Bologna. Bartels, D., and F. Salamini. 2001. Desiccation tolerance in the resurrection plant Craterostigma plantagineum: a contribution to the study of drought tolerance at the molecular level. Plant Physiol. 127:1346–1353. Chen, X., F. Salamini, and C. Gebhardt. 2001. A potato molecular function map for carbohydrate metabolism and transport Theor. Appl. Genet. 102:284–295. Gebhardt, C., E. Ritter, and F. Salamini. 2001. RFLP map of the potato. In: R.L. Phillips I.K. Vasil (eds.), DNA-based markers in plants, 2nd ed., Advances in cellular and molecular biology of plants, 6:319–336. Kluwer Academic Publ. Dordrecht, the Netherlands. Schneider, K., B. Weisshaar, D.C. Borchardt, and F. Salamini. 2001. SNP frequency and allelic haplotype structure of Beta vulgaris expressed genes. Mol. Breed. 8:63–74. Varotto, C., E. Richly, F. Salamini, and D. Leister 2001. GST-PRIME: A genome-wide primer design software for the generation of gene sequence tags. Nucleic Acids Research 29:4373–4377. Pesaresi, P., C. Varotto, E. Richly, J. Kurth, F. Salamini, and D. Leister 2001. Functional genomics of Arabidopsis photosynthesis. Plant Physiol. Biochem. 39:285–294. Mu¨ller, J., Wang, Y., R. Franzen, L. Santi, F. Salamini, and W. Rohde 2001. In vitro interactions between barley TALE homeodomain proteins suggest a role for proteinprotein associations in the regulation of Knox gene function. Plant J. 27:12–23. Pesaresi, P., C. Varotto, J.- Meurer, P. Jahns, F. Salamini, and D. Leister 2001. Knock-out of the plastid ribosomal protein L11 in Arabidopsis: Effects on mRNA translation and photosynthesis. Plant J. 27:179–189. Schneider, K., D.C. Borchardt, D. Bellin, and F. Salamini. 2001. Ansa¨tze zur Verknu¨pfung von struktureller und funktionaler Genomanalyse in der Zuckerru¨be (Beta vulgaris L.). Vortr. Pflanzenzu¨chtung 52:61–68. Gebhardt, C., R. Scha¨fer-Pregl, X. Chen, and F. Salamimi. 2001. Without a map you cannot navigate: DNA markers for sailing the genome of potato (Solanum tuberosum), pp. 195– 207. In: R.G. van den Berg, G.W.M. Barendse, G.M. van der Weerden, and C. Mariani, (eds.), Solanaceae V., Advances in taxonomy and utilization. Nijmegen Univ. Press., Nijmegen.The Netherlands. Maiwald, D., C. Varotto, M. Weigel, E. Richly, A. Biehl, P. Pesaresi, J. Kurth, F. Salamini, and D. Leister. 2001. Higher plant photosynthesis and related genomics. Vortr. Pflanzenzu¨cht 52:27–35.


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Leister D., A. Kurth, E. Biehl, J. Richly, P. Pesaresi, M. Weigel, C. Varotto, D. Maiwald, and F. Salamini. 2001. Photosynthesis and related genomics. PS2001 3:1–6. Proc. 12th Int. Congr. Photosynthesis. CSIRO Publ., Melbourne, Australia. Salamini, F. 2002. Geography and genetics of wild cereal domestication in the Near East. In: Eucarpia, Cereal Section Meeting, 21–25 November, From biodiversity to genomics: breeding strategies for small grain cereals in the third millennium, pp. 3–6. Salamini, F. 2002. A path towards agricultural productivity: cereal genomics. Plant Mol. Biol. 48:443–444. Salamini, F., and K. Schneider. 2002. Integration of new genomic technologies in variety development. Vortr. Pflanzenzu¨chtung 54:189–193. Salamini, F., A. Sohn, and H. Thomas. 2002. Biotechnology development in Germany: The case of Nordrhein Westfalen. pp. 239–248. In: A. Quadrio Curzio and M. Fortis (eds.), Complexity and industrial clusters. dynamics and models in theory and practice. Physica-Verlag/Springer-Verlag, Heidelberg. ¨ zkan, A. Brandolini, R. Scha¨fer-Pregl, and W. Martin. 2002. Genetics and Salamini, F., H. O geography of wild cereal domestication in the Near East. Nature Reviews Genetics 3:429–441. Zhang, Z.-Y., F. Salamini, and R. Thompson. 2002. Fine mapping of the defective endosperm maize mutant RGF1 using different DNA pooling strategies and three classes of molecular markers. Maydica 47:277–286. ¨ zkan, H., A. Brandolini, R. Scha¨fer-Pregl, and F. Salamini. 2002. AFLP analysis of a O collection of tetraploid wheats indicates the origin of emmer and hard wheat domestication in southeast Turkey. Mol. Biol. Evolution 19:1797–1801. Kurth, J., C. Varotto, P. Pesaresi, A. Biehl, E. Richly, F. Salamini, and D. Leister. 2002. Gene-sequence-tag expression analyses of 1,800 genes related to chloroplast functions. Planta 215:101–109. Schneider, K., R. Scha¨fer-Pregl, D.C. Borchardt, and F. Salamini. 2002. Mapping QTLs for sucrose content, yield and quality in a sugar beet population fingerprinted by ESTrelated markers. Theor. Appl. Genet 104:1107–1113. Deng, X., J. Phillips, A.H. Meijer, F. Salamini, and D. Bartels. 2002. Characterization of five novel dehydration-responsive homeodomain leucine zipper genes from the resurrection plant Craterostigma plantagineum. Plant Mol. Biol. 49:601–610. Ballvora, A., M.R. Ercolano, J. Weib, K. Meksem, C.A. Bormann, P. Oberhagemann, F. Salamini, and C. Gebhardt. 2002. The R1 gene for potato resistance to late blight (Phytophthora infestans) belongs to the leucine zipper/NBS/LRR class of plant resistance genes. Plant J. 30:361–371. Grabes, T., P. Pesaresi, F. Schiavon, C. Varotto, F. Salamini, P. Jahns, and D. Leister. 2002. The role of DpH-dependent dissipation of excitation energy in protecting photosystem II against light-induced damage in Arabidopsis thaliana. Plant Physiol. Biochem. 40:41–49. Phillips, J.R., T. Hilbricht, F. Salamini, and D. Bartels. 2002. A novel abscisic acid- and dehydration-responsive gene family from the resurrection plant Craterostigma plantagineum encodes a plastid-targeted protein with DNA-binding activity. Planta 215:258– 266. Rodrigo, M.-J., J. Moskovitz, F. Salamini, and D. Bartels. 2002. Reverse genetic approaches in plants and yeast suggest a role for novel, evolutionarily conserved, selenoprotein-related genes in oxidative stress defense. Mol. Genet. Genom. 267:613–621. Hilbricht, T., F. Salamini, and D. Bartels. 2002. CpR18, a novel SAP-domain plant transcription factor, binds to a promoter region necessary for ABA mediated expression of

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the CDeT27-45 gene from the resurrection plant Craterostigma plantagineum Hochst. Plant J. 31:293–303. Varotto, C., D. Maiwald, P. Pesaresi, P. Jahns, F. Salamini, and D. Leister. 2002. The metal ion transporter IRT1 is necessary for iron homeostasis and efficient photosynthesis in Arabidopsis thaliana. Plant J. 31:589–599. El Rabey, H.A., A. Badr, R. Scha¨fer-Pregl, W. Martin, and F. Salamini. 2002. Speciation and species separation in Hordeum L. (Poaceae) resolved by discontinuous molecular markers. Plant Biol. 4:567–575. Velasco, R., C. Korfhage, A. Salamini, E. Tacke, J. Schmitz, M. Motto, F. Salamini, and H.-P. Do¨ring 2002. Expression of the glossy2 gene of maize during plant development. Maydica 47:71–81. Taenzler, B., R.F. Esposti, P. Vaccino, A. Brandolini, S. Effgen, M. Heun, R. Scha¨fer-Pregl, B. Borghi, and F. Salamini. 2002. Molecular linkage map of Einkorn wheat: Mapping of storage-protein and soft-glume genes and bread-making quality QTLs. Genetical Res. Camb. 80:131–143. Pesaresi, P., C. Lunde, P. Jahns, D. Tarantino, J. Meurer, C. Varotto, R.D. Hirtz, C. Soave, H.V. Scheller, F. Salamini, and D. Leister. 2002. A stable LHCII-PSI aggregate and suppression of photosynthetic state transitions in the psae1-1 mutant of Arabidopsis thaliana. Planta 215:940–948. Salamini, F. 2003. Hormones and the green revolution. Science 302:71–72. Salamini, F. 2003. Le biotecnologie vegetali. pp. 37–56. In: T. Fanfani and F. Ghelli (eds.), Quaderni Fondazione Piaggio. Nuova serie. La Tipografica Varese, Firenze. Salamini, F., and T. Maggiore. 2003. Il miglioramento genetico in agricoltura: dalla selezione massale ai transgeni. pp. 43–67. In: G. Bretoni (ed.), l’uomo e l’agricoltura: quale futuro? Treviso, Casa dei carraresi. Vita e Pensiero, Universita` Cattolica, Milano. Salamini, F., et al. 2003. Plant biotechnology and GM varieties. Committee of the Italian National Academies of Lincei and of the Sciences. Nat. Acad. Sci., Roma. Morandini, P., and F. Salamini. 2003. Plant biotechnology and breeding: Allied for years to come. Trends Plant Sci. 8:70–75. Morgante, M., and F. Salamini. 2003. From plant genomics to breeding practice. Current Opinions in Biotechnology 14:214–219. Heibges, A., and F. Salamini, C. 2003. Gebhardt. Functional comparison of homologous members of three groups of Kunitz-type enzyme inhibitors from potato tubers (Solanum tuberosum L.). Mol. Gen. Genomics 269:535–541. Santi, L., Y. Wang, M.R. Stile, K. Berendzen, D. Wanke, C. Roig, C. Pozzi, K. Mu¨ller, J. Mu¨ller, W. Rohde, and F. Salamini. 2003. The GA octodinucleotide repeat binding factor BBR participates to the transcriptional regulation of the homeobox gene Bkn3. Plant J. 34:813–826. Menendez, C.M., E. Ritter, R. Scha¨fer-Pregl, B. Walkemeier, A. Kalde, F. Salamini, and C. Gebhardt 2003. Cold-sweetening in diploid potato. Mapping QTL and candidate genes. Genetics 162:1423–1434. Pesaresi, P., C. Varotto, E. Richly, A. Lebnick, F. Salamini, and D. Leister. 2003. Proteinprotein and protein function relationships in Arabidopsis photosystem I: Cluster analysis of PSI polypeptide levels and photosynthetic parameters in PSI mutants. J. Plant Physiol. 160:17–22. Hunger, S., G. DiGaspero, S. Mo¨hring, D. Bellin, R. Scha¨fer-Pregl, D.C. Borchardt, C.E. Durel, M. Werber, B. Weisshaar, F. Salamini, and K. Schneider. 2003. Isolation and linkage analysis of expressed disease-resistance gene analogues of sugar beet (Beta vulgaris L.). Genome 46:70–82.


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Pozzi, C., D. di Pietro, G. Halas, C. Roig, and F. Salamini. 2003. Integration of a barley (Hordeum vulgare) molecular linkage map with the position of genetic loci hosting 29 developmental mutants. Heredity 90:390–393. Richly, E., A. Dietzmann, A. Biehl, J. Kurth, C. Laloi, K. Apel, F. Salamini, and D. Leister. 2003. Co-variations in the nuclear chloroplast transcriptome reveal a regulatory master switch. EMBO Reports 4:491–498. Weigel, M., C. Varotto, P. Pesaresi, F. Salamini, and D. Leister 2003. Plastocyanin is indispensable for photosynthetic electron flow in Arabidopsis thaliana. J. Biol. Chem. 278:31286–31289. Smith-Espinoza, C.J., A. Richter, F. Salamini, and D. Bartels. 2003. Dissecting the response to dehydration and salt (NaCl) in the resurrection plant Craterostigma plantagineum. Plant, Cell Environ. 26:1307–1315. Heibges, A., H. Glaczinski, A. Ballvora, F. Salamini, and C. Gebhardt. 2003. Structural diversity and organization of three gene families for Kunitz-type enzyme inhibitors from potato tubers (Solanum tuberosum L.). Molecular Gen. Genom. 269:526–534. Maiwald, D., A. Dietzmann, P. Jahns, P. Pesaresi, A. Joliot, J.Z. Levin, F. Salamini, and D. Leister. 2003. Knock-out of the genes coding for the Rieske protein and the ATPsynthase delta-subunit of Arabidopsis. Effects on photosynthesis, thylakoid protein composition and nuclear chloroplast gene expression. Plant Physiol.133:191–202. Pesaresi, P., N.A. Gardner, S. Masiero, A. Dietzmann, L. Eichacker, R. Wickner, F. Salamini, and D. Leister. 2003. Cytoplasmic N-terminal protein acetylation is required for efficient photosynthesis in Arabidopsis. Plant Cell 15:1817–1832. Salamini, F. 2004. Per gli ogm, come Alice nel paese delle meraviglie. Avvenire 21(10):3. Salamini, F. 2004. Piante transgeniche:dal laboratorio al campo. pp. 27–52. In: Camera di Commercio di Ravenna (eds.), Organismi geneticamente modificati. Nuove opportunita` o potenziale a rischio? Franco Angeli, Milano. Salamini, F. 2004. Presentazione. Agroindustria 3:67. Salamini, F., M. Heun, A. Brandolini, H. Ozkan, and J. Wunder. 2004. Comment on ‘‘AFLP data and the origins of domesticated crops.’’ Genome 47:615–620. Maggiore, T., and F. Salamini. 2004. Innovazione in agricoltura. Produzioni vegetali. Informatore Fitopatologico 12(12):7–15. Pozzi, C., L. Rossini, A. Vecchietti, and F. Salamini. 2004. Gene and genome changes during domestication of cereals. In P.K. Gupta and R.K. Varshney (eds.), Cereal genomics. Kluwier Academic Pub., London. Ercolano, M.R., A. Ballvora, J. Paal, H.-H. Steinbiss, F. Salamini, and C. Gebhardt. 2004. Functional complementation analysis in potato via biolistic transformation with BAC large DNA fragments. Mol. Breed. 13:15–22. Ihnatowicz, A., P. Pesaresi, C. Varotto, E. Richly, A. Schneider, P. Jahns, F. Salamini, and D. Leister. 2004. Mutants for photosystem I subunit D of Arabidopsis thaliana: Effects on photosynthesis, photosystem I stability and expression of nuclear genes for chloroplast functions. Plant J. 37:839–852. Paal, J., H. Henselewski, J. Muth, K. Meksem, C. Meneńdez, F. Salamini, A. Ballvora, and C. Gebhardt. 2004. Molecular cloning of the potato Gro 1-4 gene conferring resistance to pathotype Ro 1 of the root cyst nematode Globodera rostochensis, based on a candidate gene approach. Plant J. 38: 285–297. Roig, C., C. Pozzi, L. Santi, J. Mu¨ller, M. Stanca, and F. Salamini. 2004. The genetics of barley hooded suppression Genetics 167:439–448. Salamini, F. 2004. Vocati all’agricoltura, ma . . . Bipielle Magazine 12:78 Salamini, F. 2005. Parco Tecnologico Padano: Da qui passa il futuro. Magazine Bipielle 13:54–56.

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Salamini, F. 2005. Un’ ondata tecnologica che modifichera`, in meglio, piante e farmaci. Magazine Bipitalia 14:58–60. Salamini, F. 2005. Ogm: La storia infinita. Inf. Agr. 50:14–15. Salamini, F. 2005 Lezione Rossi-Doria. Innovazione in agricoltura, sviluppo rurale e il problema ambientale. Associazione per studi e ricerche Manlio Rossi-Doria, Portici. Borghi, B., and F. Salamini. 2005. I seminativi tra passato glorioso e futuro incerto. Inf. Agr. 40:64–68. Morandini, P., F. Salamini, and P. Gantet, 2005. Engineering of plant metabolism for drug and food. Curr. Med. Chem. 5:103–112. Mo¨hring, S., F. Salamini, and K. Schneider. 2005. Multiplexed, linkage group-specific SNP marker sets for rapid genetic mapping and fingerprinting of sugar beet (Beta vulgaris L.). Mol. Breed. 4:475–488. Smith-Espinoza, C.J., J.R. Phillips, F. Salamini, and D. Bartels. 2005. Identification of further Craterostigma plantagineum cdt mutants affected in abscisic acid mediated desiccation tolerance. Mol Genet Genom. 274:364–72. Biehl, A., E. Richly, C. Noutsos, F. Salamini, and D. Leister. 2005. Analysis of 101 nuclear transcriptomes reveals 23 distinct regulons and their relationship to metabolism, chromosomal gene distribution and co-ordination of nuclear and plastid gene expression. Gene 344:33–41. Hackbusch, J., K. Richter, J. Mu¨ller, F. Salamini, and J. Uhrig. 2005. A central role of Arabidopsis thaliana ovate family proteins in networking and subcellular localization of 3-aa loop homeodomain proteins Proc. Nat. Acad. Sci. (USA) 102:4908–4912. ¨ zkan, H., A. Brandolini, C. Pozzi, S. Effgen, J. Wunder, and F. Salamini. 2005. A O reconsideration of the domestication geography of tetraploid wheats. Theor. Appl. Genet. 110:1052–1060. Pesaresi, P., C. Lunde, P. Jahns, D. Tarantino, J.- Meurer, C. Varotto, R.D. Hirtz, C. Soave, H.V. Scheller, and D. Leister, 2002. A stable LHCII-PSI aggregate and suppression of photosyntetic state transitions in the psae1-1 mutant of Arabidopsis thaliana. Planta 215:940–948. Sturaro, M., H. Hartings, E. Schmelzer, R. Velasco, F. Salamini, and M. Motto. 2005. Cloning and characterization of glossy 1, a maize gene involved in cuticle membrane and wax production. Plant Physiol.138:478–489. Salamini, F. 2006. Requiem per una Riforma, Spazio Rurale 1:18–19. Salamini, F. 2006. Nota introduttiva. In: R. Groppali (Ed.), Atlante della biodiversita` del Parco Adda Sud, 4: 9–10. Salamini, F. 2006. Agricoltura sostenibile e microrganismi utili. pp. 193–203. In: M. Iaccarino (Ed.)., Microrganismi benefici per le piante. Idelson-Gnocchi, Napoli. Lorenzoni, C., and F. Salamini. 2005. Angelo Bianchi: An appreciation. Maydica 50:181– 183. Rossini, L., A. Vecchietti, L. Nicoloso, N. Stein, S. Franzago, F. Salamini, and C. Pozzi. 2006. Candidate genes for barley mutants involved in plant architecture: an in silico approach. Theor. Appl. Genet. 112:1073–85. Sgaramella, V., and F. Salamini. 2006. Il genoma umano e l’arte di arrangiarsi. Darwin 14:48–53. Sgaramella, V., and F. Salamini. 2006. Gene paucity, genome instability, clonal development: Has an individual genome the potential to encode for more than one brain? DNA Repair 10:531–533. Pesaresi, P., S. Masiero, H. Eubel, H.P. Braun, S. Bhushan, E. Glaser, F. Salamini, and D. Leister. 2006. Nuclear photosynthetic gene expression is synergistically modulated by rates of protein synthesis in chloroplasts and mitochondria. Plant Cell. 18:970–91.


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¨ zkan, B. Kilian, and F. Salamini. 2006. Brandolini, A., P. Vaccino, G. Boggini, H. O Quantification of genetic relationships among A genomes of wheats. Genome 49:297– 305. ¨ zkan, J. Kohl, A. von Haeseler, F. Barale, O. Deusch, A. Brandolini, C. Kilian, B., H. O Yucel, W. Martin, and F. Salamini. 2006. Haplotype structure at seven barley genes: Relevance to gene pool bottlenecks, phylogeny of ear type and site of barley domestication. Mol. Genet. Genom. 276:230–241. Abbo, S., A. Gopher, Z. Peleg, Y. Saranga, T. Fahima, F. Salamini, and S. Lev-Yadun. 2006. The ripples of ‘‘the Big (agricultural) Bang’’: The spread of early wheat cultivation. Genome/Geńome 49:861–863. ¨ zkan, O. Deusch, S. Effgen, A. Brandolini, J. Kohl, W. Martin, and F. Kilian. B., H. O Salamini. 2006. Independent wheat B and G genome origins in outcrossing Aegilops progenitor haplotypes. Mol. Biol. Evol. 23:1–11.

2 Epigenetics and Plant Breeding Athanasios S. Tsaftaris Department of Genetics and Plant Breeding Auth Thessaloniki 54124 and Institute of Agrobiotechnology Certh Thermi 57001 Greece Alexios N. Polidoros, Aliki Kapazoglou, and Eleni Tani Institute of Agrobiotechnology Certh Thermi 57001 Greece Nives M. Kovacˇevic´ Department of Agronomy University of Wisconsin-Madison 1575 Linden Drive Madison, WI 53706, USA

I. INTRODUCTION II. MOLECULAR EPIGENETIC MECHANISMS A. DNA Methylation B. Modifications of Histones C. RNA-based Control Mechanisms D. The Triangle of Interactions of Epigenetic Mechanisms E. Methods of Studying of Epigenetic Mechanisms III. PLANT BIOLOGICAL PHENOMENA INVOLVING EPIGENETIC MECHANISMS A. Epigenetic Control of Transposable Elements and Repeats B. Epigenetic Mechanisms and Stress C. Paramutation in Plants D. Parental Imprinting E. Viral-induced Silencing F. Transgene Silencing IV. EPIGENETIC MECHANISMS AND PLANT DEVELOPMENT A. Plant Development B. Shoot and Leaf Formation and Development C. Flowering D. Seed Development V. IMPLICATIONS IN PLANT BREEDING A. Genetic and Epigenetic Variation B. Improving Plant Stress Tolerance C. Epigenetic Mechanisms, Yield, and Heterosis D. Changes in Plant Development and Architecture Plant Breeding Reviews, Volume 30. Edited by Jules Janick Copyright © 2008 John Wiley & Sons, Inc.

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E. Manipulating Parental Imprinting F. Improving Plant Resistance to Viruses and Other Parasites G. The Use of RNAi for Crop Improvement H. Securing Stability of Transgenes VI. OUTLOOK VII. ACKNOWLEDGMENTS VIII. LITERATURE CITED

ABBREVIATIONS 5-AzaC 5mC 8-oxoG CHS CLF CMT CMV CTR DCL DME DnMT DRM dsDNA dsRNA EIS Esc E(z) FBP26 FIE FIS FLC GMPs GUS H3-K9 HATs HcPro HPLC HTH KYP MARs MBPs MEA

5-azacytidine 5 methyl cytosine 8-hydroxyguanisine chalcone synthase curly leaf chromomethylase (chromomelthyltransferase) cucumber mosaic virus constitutive triple response Dicer-like protein Demeter DNA methyl transferase domain-rearranged methylase (methyltransferase) double-stranded DNA double-stranded RNA epigenetic information systems extra sex combs enhancer of Zeste flowering binding protein 26 fertilization-independent endosperm fertilization-independent seed Flowering Locus C genetically modified plants b-glucuronidase histone 3-lysine 9 histone acetyltransferases Hc-protease high-performance liquid chromatography hothead kryptonite matrix attachment regions methylation binding proteins MEDEA

2. EPIGENETICS AND PLANT BREEDING

MET1 mRNA MSI1 MT or Mtase PcG PDR PFG PHE PSTVd PTGS Rboh RdDM RdRP RE RISC RNAi ROS S-AdoMet siRNAs sRNAs ssDNA ssRNA Su(z)12 TE TGS VRN VIGS VIN3

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methyltransferase1 messenger RNA multicopy suppressor of ira methyltransferase polycomb group pathogen-derived resistance petunia flowering gene pheres potato spindle tuber viroid post transcriptional gene silencing respiratory burst oxidase homolog RNA-directed DNA methylation RNA-dependent RNA polymerase repeated element RNA-induced silencing complex RNA interference repressor of silencing or reactive oxygen species S adenosylmethionine small interfering RNAs small RNAs single-stranded DNA single-stranded RNA suppressor of Zeste transposable element transcriptional gene silencing vernalization virus-induced gene silencing Vernalization-insensitive 3

I. INTRODUCTION The concept of epigenesis has its roots in ancient Greece, where it was first proposed by Aristotle to differentiate from preformation, a theory favored by other philosophers, such as Democritus and Leucippus, in order to explain the development of a new organism. According to preformation, in the beginning there was an individual of each species of animal or plant that contained, within it, as nested miniatures, all the other individuals of the species that would ever live. In epigenesis, there were not such preformed miniatures and new structures arose progressively by a process that metaphorically was linked by Aristotle

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to the ‘‘knitting of a net.’’ Preformation was the prevailing theory for millennia until the invention of the microscope and the discovery that living organisms, including embryos, are composed of cells. Development was then recognized as being epigenetic since during this process, division of the egg generates many new cells that differentiate to form different tissues. Later, in the middle of the twentieth century, Waddington (1942) developed the term epigenetics, a derivative of Aristotle’s epigenesis, aiming to synthesize preformation and epigenesis into a single theory by combining genetics—the study of the hereditary material (preexisting complexity) found in the zygote—with developmental biology—the study of changes undergone by the zygote (epigenesis). Holliday (1994), describing in molecular terms DNA methylation as an epigenetic mechanism, defined epigenetics as the study of the changes in gene expression, which occur in organisms with differentiated cells, and the mitotic inheritance of given patterns of gene expression. Following the proof that epigenetic mechanisms, leading to changes of gene functions, are not only mitotically stable during development but are also meiotically heritable led to the definition of epigenetics as the study of mitotically and meiotically heritable changes in gene expression that do not entail a change in DNA sequence (Wu and Morris 2001). As mentioned by Zilberman and Henikoff (2005), the major reason for changing views of epigenetics is that the original concept was meant to be a broad theoretical framework to guide studies in an area in which not only were the mechanisms not understood but the nature of the processes was unknown. In this spirit, Timothy Bestor (Zilberman and Henikoff 2005) offered the following light-hearted description at the 1995 Gordon Research Conference on Epigenetics: molecular biology: known gene, known product; genetics: known gene, unknown product; biochemistry: unknown gene, known product; epigenetics: unknown gene, unknown product. In recent years, both genetic and biochemical studies have greatly advanced our knowledge of epigenetic processes involving sRNAmediated gene silencing and chromatin-based inheritance of gene activity states through DNA methylation and/or modifications of histones. What has begun to emerge under the almost mystical epigenetic umbrella is a picture of an ancient system of cellular and genomic immunity predating the divergence of plants, ciliates, animals, and fungi. Much of the epigenetic machinery of higher eukaryotes, including plants, appears to be directed at silencing viruses, TEs, and other REs, with epigenetic developmental regulation of endogenous genes being mostly derived later from such processes (Martienssen 1996; Lippman


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Fig. 2.1. The triangle of the three epigenetic information systems (EIS) and their interactions. DNA methylation is required for chromatin modifications and vice versa, and sRNA-based mechanisms (RNAi) regulate both.

et al. 2004). The field of epigenetics is currently enjoying a meteoric rise, after the successful sequencing of a plethora of different genomes and the turn toward functional genomics. In order to understand the role of different parts of the genome, epigenetics definitely appears to hold one of the master keys to unlock and understand these roles (Beck and Oleck 2003; Mattick 2004). Three such epigenetic information systems (EIS)—DNA methylation, histone modification, and RNA interference—have been described so far, along with their mechanisms of action (Fig. 2.1). DNA methylation was historically the first and more extensively studied chromatin epigenetic mark, and covalent modifications of the different histones were defined as major epigenetic marks conveying epigenetic information by changing chromatin states and consequently DNA function. While cytosine methylation is the only epigenetic mark in DNA, histone modifications are numerous involving methylation, acetylation, phosphorylation, ribosylation, ubiquitation of mostly H3 histone, but H4, histones H2A, H2B, H1 as well. This constitutes the ‘‘histone code’’ of chromatin epigenetic marks (Turner 2002). Finally, during the last few years, arguably the most important advance in biology has been the discovery that small RNA molecules can regulate the expression of genes. For years, RNAs were

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thought to have just two broad functions in cells. Single-stranded mRNAs were considered vital intermediaries in gene expression, transmitting information between DNA and protein, while ribosomal and transfer RNAs were playing structural and information-decoding roles in the process of protein synthesis. This dogma is largely true for bacterial cells with their compact genome (Mattick 2004). This picture started changing after the discovery that RNAs could play different catalytic roles (Chech 1986). Furthermore, data from recent studies in different fields, including among others viral resistance, animal and plant development, and transgene silencing in plants, supported additional roles for RNA molecules. These data showed that sRNA molecules of 21 to 24 nucleotides long could interfere with the translation or the degradation of complementary mRNA molecules. Drs. Melo and Fire were honored with the 2006 Nobel Prize in physiology or medicine for their contribution in understanding the role of sRNA. In addition, sRNA molecules could lead to chromatin modifications of both DNA and histones, leading to transcriptional arrest of certain partially complementary genes. This phenomenon, termed RNA interference (RNAi), placed sRNAs in a central role among epigenetic mechanisms and was recognized as a third EIS. According to Mattick (2004), the current conception of how genetic information is encoded and transmitted in higher organisms will need to be reassessed. The main output of the genome of complex organisms is genetically active but non-protein coding RNA. Researchers now realize that there are numerous layers of biological information in DNA, interspersed between, or superimposed on, the passages written in the triplet code of protein coding (Pearson 2006). The three EIS are not independent from each other; rather they form a triangle of interdependent interactions. Unraveling the relationships between these epigenetic components led to rapidly evolving new concepts that reveal how, by interacting, they complement, reinforce, and stabilize the effects of each other. Histone deacetylation and other modifications, particularly the methylation of lysine 9 within histone H3 (H3K9) of histone tails, cause chromatin condensation and block transcription initiation. In addition, histone modification can also attract DNA MTases to initiate 5mC formation, which in turn can reinforce histone modification patterns conducive to silencing (Fuks et al. 2003b; Tariq and Paszkowski 2004). The opposite is also true; that is, DNA methylation reinforces silencing by the attraction of histone modifications (methylation and deacetylation), leading to a more compact and thus inactive chromatin (Tamaru and Selker 2001; Tariq and Paszkowski 2004). With the recent advent of RNA interference in the epigenetic field, it became clear almost from the start that among their other roles, sRNAs also provide a common denominator of both DNA


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methylation and/or histone modifications. Experiments in plants have clearly shown the involvement of sRNAs in the establishment of heterochromatic states and silencing (Wassenegger et al. 1994; Matzke et al. 2001). Disruption of one of these interacting systems can lead to expression or silencing of genes, resulting in epigenetically controlled phenotypes. Thus, their significance as new sources of epigenetically controlled variation with multiple concomitant implications in conventional plant breeding and modern genetic engineering is consolidated. The epigenetic mechanisms and molecular machineries of their manifestation will be described in Section II. Recent studies suggest that far more numerous pathways involved in the manifestation of epigenetic phenomena and their developmental role is by far more critical than it was anticipated even a few years ago. In particular, research on the mechanisms of RNA interference proceeded at a staggering pace. It is now clear that this is part of an evolutionarily ancient mechanism of genome defense against any ‘‘parasitic’’ nucleic acid, both in the cytoplasm and in the nucleus. In parallel, RNAi became an extremely useful experimental tool not only for learning what genes do by knocking out their activity but also for generating useful genetic plant variants. The three EIS will be described briefly since extended recent reviews on each of them have been presented (Tsaftaris and Polidoros 2000; Matzke et al. 2001; Bender 2004; Bannister and Kouzarides 2005; Tsaftaris et al. 2005). The emphasis in Section II will be the integration of very recent data and the description of their interrelationships. Section III describes epigenetic involvement in a number of biological phenomena in plants, some that were known for many years, such as paramutation, parental imprinting, and transposition of elements, amd more recently recognized ones, such as transgene silencing, viralinduced silencing, and genomic effects of different biotic and abiotic stresses. Studies of these phenomena are revealing not only for the molecular mechanisms involved but also for discovering and understanding the details of all three EIS. The involvement of epigenetic mechanisms in controlling plant development is the subject of Section IV. It is now well established that epigenetic mechanisms are involved in the control of major developmental pathways in plants, the function of meristems, the formation of different organs, and the genomic response of plants to different environmental conditions. Section V describes the multiple implications of EIS in conventional plant breeding and modern genetic engineering. Emphasis is given to their role and involvement as an extra source of polymorphism-generating epialles and useful variation that is inherited between generations.

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The particular involvement of EIS in plant genomic responses (in addition to the known physiological responses) to different kinds of abiotic and biotic stresses, and in hybrid vigor of the modern F1 hybrids, is described. Robustness—that is, stable yield and better exploitation of resources—distinguish modern successful F1 hybrids and other recently released cultivars (Janick 1999; Duvick et al. 2001, 2004; Fasoula and Fasoula 2002; Tollenaar and Lee 2002; Tsaftaris et al. 2005). Understanding the molecular mechanisms behind these agronomic characteristics of elite cultivars will help to increase plant breeding efficiency in conventional breeding and assist conventional breeding with the use of molecular markers or transgenic technologies. Different aspects of the roles of EIS in modern genetic engineering are discussed. Understanding and avoiding stress-induced transgene silencing in certain transgenic cultivars when genetically modified plants (GMPs) are transferred out of the laboratory or greenhouse into the open field is one of the many reasons for serious investments in EIS research. The involvement of EIS in issues related to human health, such as stem cells and stem cell therapy (Sell 2006), cancer and other human diseases is another (see Walter and Paulsen 2003; Jiang et al. 2004; Fenech 2005). In addition, following the developments of RNAi use as an experimental tool, efforts to knock out individual genes, and mainly parallel efforts and results to generate new plant genotypes using this technology, are also presented. This review has three main objectives. The first is to briefly review recent progress with the three EIS—particularly their interactions—and expose the agricultural community, particularly plant breeders, to this new and fast-evolving field of epigenetic research, the molecular mechanisms involved, and the methodologies used. It is now certain that many of the open questions, problems, and goals of plant agronomists and breeders—such as viral diseases, tolerance of plants to certain viruses and other pathogens, plant tolerance to abiotic stresses, somaclonal variation, polyploidy, interspecific hybridization, and robustness, homeostasis or stability of performance of conventional or genetically modified cultivars, as well as numerous plants biological phenomena such as paramutation, cycling of transposable elements (TEs), parental imprinting, transgene silencing—have an epigenetic dimension in addition to a genetic one. Understanding, for instance, transcriptional and posttranscriptional silencing is not only a matter of basic research but is also of special interest to the agrobiotech industry after the release of a GMP. The second objective is to make familiar the problems, questions, and challenges of the agricultural and plant breeding community to plant


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molecular biologists, particularly those working in molecular epigenetic mechanisms. Of paramount importance is the exploration of the possibilities offered by new knowledge and methods, particularly of modern genomic approaches (Zhang et al. 2006) for understanding these phenomena and helping to overcome the problems and challenges faced. In return, the numerous genotypes or cultivars, both domesticated and bred by many years of work by farmers and breeders, could be very useful materials for molecular epigenetic analysis, study, and understanding of EIS. In other words, this second aim is to interconnect the communities of plant molecular epigenetics and breeding. The third objective is to uncover the role of EIS in plant domestication and plant breeding. Research on evolutionary developmental genetics (Evo-Devo) has revealed that the amazing diversity of multicellular organisms is the result of flexibility of small number of building blocks used during development, connecting evolution with development. Hence, the last objective of this review is to extend the intercalation of plant development and developmental genetic mechanisms to plant domestication and breeding research. For some topics or subtopics of this review, there are excellent recent review articles: Matzke et al. 2001; Bender 2004; Ringrose and Paro 2004; Bannister and Kouzarides 2005; Tsaftaris et al. 2005. Journal issues include FEBS Letters 579(26) edited by S.-W. Ding; and books: Matzke and Matzke 2000; Engel and Antonarakis 2002; Beck and Olek 2003; Galun 2003, 2005; Hannon 2005; Jablonka and Lamb 2005. However, this is the first integrated examination of the role and significance of the different EIS and their concerted interaction in plant gene interaction and plant development and of their multiple implications in plant breeding. A glossary of terms used in this review is included in Table 2.1. Certain statements made by Eva Jablonka and Marion Lamb (2005) in ‘‘Evolution in Four Dimensions’’ reflect the philosophy of this article: ‘‘There is more to heredity than genes. . . . Organisms have at least two systems (genetic and epigenetic) of heredity. . . . EIS play a double role, being both response systems and systems of transmission. . . . They are additional transmission technologies, transmitting interpretations of information in DNA.’’ II. MOLECULAR EPIGENETIC MECHANISMS A. DNA Methylation DNA methylation is a conserved heritable epigenetic modification resulting from the enzymatic addition of a methyl moiety to DNA.

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Table 2.1. Glossary of terms ARGONAUTE (AGO). A family of evolutionarily conserved genes; their protein products are involved in various RNA interference (RNAi) processes being part of the RNA interference and silencing complex (RISC). Chromatin remodeling. A change in chromatin structure that is achieved by the action of ATP-dependent remodeling complexes. CpG site. A DNA site at which C (cytosine) is followed by G (guanine); p denotes the phosphate group, so that C is at the 5’ position relative to G. Dicer. The term was initially coined by Greg Hannon to describe a drosophila multidomain enzyme of the RNase III family. Drosophila Dicer cuts long dsRNA into small dsRNAs of 21 to 23 nucleotides with a 30 overhang, known as siRNAs. In plants, at least two types of detectable Dicer activity are responsible for the production of siRNAs with distinct sizes of 21 and 24 nucleotides. These plant enzymes are known as Dicer-like enzymes. Because plants have several Dicer homologs, they are sometimes referred to individually as Dicer-like-I, Dicer-like-II. Epigenetic information systems. A mitotically and/or meiotically heritable trait that is not accompanied by a change in the DNA sequence. Epigenetic mark. Covalent modifications of DNA or chromatin proteins that affect gene expression, which is mitotically and/or meiotically heritable. Epimutation. A heritable change in phenotype that is not the result of an altered DNA base sequence. Homeostasis. The maintenance of relatively steady states in an organism through internal regulatory mechanisms, despite variations in internal and external conditions. Imprinting. An epigenetic mechanism that determines expression or repression of genes according to their parental origin. Methyl-transferase (MT). The enzyme that catalyses the addition of methyl groups to DNA. MicroRNA (miRNA) pathway. miRNAs are an abundant class of noncoding small RNAs (of 21 to 24 nucleotides) that are present in diverse eukaryotes and formed by Dicer or Dicer-like enzymes. In animals, most miRNAs function by repressing the translation of specific target mRNAs, but most plant miRNAs function like natural siRNAs to target specific mRNAs for cleavage. The only difference between plant and animal miRNAs is the extent of their complementarity to target mRNAs. Whereas plant miRNAs have extensive complementarity to their target mRNAs, animal miRNAs are much less homologous and, when paired to their targets, form bulges that are proposed to block the translation of the target mRNA. miRNA precursor. An imperfect stem–loop RNA structure generated from a long transcript, known as primary miRNA, that is encoded by a nonprotein coding region in the genome. Dicer or Dicer-like enzyme cleaves a miRNA precursor and produces a mature miRNA. Paramutation. A term coined by Brink for the process whereby one allele in a heterozygote alters the heritable properties of the other allele. Post Transcriptional Gene Silencing (PTGS). A targeted RNA degradation mechanism in plants. PTGS may be induced by transgenes or viral infection and causes the degradation of RNAs with homology or complementarity to the transgene transcript or viral genome. The signal to degrade the specific RNA sequence is transmitted throughout the plant.


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Table 2.1. (continued) RNA-dependent RNA polymerase (RdRP). An RNA polymerase that is involved in RNA silencing in plants, worm, Neurospora and Dictyostelium, but not in drosophila or human cells. RdRPs have been proposed to use cellular aberrant RNAs as templates and to copy them into cRNAs to form dsRNA. This newly synthesized dsRNA is thought to act as a substrate for Dicer-like enzymes. RNA-induced silencing complex (RISC). A siRNA– or miRNA–protein complex that acts as an endonuclease and cleaves the complementary target mRNA, or as a repressor and blocks the target mRNA translation. RNA interference (RNAi). A specific form of RNA silencing that reflects posttranscriptional RNA degradation induced by exogenous dsRNA. The term RNAi is now used widely to describe RNA silencing in both plants and animals. RNA silencing. A group of related phenomena in diverse eukaryotes in which aberrant or dsRNA triggers a marked reduction in either transcription of the corresponding gene or direct degradation of the corresponding mRNA. Small interfering RNA (siRNA). Small RNAs (21 to 25 nucleotides) that are produced from long dsRNA by Dicer or Dicer-like enzymes or chemically synthesized, and can be recruited by multiple cellular proteins to form an RNA-induced silencing complex (RISC) that interferes with mRNA stability or mRNA translation. SUPERMAN. A floral homeotic gene, mutations of which affect flower development in Arabidopsis. Hypermethylated SUPERMAN alleles clarkkent (clk) were used for genetic screens that led to the isolation of mutations in KRYPTONITE (KYP), CHROMOMETHYLASE3 (CMT3), and domain-rearranged methyltransferases (DRMs). Transcriptional Gene Silencing (TGS). Epigenetic silencing of transgenes or endogenous genes at the level of transcription. Transgene/transgenic organism. Newly integrated DNA within an organism is a transgene, while the transformed organism is a transgenic organism.

The methyl moiety on a DNA base generally contributes to transcriptional repression by preventing activators from binding to their target or favoring the formation of inactive chromatin. In eukaryotes, DNA methylation that occurs on cytosine plays important roles in gene repression, genome organization and stability, heterochromatin formation, transgene silencing, genomic imprinting, X-chromosome inactivation, inactivation of TEs and REs, and stem or meristematic cell formation or maintenance, differentiation, and developmental control. Aberrant methylation patterns of tumor suppressor genes leading to their silence constitute a common feature of many human and animal cancers (Jiang et al. 2004). Methylation is primarily found as part of host defense systems in prokaryotes and plays a role in repair and replication. Methylation is detected in all four groups of eukaryotes, namely protists, plants, fungi, and animals, although it is not constantly detectable in every organism. In general, methylation is preferentially targeted

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to heterochromatin consisting of RE including centromere-associated repeats, ribosomal RNA-encoding repeats, and TE sequences (Lippman et al. 2004). Levels of methylation vary greatly between organisms that display this modification. This can be attributed to several factors influencing 5mC content, including the RE and TE content of the genome. Thus, the overall 5mC content of a plant genome correlates with the repeated sequence content of that genome. As mentioned, the majority of methylated residues in plants are found in repetitive DNA associated with heterochromatin, but several genes in euchromatic regions have also been shown to be methylated. Although methylation at symmetric cytosines CpG and CpNpG is most common in plants, it can occur in any sequence context, particularly by RNA-directed DNA methylation (Matzke et al. 2001). Repeated HPLC measurements of different parental inbreeds and hybrids in maize show that 22 to 25% of the cytosines in the 2500 Mb maize genome are methylated (Tsaftaris et al. 1999; Tsaftaris and Polidoros 2000). Vertebrates are depleted in CpG dinucleotides and consequently show low levels of 5mC (Razin and Riggs 1980). The distribution of methylation on DNA sequences also varies between eukaryotes. Mammals and other vertebrates display a genome– widespread pattern of methylation including coding sequences of genes (Lander et al. 2001). In contrast, invertebrates, fungi, plants, and protists appear to show primarily a fractional pattern of methylation confined to only part of the genome, and almost invariably located outside cellular genes (Bennetzen et al. 1998). The distinction of two patterns of genomic methylation between animals and plants could be the consequence of the preference of animal TEs to transpose mainly within the transcribed part of the genes, primarily inside introns, while in plants, TEs belonging to different families show a preferential transposition outside the transcribed part of genes, sometimes in their regulatory vicinity and preferentially within each other (Bestor 2003). Recently Tran et al. (2005) found a new type of DNA methylation in Arabidopsis, which consisted of dense CG methylation clusters found at scattered sites throughout the genome. These clusters lack non-CG methylation and are preferentially found in genes, although they are relatively deficient toward the 5’ end. CG methylation clusters are present in lines derived from different accessions and in mutants that eliminate de novo methylation, indicating that CG methylation clusters are stably maintained at specific sites. Because 5mC is mutagenic, the appearance of CG methylation clusters over evolutionary time predicts a genome-wide deficiency of CG dinucleotides and an excess of C(A/T)G trinucleotides within transcribed regions, implying that CG methylation clusters have


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contributed profoundly to plant gene evolution. The authors suggest that CG methylation clusters silence cryptic promoters that arise sporadically within transcription units. Lippman et al. (2004), using microarray analysis, also showed earlier that sporadic TE-derived tandem repeats within euchromatic areas could provide targets of DNA methylation (through siRNA originated from the repeats, see Section III.D). They also showed that TE could regulate genes epigenetically when they are inserted within or near the promoter of a gene. Finally, variations in methylation are observed in response to endogenous or exogenous cues. In mammals, for example: (1) a highly regulated developmental process first erases and then resets the genome-wide methylation pattern during early embryogenesis, (2) many tissue-specific genes undergo demethylation during tissue differentiation, and (3) differences in methylation exist in sperm and oocyte (Brandeis et al. 1993). Similarly in plants, tissue- and developmental stage–specific as well as stress-induced variation in DNA methylation has been recorded (Messeguer et al. 1991; Tsaftaris and Polidoros 1993; Kovacˇevic´ et al. 2005). Unlike mammals, which erase and reset genomic methylation patterns early in embryogenesis, plants can inherit epigenetic changes through meiosis. ‘‘Epiallels"—that is, phenotypic variants that are epigenetically rather than genetically different from their parents—are frequently discovered in plants and in certain cases found to be inherited to the next progeny generation (Martienssen and Colot 2001; Kakutani 2002; Tani et al. 2005). Perhaps what is revealing for the inherited stability of epialles is the peloric mutant (change from bilateral to radial flower symmetry), found both in Antirrhinum and Linaria, which was originally described by Linnaeus and whose phenotype remained stable for 250 years. Findings by Hoekenga et al. (2000) suggest that changes in genomic DNA methylation and local chromatin structure can also be due to developmental changes. Therefore, the vegetative phase seems to regulate in a highly specific manner the methylation changes of the maize epigenetic allele Pl-Blotched. Furthermore, during the juvenile- to-adult vegetative transition, the level of DNA methylation and the extent of the compact chromatin domain increase and reach their maximum in adult leaves. Similar to the vegetative phase change, the methylation changes and the modifications in chromatin structure of this locus are reset at each generation. These findings,led Hoekenga et al. (2000) to propose that the developmental regulation of Pl-Blotched is controlled by signals that also control vegetative phase change. DNA methylation is catalyzed by a family of conserved DNA MTases, which points to an ancestral origin of this form of DNA modification

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(see Goll and Bestor 2005 for a review). Two different types of DNA MTases activities are establishing DNA methylation patterns: (1) maintenance MTases, which maintain stable 5mC patterns through successive generations, methylating C in proximity with 5mC on the complementary strand: and (2) de novo MTases that are able to transfer methyl groups to C of completely unmethylated double stranded DNA. On the basis of sequence and structural similarities, four groups of DNA MTases have been recognized: Dnmt1, Pmt1/Dnmt2, Dnmt3 and CMT. The mammalian Dnmt3, fungal Masc1, and plant DRMs have been shown to encode for de novo MTases. The Arabidopsis genome encodes for two related de novo MTases genes, DRM1 and DMR2. The drm1/ drm2 double mutants are blocked in TGS at some loci and completely abolish de novo methylation at CG, CNG, and asymmetric sites. Members of the mammalian Dnmt1 and plant MET1 class of enzymes serve primarily as maintenance MTases. Mutations in the Arabidopsis MET1 gene cause a global reduction of 5mC throughout the genome and a number of developmental abnormalities. Loss of CG methylation in met1 has also been shown to abolish the heterochromatic mark H3K9 at loci that remain transcriptionally silent (Tariq and Paszkowski 2004). The CMT class of enzymes appears to be specific to plants. Methylation profiling of mutants suggested that CMT3 preferentially methylates TErelated sequences. Interestingly many of these targets are shared between CMT3 and MET1, suggesting that CG and non-CG methylation systems might function redundantly for regulating TEs. CMT3 and DRMs also act in a partially redundant and locus-specific manner to control asymmetric and CNG methylation (Tariq and Paszkowski 2004). It was shown recently that loss-offunction mutations in MET1 and CMT3 lead to arabidopsis embryos with abnormal development and reduced viability (Xiao et al. 2006), pointing to the critical role of DNA methylation in proper embryogenesis. Methylation can be removed from DNA by either passive or active mechanisms. Passive demethylation can occur when 5mC is replaced with nonmodified cytosines during DNA replication (Tsaftaris and Polidoros 2000). Apparently this is a dynamic process. As DNA replication, DNA remethylation and cell division have their own duration time. Factors speeding up or slowing down these procedures will have an effect on the capacity of MTases to complete their job in preserving DNA methylation patterns. For instance, conditions promoting fast DNA synthesis and cell division, such as plant cell cultures in vitro, plant cell division in meristems or in mammalian cancers, could affect the time and efficiency of preserving DNA methylation patterns (Klein 2005). Thus, for example, numerous somaclonal variants involving


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changes in DNA methylation have been described in asexually propagated progeny plants that originated from in vitro cell cultures (see Section III.C) while significant changes in DNA methylation have been reported for many human cancers (Walter and Paulsen 2003; Jiang et al. 2004). Furthermore, cytosine MTases, described above for DNA methylation, and histone MTases required for methylating new nucleosomal histones after DNA replication both use S-AdoMet as their methyl donor. Plants, in particular, require extra amounts of this important compound since S-AdoMet serves also as methyl donor for their cellwall lignin biosynthesis. Consequently, at a certain point of their cell cycles, cells require relatively high amounts of S-AdoMet or of its precursor methionine and by extension other molecules involved in S-AdoMet metabolism, in order to preserve in a timely fashion both DNA and histone methylation marks (Paz et al. 2002; Fenech 2003). According to Mull et al. (2006), for instance, a histone methylationdependent DNA methylation pathway is uniquely impaired by deficiency in arabidopsis 5-adenosylhomocysteine hydrolase activity needed to metabolize the by-product of trans- methylation reactions. While the availability of these cofactors during plant growth, particularly under different kinds of stresses, remains to be studied, parallel studies in mammalian cancers revealed the significant role played by S-AdoMet metabolism in preserving methyl marks in both DNA and histones (Fenech 2003; Jiang et al. 2004) and as a consequence a whole new field of epinutrition, based on epigenetic-related aspects of nutrition that is emerging (Aggarwal and Shishodia 2006). In active demethylation, the participation of specific enzymes demethylating DNA sequences is anticipated. Removal of 5mC as a free base from DNA by the enzyme 5mC-DNA glycosylase has been reported in mammals and birds (Vairapandi and Duker 1993; Jost et al. 1999). Also, the excision of nucleotides containing 5-mC from DNA has been reported (Swisher et al. 1998). Either mode of removal of 5mC from DNA would, of necessity, be followed by synthesis of a DNA repair patch containing unmethylated cytosines. A link between these mechanisms, with removal of 5mC by a DNA glycosylase, requiring an RNA cofactor that presumably guides the enzyme to the specific substrate locus, has been demonstrated in the chick embryo system (Jost et al. 1999). Until recently there was no direct in vivo evidence for active demethylation in plants. Two Arabidopsis mutations, the one identified by inducing TGS of transgenes and of endogenous homologous loci (Gong et al. 2002) and the other, by its interference with maternal expression of an imprinted gene (Choi et al. 2002), were mapped to genes that encode the DNA glycosylases, repressor of silencing (ROS1) and Demeter

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(DME), respectively. According to their catalytic activity, DNA glycosylases can be classified into two subgroups: monofunctional DNA glycosylases that catalyze only hydrolysis of the glycosylic bond or bifunctional DNA glycosylase/lyase with associated lyase activity that cleaves the DNA backbone at the site where the base has been removed. The bifunctional DNA glycosylase/lyases belong to two broad classes, based on their reaction mechanisms. ROS1 encodes a nuclear protein of that induces strand breaks in DNA containing 5mC, suggesting that ROS1 may be directly involved in DNA demethylation through a base excision repair mechanism. Kapoor et al. (2005) isolated the ROS1 gene and showed that it encodes a nuclear protein with bifunctional DNA glycosylase/lyase activity against methylated but not unmethylated DNA. ROS1 participates in active DNA demethylation by a base-excision pathway, suggesting that active DNA demethylation is important in pruning the methylation patterns of the genome, and even the normally ‘‘silent’’ transposons are under dynamic control by both methylation and demethylation (Zhu et al. 2007). This dynamic control may be important in keeping the plant epigenome plastic so that it can efficiently respond to developmental and environmental cues. The genome of Arabidopsis encodes several other proteins belonging to family of DNA glycosylases, all of them with similar DNA repair activities to homologs found in bacteria, fungi or animals. The second glycosylase gene is DME. cDNAs (of this gene) from Arabidopsis and rice have been isolated and encode 1729 and 1952 aa proteins, respectively (Choi et al. 2002), and a cDNA of the same gene from barley has been cloned in our laboratory (Kapazoglou et al. 2004). Choi et al. (2002) found that DME is expressed primarily in the central cell of the female gametophyte, the progenitor of endosperm. DME is required for maternal allele expression of the imprinted gene MEDEA (MEA), a PcG gene encoding a histone 3 lysine 27 (H3K27) methyltransferase enzyme in the central cell (described in detail in the next subtopic). Ectopic DME expression in endosperm activates expression of the normally silenced paternal MEA allele. In leaf, ectopic DME expression induces MEA and nicks the MEA promoter. According to Choi et al. (2002), DME activates maternal expression of imprinted genes like MEA in the central cell. The active MEA, in turn, controls the expression of the type I MADS-box gene PHERES1 (PHE1) presumably by H3K27 methylation of its histones. Suppressing PHE1 ensures proper central cell and endosperm development (Ko¨hler et al., 2003b). (see Section IV.D for more details of the interrelationships of the two epigenetic mechanisms and Section III.D for parental imprinting.) Another studied target of DME in the endosperem is the FWA gene (Kinoshita et al. 2004). FWA was initially


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identified in Arabidopsis from late-flowering mutants that show ectopic FWA expression due to heritable hypomethylation of repeats around transcription start site. FWA, like MEA, displays maternal imprinting in the endosperm with its imprinting depending on DNA methyltransferase MET1. But as reported by Kinoshita et al. (2004), its specific maternal gametophyte activation, like the activation of MEA, depends on the activity of DME in the central cell. It is not clear how DME is regulated. There is a wealth of evidence that miRNAs regulate a plethora of genes involved in developmental processes. A search for putative miRNA targets along the sequence of the Arabidopsis DME mRNA detected six different miRNAs targets. The presence of potential miRNA targets in the barley DME is also being studied (Kapazoglou et al. 2004). The first genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis has ben reported by Zhang et al. (2006). Their results show that pericentromeric heterochromatin, repetitive sequences, and regions producing small interfering RNAs are heavily methylated. Unexpectedly, over one-third of expressed genes contain methylation within transcribed regions, whereas only 5% of genes show methylation within promoter regions. Interestingly, genes methylated in transcribed regions are highly expressed and constitutively active, whereas promoter-methylated genes show a greater degree of tissue-specific expression. Indicative of the specific importance of the role of promoter methylations in regulating the expression of downstream structural genes, whole-genome tiling-array transcriptional profiling of DNA methyltransferase null mutants identified hundreds of genes and intergenic noncoding RNAs with altered expression levels, many of which may be epigenetically controlled by DNA methylation. In conclusion, it appears that 5mC was initially and still used as a heterochromatic mark to suppress different forms of genomic ‘‘parasitic’’ DNA or to lower transcriptional noise, alleviating in part the enormous problem of complexity in transcription regulation. Thus it contributes in this way to the genome robustness and stability of performance at genome level. It seems that later this mark was adapted to a fast, reversible, and influenced by both external and internal signals, system of gene regulation (see Bestor 2003 for details on DNA methylation and epigenetics). DNA methylation, in addition to being the cause of epigenetic variation, is the cause of mutations and the generation of genetic variation. Methylated C is a hot spot for mutations since 5mC frequently deaminates to T (Coulondre et al. 1978; Jones et al. 1992). The mutability of 5mC was first demonstrated in E. coli (Coulondre et al. 1978). C bases that were methylated in the lacI gene of E. coli were found to be hot spots

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for spontaneous base substitution mutations, and the hot spots disappeared when the same sites were unmethylated, which was the reason for this increase. Whereas C deaminates to uracil (U), 5mC deaminates to T, which is a normal DNA base and therefore inherently more difficult to repair (Duncan and Miller 1980). In vertebrates, the presence of high levels of CG methylation was associated with significant deamination of 5mC to T, a change that was incompletely or inefficiently repaired (Bird 1986; Shen et al. 1994). Thus, where a 5mCG dinucleotide pair was initially present in a gene, the deamination process would convert this into a TG/CA dinucleotide pair. Currently, mutation at CG sites continues to play a significant role in the formation of new germ-line mutations contributing to genetic disease. Cooper and Krawczak (1990), in a survey of a wide variety of genetic diseases, found that 44 of 139 (32%) point mutations were C to T or G to A transitions occurring at CG dinucleotides. The isolation of tumor-suppressor genes and the detection of mutations within them in somatic cells has led to the realization that 5mC is a frequent contributor to mutations relevant to human carcinogenesis (Jones et al. 1992). Moreover, as stressed by Matzke and Matzke (1996), the C methylation pathway is inherently mutagenic, particularly under conditions in which the methyl donor S-AdoMet is limiting. Such a low in S-AdoMet environment permits the accumulation of an intermediate in the C methylation pathway (5,6-dihydrocytosine), which has a 104-fold higher rate deamination than 5-methylcytosine, implying that epinutrition in organisms is not only related to epigenic but extends to genetic (mutational) effects too. B. Modifications of Histones Histone modifications have also been defined as epigenetic modifiers. Certain histone marks disseminate heterochromatin, the condensed and ‘‘transcriptionally’’ silent chromatin in eukaryotic genomes along with the presence or absence of C methylation, while others relate to euchromatic status (Grewal and Jia 2007). In its ‘‘naked’’ form, DNA is unwieldy and unmanageable for a cell to package. A eukaryotic genome that is average in size is approximately 2m of DNA, which needs to be constrained in a typical 10çm (i.e. 10-6m) diameter nucleus; it also needs to be functional. This problem is solved by histones, which compact and control DNA. Histone octamers, consisting of two subunits each of histones H2A, H2B, H3, and H4, comprise the fundamental protein unit of chromatin that facilitates compaction of DNA into the nucleus. Approximately 150 bp of DNA wraps around the histone octamer to form the nucleosome, and then this DNA/protein complex is further


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compacted by higher-order folding. Reasoning from first principles, it seems extremely unlikely that cells would have evolved systems to accomplish this extraordinary feat of packaging were it not at the same time used to regulate the function of DNA. Indeed, it is difficult to imagine a packaging system that would not inevitably exert a major functional effect. There is now a rapidly growing body of experimental evidence to show that packaging of DNA and its organization within the cell nucleus play central, sometimes dominant, roles in regulating its different functions (Fuchs et al. 2006). Many histone sites, particularly at the N-terminal tails extending outward from the nucleosome core, can be posttranslationally modified at several different lysine, serine, and arginine residues. Keeping in mind the eight histone molecules, the several different lysine, serine, or arginine residues that could be modified, and the many different types of modifications like methylations, acetylations, phosphorylations, and ubiquitinations, the number of different combinations of histone modifications becomes enormous and the different ways of controlling the status of DNA unimaginable (Bannister and Kouzarides 2005). Counting only the methylation of histones, Bannister and Kouzarides (2005) calculated 3 1011 distinct methylation states, taking into consideration the 24 known methylation sites (17 lysine and 7 arginine residues) and the fact that lysine residues may be mono-, di-, or trimethylated whereas the arginine chain may be mono- or (symmetrically or asymmetrically) dimethylated. Altogether the many different types of histone modifications constitute the newly emerged epigenetic ‘‘histone codes,’’ which suggests that specific combination of histone modifications dictate specific transcriptional responses and cellular functions (Turner 2002). The detailed analysis of these modifications, their metabolism, and the enzymatic mechanisms involved is enormous and out of scope for this review, and the reader should refer to elsewhere (Turner 2002; Fischle et al. 2003; Rusche et al. 2003; Fuchs et al. 2006). In this subsection we focus primarily on histones H3 and H4 methylation marks and secondly on their acetylation. The modifications of these two histones demonstrated the power of changes onto DNA-based functions, regulating fundamental processes such as gene transcription, DNA repair, replication, and recombination (Bannister and Kouzarides 2005) and also extend to DNA methylation. The most studied methylation marks of H3 are lysine 4 (H3K4), lysine 9 (H3K9), lysine 27 (H3K27), and lysine 79 (H3K79). For H4, lysine 20 (H4K20) is the more thoroughly studied, not only in yeast, drosophila, and mammals including humans, but also in plants (Rusche et al. 2003; Ringrose and Paro 2004).

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Chromatin modulation by histone methylation involves multimeric protein–protein complexes containing, among other proteins, enzymes with histone MTase activity. All MTases contain a SET domain, a 130to 160-amino acid motif named after the gene (Su-var3–9), Enhancer of zeste (E-z), and Trithorax (Trx) (see Section II.B). Perhaps what could be indicative for the significant role played by these enzymes is the finding of Baumbusch et al. (2001) that even the small genome of Arabidopsis contains at least 29 active genes encoding SET-domain proteins that can be assigned to four evolutionarily conserved classes. This high number of SET-domain genes stresses their significant epigenetic control on chromatin functions during plant development. The identification of putative nuclear localization signals and AT-hooks in many of the SET proteins studied supports their epigenetic role in the nucleus. The finding by these researchers that eight of the Su-var3–9 type genes lack introns indicates evolution of new SET genes by retrotransposition. Generally speaking, inactive chromatin (heterochromatin) is more methylated in both histones and DNA in comparison to active euchromatin, which is generally less methylated. This is particularly true for H3K9, H3K27, H3K79, and H4K20 methylations (Fig. 2.2). Then there are differences in heterochromatization of chromosomal areas depending

Fig. 2.2. Different modifications of histones lead to different states of chromatin architecture, which affects gene expression.


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on the number of methylated histones, the number of methylated lysines in each histone, and the degree of their methylation (mono-ditri-), as well as the extent to C methylation of the nucleosomal DNA. But the general rule holds true with the exception of H3K4. Methylation of H3K4, and particularly the trimethylated form of this lysine, leads to activated chromatin (Lachner and Jenuwein 2002). In fact, this methyl marks act in synergy with histone acetyltransferases (HATs), pointing to the convergence of the two histone epigenetic marks leading to euchromatic status. In summary, it is well established today, in all higher eukaryotes, that transcriptionally active, euchromatic regions are marked by hyperacetylation of all four histones and di-or-tri methylation of H3K4; on the contrary, gene-poor, transcriptionally inactive heterochromatin regions exhibit (1) hypoacetylation generated by HDACs, (2) demethylation of H3K4, and (3) hypermethylation of the rest of the targets mentioned (Vaquero et al. 2003; Dou et al. 2005). It is possible that histone methylation can induce alterations in chromatin architecture, either condensing or relaxing its structure. However, a methyl group is relatively small and its addition to lysine or arginine residues does not neutralize their charge, so it is unlikely that methylation alone will significantly affect chromatin structure. It is more likely that it creates binding sites for regulatory proteins that contain specialized binding domains that recognize methylated lysines or arginines. Indeed, it is found that repressive proteins, such as heterochromatin protein 1 (HP1), not only depend on chromatin condensing, it also attracts cytosine MTases leading to DNA methylation (Tamaru and Selker 2001; Jackson et al. 2002; Malagnac et al. 2002). Conversely, DNA methylation can also trigger H3K9 methylation (Johnson et al. 2002; Soppe et al. 2002; Lehnertz et al. 2003; Tariq et al. 2003). It has also been found that HDACs, histone MTases, and methylhistone-binding proteins lead to the recruitment of DNA MTases (Nan et al. 1998; Fuks 2003a, b; for a detailed genome-wide analysis of HDACs, see Ekwall [2005]). In conclusion, this not only links the two histone-based mechanisms of epigenetic marks (histone H3K9 methylation and deacetylation) with DNA methylation, but also indicates their convergence and cooperation for a tighter and perhaps more permanent and secure silencing of the DNA under these epigenetic controls. Multimeric protein complexes have been identified that function as ‘‘cellular memory keys’’ that ‘‘lock’’ gene expression states and enable their inheritance over many cell mitotic or meiotic division cycles (Narlikar et al. 2002). The multiprotein complexes, involved in the regulation of developmental stages, were originally identified and studied in drosophila, where two such main complexes were originally

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described in this insect: the Polycomb (PcG) and the Trithorax (Trx) complexes (Mahmoudi and Vernijzer 2001). Both are known for their role in controlling the expression of homeotic genes but with opposite effects; PcG maintains them in an inactive state while the Trx leads them to an active state. Similar complexes were recently identified to execute conserved functions in plants, with the PcG protein group in plants being the most studied (Alvarez-Venegas et al. 2003). The PcG complex is involved in the control of the expression of homeotic genes that are essential for proper plant development transcription factors (primarily plant MADS-box genes). Although there is no structural relevance between the target genes of PcG complexes in animals (Homeobox genes) and plants (MADS-box genes), the PcGs are functionally similar in controlling developmental processes (Fig. 2.3). Built on a skeletal protein with many WD-40 repeats (WD-40 repeats facilitate

Fig. 2.3. A scheme showing a model of similar PcG complexes acting at different stages in plant development. The drosophila PcG counterpart is shown boxed in the middle. FIE: fertilization-independent endosperm; MEA: Medea; FIS2: fertilization-independent seed 2; MSI1: multicopy supressor of IRA; EMF: embryonic flower; CLF: curly leaf; SWN: Swinger; VRN2: Vernalization 2; ESC: extra sex combs; E(Z): enhancer of Zeste; SU(Z)12: suppressor of Zeste 12. Plant FIE and its drosophila homolog, ESC, comprise the core subunit of the complex around which the other PcG subunit homologs are bound.


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protein–protein interactions taking a propeller-type form), thus allowing many different proteins operating on chromatin, such as the H3K27 MTases MEA, and HDACs among others, to form different PcG complexes. Through the H3K27 methylation and HDAC activities that are associated with DNA methylation, PcGs keeps the chromatin condensed and any respective gene included in the area inactive, thus silent. Arabidopsis MEA was the first plant H3K27 MTase homolog of the PcG H3K27 MTase proteins to be described (Grossniklaus et al. 1998; Kinoshita et al. 1999; Kiyosue et al. 1999; Makarevich et al. 2006). Later homolog genes like curly leaf (CLF) (Goodrich et al. 1997) have been described also from Arabidopsis and other plants operating in different tissues or developmental stages (Springer et al. 2002). In conclusion, different PcG complexes have been described, operating in different tissues and/or developmental stages (Levine et al. 2004). Built on WD-40 PcG complexes, they differ in their H3K27 MTase, HDAC, and other components showing different specificities and thus are targeted to different target homeotic genes destined to operate in different developmental stages. It should be stressed again that PcG is a suppressive complex operating to keep silenced genes in the quiescent state during early embryonic development until the activation time. Despite the fact that all PcG genes have been identified from mutations inactivating the coded protein (leading to reactivation of the silenced loci), normally wild-type PcG genes maintain chromatin in their compact, inactive state, leading to transcription inactivation (Schwartz and Pirrotta 2007). Inactivation through H3K27 methylation, is reversible and not permanent (Matzke et al. 2004). In the case it is accompanied by methylation of H3K9 (and of other sites as well), histone deacetylation and DNA methylation, then chromatin is becoming progressively more and more condensed and heterochromatic. This leads to more permanent (cemented) inactivation that is almost irreversible during development (Tariq and Pazworski 2004). This characterizes the majority of the heterochromatic genome consisting of REs and TEs. Contrary to the view that methylation of different lysines, in cooperation with DNA methylation, leads to more compact, more heterochromatic, thus inactive chromatin, as mentioned already, methylation of the outermost lysine (K4) of H3 (H3K4) leads to gene activation by chromatin relaxation. This is accomplished by the Trx complex involving H3K4 MTase activity. Thus Trx acts antagonistically to PcG. Alvarez-Venegas et al. (2003) have cloned plant homologs of Trx from Arabidopsis (ATX-1). ATX-1 found to contain a H3K4 MTase, like any other MTase, leading to activation of flower homeotic genes. Additional

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ATP-dependent chromatin-activating complexes of Trx type, identified mostly in yeast (SWI2/SNF2, ISWI, Mi2/CHD, and INO80) with homologous structures found in drosophila and humans, are currently under intense investigation in plants too. Plants have a remarkably high number of ATPases with homology to ATPases participating in chromatin remodeling (Wagner 2003). From 42 putative Arabidopsis SNF2-like ATPases (see the Plant Chromatin Database at http://chromdb.org), 4 belong to the canonical SWI2/SNF2 subfamily (Verbsky and Richards 2001). However, only 1 of these, BRAHMA (At2g46020), carries a Cterminal region resembling a chromodomain, a hair mark for binding acetylated lysine residues of histone tails (Hudson 2000; Brzeski and Jerzmanowski 2003). In contrast to the multiplicity of SNF2-like ATPase proteins, Arabidopsis has only one gene, BUSHY (BSH), coding for a structural and functional homolog of SNF5 (Brzeski et al. 1999), but contains four genes encoding SWI3 homologs. Sarnowski et al. (2005) described also the characterization of knockout mutations in all four Arabidopsis ATSWI3. ATSWI3A and ATSWI3B cause similar blocks of embryo development at the early globular stage. However, unlike atswi3a, the atswiSb mutations result in aberrant segregation of progeny with arrested ovules, which suggests a possible role for ATSWISB in imprinting (see Sarnowski et al. 2005 for detailed analysis of the plant SNF2 complexes). Dou et al. (2005) made the significant observation that H3K4 MTase is physically associated with H4K16 HAT, a known gene activator, since many transcriptional co-activators proved to be HATs (Carrozza et al. 2003). The physical association between H3K4 MTase, a known chromatin activator (Hayashi et al. 2005), and H4K17, another chromatin activator, leads to direct synergy of two chromatin activation mechanisms operating through histones, in reversing silenced chromatin by antagonizing the silencing activity of H3K9, H3K27 and H3K79 MTases, which also operate through histones. Until very recently, the methylation of histones was thought to be an irreversible process, despite the fact that for more than 30 years, a demethylase activity was described (Paik and Kim 1973). The reversibility of histone methylation became apparent a few years ago when antibodies against methylated lysine or methylated arginine residues were used in ChIP assays. These experiments revealed that the methylation of histone residues appeared to be reduced under certain conditions (Martienssen and Colot 2001). This prompted the idea that histone demethylation was a likely possibility, and indeed an amine oxidase specific for removing methyl groups from histones in actively transcripted H3K4 chromatin was described, leading in this case to


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suppression (see Bannister and Kouzarides 2005 for review of histone demethylation). Perhaps what is more revealing for the scope of this article is the link between histone methylations–demethylations, and the concomitant effects on DNA methylations are the recent findings that proteins with a Tudor domain are binding to methylated H3K79 or to H4K20. This is of particular importance because these proteins are involved in binding to damaged DNA and DNA recombination. Many different kinds of stresses lead directly (radiations) or indirectly (other abiotic and biotic stresses) to damages of proteins and DNA by oxygen radicals (see Section V.B). Efficiency of removal of damaged DNA bases, refilling the respective methyl groups on C after this repair, and reestablishment of histone modifications will drastically affect silencing. In this way, epigenetic mechanisms and different kinds of stresses on one hand and these processes with genome plasticity on the other are interconnected (see Section V.B). C. RNA-based Control Mechanisms For a long time, RNA has lived in the shadow of DNA and proteins. The shadow has been so obscuring that a whole universe of small RNAs, primarily non-coding for proteins, has been hidden from view until recently. This was due to the methodologies applied in studying mainly the messenger role of large RNAs. The situation changed dramatically in the last few years after the discovery of the significant roles played by small RNA (sRNA). sRNAs were found to interfere (thus the term RNAi for these mechanisms) with many aspects of gene actions and genome organization and, as consequence, to control significant steps in cell-cycle and the development of eukaryotic organisms. As Karp and Ambros (2005) noted, it came as a huge surprise that a major level of generegulation was completely unknown until the recent discovery of sRNAs. Like many other significant discoveries, revolutionary discoveries concerning this newly emerging field of RNAi were also made from research groups working with plants. In one case, attempts to boost the expression of an endogenous gene with an extra transgene copy eliminated all expression from both genes: the endogenous gene and the transgene. In this experiment, efforts to enhance floral coloration in petunia by overexpressing a transgene encoding a protein involved in pigment synthesis led to partial or complete loss of color. This resulted from coordinate silencing (‘‘cosuppression’’) of both the transgene and the homologous plant gene (Napoli et al. 1990; Van der Krol et al. 1990), which later was shown to occur at the posttranscriptional level (De Carvalho et al. 1992; Van Blokland et al. 1994). PTGS was discov-

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ered from another experiment designed to evaluate antisense suppression, which was a promising approach at the time for selectively silencing plant gene expression. In theory, antisense RNA encoded by a transgene should base pair to the complementary mRNA of a plant gene, preventing its translation into protein. Although the control ‘‘sense’’ transgene RNAs are unable to base pair to mRNA, and hence should not induce silencing, they often inexplicably did (Smith et al. 1990). A study by Lindbo et al. (1993) showed that plant RNA viruses could be both initiators and targets of PTGS. Plants expressing a transgene encoding a truncated viral-coat protein became resistant to the corresponding virus, a state achieved by mutual degradation of viral RNA and transgene mRNA. In addition to forging a link between RNA virus resistance and PTGS, this study included a remarkably prescient model for PTGS that featured an RNA-dependent RNA polymerase (RDR), dsRNA, and small RNAs, all of which were later found to be important for the RNAi. In a second approach, plants that recovered from one viral infection were resistant against an unrelated virus, provided it carried a small sequence insert from the first virus (Ratcliff et al. 1997). Finally, replicating viroids or viruses that propagated entirely through RNA intermediates left behind an imprint of methylation on homologous DNA. The discovery of silencing-associated sRNAs by Hamilton and Baulcombe (1999) gave this diverse set of homology-dependent events a common specificity determinant and pointed the way to a densely populated sRNA world. Finally, an influential paper by Wassenegger et al. (1994) reported the discovery of RNA-directed DNA methylation in transgenic tobacco plants (see Section II.C). The repression was dependent on partial sequence complementarity between the lin-4 21-nt RNA and the 3’ (UTR) of lin-14 mRNA. (For a more detailed history of the discoveries involving sRNAs, see Matzke et al. [2004]); Zamore and Haley [2005]; and the book by Galun [2005].) Double-strand RNAs could be generated in the cell from many different sources and using different molecular mechanisms: (1) sense together with antisense transcription that has been documented from EST sequencing efforts in a much higher frequency than ever thought; (2) RNA duplication of RNA molecules from different sources, including RNA molecules from RNA viruses and viroids through the action of RNA directed RNA polymerase; (3) transcription of TE and RE with direct repeats in their sequence leading to different intramolecular dsRNA motives; (4) transcription of different primarily intergenic DNA sequences coding for larger precursor RNA molecules like the precursors of mRNAs, which are also cupped and polyadenylated in their 5’ and 3’ ends, respectively, and are processed down to the


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production of sRNAs called miRNAs. dsRNA, the central player in RNA-mediated gene silencing, is chopped into small RNAs by the enzyme Dicer. Farther down sRNAs become associated and guide their own further amplification to many more copies and join an RNAinduced silencing complex (RISC), leading either to mRNA clearage or translational inhibition. In order to guide suppression of homologous targets, the sRNA duplex must become single-stranded. The strand with the 5’ phosphate at the less stable end of the helix is incorporated as a guide RNA into an effector complex containing an Argonaute (AGO) protein. Two types of effector complexes have been described. The cytoplasmic complex, RISC, mediates both mRNA cleavage and translational inhibition. Additionally, the RNA-induced transcriptional genesilencing complex (RITS) operating in the nucleus leads to chromatin modifications and DNA methylation (Matzke et al. 2002). Since we now have a good understanding of the RNAi pathway, current interest is turning to variations on the basic mechanism. Recent studies involving plants and the nematode Caenorhabditis elegans (Pak and Fire 2007; Sijen et al. 2007) deal with the amplification of silencing-related RNA and explain how strong, persistent silencing can be initiated with small amounts of ‘‘initiator’’ double-stranded RNA. The amplification process has implications for application of RNA interference to control gene expression in biotechnology and for understanding the effects of silencing RNAs on cell function and organism development (Baulcombe 2007). sRNAs can follow a non-cell autonomous travel throughout the plant organisms using different proteins as carriers (Lucas and Lee 2004) in plants and in some animals; the effects of posttranscriptional RNA silencing can extend beyond its sites of initiation, owing to the movement of signal molecules. Breakthrough grafting experiments indeed demonstrated 100% transmission of the nitrate reductase (Nia) cosuppressed state from silenced rootstocks to nonsilenced transgenic scions (Palauqui et al. 1997). Shortly after this discovery, leaf-infiltration of recombinant Agrobacterium cultures (agro-infiltration) into transgenic Nicotiana benthamiana expressing the green fluorescent protein (GFP) was found to trigger a systemic, sequence-specific loss of GFP expression (Voinnet and Baulcombe 1997). Since then, non-cell autonomous silencing has been documented in several additional plant species, including sunflower, curcubits, Arabidopsis, and, to a lesser extent, roots of the legume Medicago; it was also recently demonstrated in fern gametophytes (Rutherford et al. 2004). (See Voinnet 2005a for a recent review on non-cell autonomous RNA silencing). With the current increasing interest in using grafted plants in vegetable production (Sapountzakis and Tsaftaris 2002;

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Grigoriadis et al. 2005), systemic RNA-silencing scion genes from rootstock RNAs (Lucas and Lee 2004; Athanasiadou et al. 2005) is under intense study as a major determinant of lowering quality characteristics in vegetable fruits. In conclusion, originating from different resources and using different molecular processes for production, numerous sRNAs are produced that after becoming part of RISC or RITS complexes fulfill divergent jobs. In this framework miRNAs, the most abundant type of endogenous sRNAs in plants and animals, have been implicated in different developmental control mechanisms by acting in trans to negatively regulate the expression of target mRNAs from other loci by blocking their translation, by interfering with their splicing, or by destructing them. They regulate others with homology within the restricted miRNA target binding site, but limited homology to the rest of the miRNA precursor. They arise from primary transcripts (pre-miRNAs) transcribed from these endogenous genes by two rounds of endoribonuclease III processing involving first Drosha, producing a hairpin-shaped pre-miRNA, and then Dicer (reviewed by Chen 2005). After Dicer processing, miRNAs emerge as siRNA-dupIex-like structures, but only one strand, the mature miRNA, is then predominantly incorporated into the RISC effector complexes. The discarded RNA strand is frequently referred to as miRNA and is degraded. In plants, miRNAs guide mRNA cleavage, and the highly complementary target sites are readily identified using bioinformatic tools (see Section II.E). In contrast, animal miRNAs preferentially target mRNAs at partially complementary yet evolutionary conserved sites, which are predominantly located within the 3’ UTR. siRNAs are produced by TEs and inverted REs transcription, upon exogenous delivery of dsRNA, transgenic expression of long dsRNA, and RNA viral infections. However, endogenous genes leading to siRNAs production are rare. Thus generally speaking, siRNA usually targets only highly homologs sequences (see the description of tasiRNAs). siRNAs serve as a defense against different ‘‘invading’’ nucleic acids such as virus, TEs, REs, transgenes, and injected dsRNA among others (Vance and Vaucheret 2001). Such ‘‘invaders’’ trigger silencing by producing long dsRNA that are cleaved into siRNA by Dicer. In this respect, TEs and REs are endogenous sources for siRNA production made from TEs and REs read through transcription, and siRNAs produced this way serve as specific guides for distinguishing specific TEs and REs for inactivation through H3K9 and DNA methylation (Lippman et al. 2004). Similarly siRNAs act as guides of RISC in destroying RNA molecules with high homology to the dsRNA trigger. This particular category of siRNAs named rasiRNAs (repeat associate small RNAs)


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produced by read-through of sense or antisense transcription of REs and TEs varies in different organisms from 24 to 27 nucleotides. The significance of the variation and the mechanism produced remains to be elucidated, but the main function of this siRNA group is its involvement in establishing and maintaining heterochromatin structure and in controlling transcripts emerging from repeat sequences. Apparently the control on TEs and REs is such that even in case of their activation, the transcript is not only immediately destroyed through PTGS, but sRNA by-products of PTGS used as guides go back to the DNA to silence and put it again under control at the chromatin level. Another category of siRNA consisting of trans-acting siRNAs (tasiRNAs) was recently identified. tasiRNAs were initially confounded with miRNAs because the same sequences were cloned multiple times, suggesting a defined register for tasiRNA excision. It was revealed later that specific miRNAs in plants target single-stranded noncoding RNA transcripts for cleavage, which then are used as template for RNA-dependent RNA polymerase (RdRP). Dicer processes the resulting dsRNA in 21-nt increments producing mature tasiRNAs, which then subject mRNAs that have one or a few tasiRNA complementary sites to degradation. tasiRNAs are found to play a critical role in controlling different developmental stages of plants, particularly in phase changing (Willmann and Poethig 2005; Kidner and Martienssen 2005, and Section IV.A). The description of tasiRNAs indicates that categorizations and different names such as miRNAs and siRNAs may not describe entirely different types of sRNAs. The field is relatively recent with no conclusive knowledge on these very important genome regulators. Their significance grows daily, and their role could be greater than expected (Vaughn and Martienssen 2005). Lu et al. (2005) identified over 1.5 million sRNAs from Arabidopsis, representing over 75,000 distinct sequences, using Massively Parallel Signature Sequencing (MPSS) technology. They report that many more genes may be under the control of sRNAs than had been previously imagined (Mattick 2004). For this model plant species, current estimates predict that 2% of genes may be under miRNA control, but the number of genes that might be regulated by siRNAs is not known. It has already been mentioned that siRNAs are known to participate in RNAi-mediated silencing of REs and TEs (Lippman and Martienssen 2004). Thus it is no surprise that the majority of sRNA sequences matched these repeated sequences. Most of the remaining sRNA sequences came from intergenic regions (IGRs), including those derived from miRNAs, which is consistent with previous studies on a much smaller scale (Gustafson et al. 2005). Interestingly, 4,000 protein-coding genes (15% of genes in the genome) and several hundred pseudogenes matched at least one sRNA perfectly,

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presumably corresponding to siRNAs (miRNAs usually match imperfectly). However, most of these genes matched only one siRNA, and only a small percent of the total sRNA sequences were from genes. It is possible therefore that many other genes may have matches that went undetected. Consistent with the idea that siRNA guides silencing as an antiviral mechanism on behalf of the plant, many plant viruses, as a counterdefense mechanism (against the plant’s silencing efforts), encode proteins that interfere with processes targeted to their inactivation at one or more steps and thereby act as supressors of their silencing (Roth et al. 2004; Silhavy and Buryuan 2004). In summary, by identifying small RNAs as agents of gene silencing that act at multiple levels throughout the cell, molecular biologists have created a new paradigm for eukaryotic gene regulation (Matzke et al. 2004). Plant scientists have played an outstanding and leading role in RNA-mediated silencing research. Their successful production of a wide variety of transgenic plants, which displayed an equally rich diversity of gene-silencing phenomena, gave the opportunity to embark in research and analyze these unexpected outcomes. Simultaneously members of the agricultural biotechnology industry avidly pursued transgenic approaches to modulate plant gene expression, to genetically engineer virus resistance, and to find ways to avoid silencing and stabilize transgene expression. The history of gene-silencing research shows once again that plants offer outstanding experimental systems for elucidating general biological principles. D. The Triangle of Interactions of Epigenetic Mechanisms By describing DNA methylation, histone modifications, and RNA interference separately, it might appear that they are independent epigenetic mechanisms. On the contrary, the functional relationships between them and their mutual reinforcement (Vire et al. 2006) are unexpectedly complex (Fig. 2.1). DNA methylation mechanisms and histone modifications that lead to chromatin compaction and gene silencing are under intense scrutiny. While results of recent studies indicate intriguing links among the three epigenetic mechanisms, there is still a long way to go before we understand the details of their interaction. For instance, starting with DNA methylation, 5mC binding proteins (5mC BP) not only recognize and bind to methylated DNA but also recruit and form complexes with HDACs and other chromatin remodeling factors (Bird 2002; Fujita et al. 2003; Fuks 2003a; Zemach and Grafi 2007). One such gene coding for a human 5mC binding protein MeCP2 was found to be associated with the human Rett syndrome (Bienvenu and Chelly 2006). In this way, DNA methylation is linked with modifications of histones.


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Sarraf and Stanchera (2004) provided results that support a model in which the binding of a 5mC binding protein, MBD1, to 5mC leads to H3K9 methylation that is also heritably maintained through DNA replication. In parallel, Tariq et al. (2003), conducting ChIP and immunostaining analysis using antibodies against H3K9 in Arabidopsis met1–3 mutants, clearly demonstrated the CG methylation (but neither CNG or CNN methylation) is a prerequisite for the maintenance of histone methylation (H3K9). To state this another way: Histone modifications are also affecting DNA methylation. Observations made in plants, fungi, and mammals highlight H3K9 methylation as a kind of ‘‘beacon’’ for DNA methylation (Fuks 2005). H3K9, H3K27, and H4K20 methylation normally leads to inactivation of chromatin, and the relatively small methyl group on histones creates binding sites for regulatory proteins that contain specialized binding domains like HP1 that also attract cytosine MTases leading to DNA methylation (Tamaru and Selker 2001; Jackson et al. 2002; Malagnac et al. 2002; Fuks et al. 2003a). HDACs, histone MTases, and methylcytosine-binding proteins (Nan et al. 1998) lead to the recruitment of DNA MTases (Fuks 2005). These data support a model in which methylated DNA is part of an epigenetic program that leads to transcriptional silencing (Fuks 2005). Methylation at H3K9 creates a foundation for the adaptor molecule HP1, which would, in turn, recruit DNA MTases. The recruited DNA MTases would catalyze CG methylation in the region of the methylated lysine. Methyl-CG-binding domains would then bind to the methylated DNA. The bound methyl-CG-binding domains would attract HDAC complexes, which, in turn, seem to be required to prepare more H3K9 methylation. Furthermore, according to the model, methyl-CpG-binding domains would also recruit H3K9 MTases. Thus, epigenetic information embodied in methylated residues would flow from histone to DNA and back. Variations of this model are possible to envisage. For example, the well-described DNA MTase-HDAC interaction might provide an alternative route for delivery of the HDAC activity that paves the way for H3K9 methylation. Further research is clearly needed to elucidate the precise sequence of events leading to epigenetic silencing. Nonetheless, this model is appealing because it implies that DNA methylation, histone deacetylation, and H3K9 methylation act in synergy to generate a self-reinforcing epigenetic cycle that maintains and perpetuates a repressed chromatin state. Such a cycle might be relevant to situations in which genes need to be ‘‘locked’’ into a permanently silenced state, as with the case of imprinted genes or of hypermethylated genes in cancer. The recent discovery of small RNA interference systems showed that sRNA molecules play a role in DNA methylation in addition to their role

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in RNA processing, translation, and destruction (Wassenerger et al. 1994). RdDM could provide a significant connection between mechanisms that govern the establishment of DNA methylation and the initiation of gene silencing. RdDM facilitates the de novo methylation of a target DNA sequence similar to the triggering double-stranded RNA molecule. During the RdDM process, cytosines become methylated regardless of their location (Mette et al. 2000). When dsRNA is directed against promoter regions, this causes their de novo methylation and the onset of TGS in plants (Mette et al. 2000; Jones et al. 2001). After removal of the trigger RNA, CG and CNG methylation is maintained but CNN methylation is reduced rapidly (Aufsatz et al. 2002). The maintenance of hypermethylation and TGS triggered by RdDM in the absence of trigger RNA requires MET1 and DDM1 activity (Jones et al. 2001). The DRMs have been shown to be the DNA MTases that are involved in the establishment of methylation by RdDM (Cao et al. 2003). Furthermore, a direct link between RNAi and DRMs could be inferred by results revealing that mutations in components of the RNAi machinery affected DRMs-dependent de novo methylation of a FWA transgene in addition to the endogenous FWA locus (Chan et al. 2004). Matzke et al. (2004) and Kanno et al. (2004) found that, in addition, HDACS and a putative chromatin protein are components of RdDM, a fact that points to the involvement of siRNA to chromatin structure (Gendrel and Colot 2005). Mette et al. (2005) reported that transcription of DNA by RNA polymerase II is required for RdDM, pointing to a direct RNA-RNA hybridization model between siRNA and nascent RNA transcripts prior to transcription completion and termination, which leads to DNA methylation and histone modifications like methylations and deacetylations leading to silencing. Thus, the nascent RNA transcript provides the necessary link through RNA-RNA hybridization (and not RNA-DNA hybridization) for a sequence-specific silencing. In parallel, from studies conducted with plants Bao et al. (2004) showed that miRNA could also trigger TGS through RNA-RNA hybridization of miRNA–nascent mRNA hybrids. Thus, miRNA is not restricting itself to PSGS but it can ‘‘mimic’’ siRNA in its TGS function, which again makes the functional distinction between siRNA and miRNA more difficult (Ronemus and Martienssen 2005).

E. Methods of Studying Epigenetic Mechanisms The techniques developed to detect DNA methylation (Lyko 2005) can be divided into three categories according to the information they provide (Table 2.2). Methods used to measure global methylation level


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Table 2.2. Methods for DNA methylation detection. Methods Methylation Status

Reference

Immunological method

Sano et al. 1980; Achwal et al. 1984; Vilpo et al. 1986 Gruenbaum et al. 1983 Gehrke et al. 1984

Nearest-neighbor analysis High-performance liquid chromatography (HPLC) Nuclear magnetic resonance (NMR) Mass spectroscopy (MS) Thin layer chromatography (TLC) GLOBAL CONTENT SssI Methyl-acceptance assay Self-primed-in situ labeling (SPRINS) Chloroacetaldehyde assay High-performance caillary electrophoresis (HPCE) Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) REGIONAL CONTENT Agarose gel electrophoresis upon digest with methylation sensitive enzymes (MSE) Chemical sequencing Cytosine-extension assay Enzymatic Regional Methylation Analysis (ERMA) SPECIFIC (INTENSITY) Methylation-sensitive restriction enzyme (MSRE) Southern Random amplification coupled with restriction enzyme digestion (CRED-RA) Direct bisulfite sequencing Methylation-sensitive arbitrarly primed PCR (MSAP-PCR) Methylation-sensitive restriction fingerprinting technique (MSRF-PCR) Methylatin-sensitive single nucleotide primer extension (MS-SNuPE) Melting curve combined bisulfite restriction analysis (COBRA) Hemi-methylation assay Bisulphite primer extension—HPLC

Wuetrich 1986 Annan et al. 1989 Wilson et al. 1986; Gowher et al. 2000; Ramsahoye et al. 2000. Wu et al. 1993, Schmitt et al. 1997 Andersen et al. 1998 Oakeley et al. 1999 Fraga et al. 2002 Cornelius et al. 2005 Song et al. 2006

Bird et al. 1979 Church and Gilbert 1984; Saluz and Jost 1986 Pogribny et al. 1999 Galm et al. 2002

Bird et al. 1978 Cai et al. 1996 Paul and Clark 1996 Gonzalgo et al. 1997a Huang et al. 1997 Gonzalgo et al. 1997b Xiong and Laird 1997 Liang et al. 2002 Matin et al. 2002 (continues )

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Table 2.2. (continued) Single nucletide polymorphism methylation (SNaPmeth) PyroMeth Quantitative multiplex-methylation-specific PCR (QM-MSP) Methylation sensitive dot blot assay (MS-DBA) Methylation-sensitive multiplex ligationdependent probe amplification (MS-MLPA) microchip electrophoresis PATTERNS OF ALLELE Hydrazine/permaganate sequencing Methylation-specific PCR (MS-PCR) Methylation sensitive PCR-in situ hybridization (MS PCR-ISH) Methylation dependent denaturating gradient gell electrophoresis (MB-DGGE) Bisulfite-DGGE Methyl Light Bisulphite-single strand conformation polymorphism Bisulphite-SSCP Methylated DNA binding column— denaturing gradient gel electrophoresis (MBDC-DNGGE) Methylation sensitive PCR Denaturing HPLC (MSP-DHPLC) In a tube fluorescence melting curve Melting curve methylation specific PCR (McMSP) CpG methylation and single nucleotide polymorphisms SNPs—DHPLC Conversion specific methyLight Con-light MSP Denaturing HPLC (DHPLC) Targeted gene methylation (TAGM) Methylation-specific PCR and PCR with confronting two-pair primers (MSP-CTPP) Real-time PCR—quantitative analysis of methylated alleles (QAMA) Restriction Landmark Genomic Scanning Methylation-sensitive amplification polymorphism (MASP) GENOME PROFILES Methylation sensitive representational difference analysis (MS-RDA)

Uhlmann et al. 2002 Uhlmann et al. 2002 Fackler et al. 2004 Clement and Benhattar 2005 Nygren et al. 2005 Zhou et al. 2005

Ohmori et al. 1978; Fritzsche et al. 1987 Herman et al. 1996 Nuovo et al. 1999 Uhlmann et al. 1999 Uhlmann et al. 1999, Aggerholm et al. 1999 Eads et al. 2000 Burri and Chaubert et al. 1999 Bianco et al. 1999 Masahiko et al. 1999

Baumer et al. 2001 Worm et al. 2001 Akey et al. 2002 Deng et al. 2002 Rand et al. 2002 Couvert et al. 2003 Jessen et al. 2004 Sasamoto et al. 2004 Zeschnigk et al. 2004 Hayashizaki et al. 1993 Vos et al. 1995, Reyna-Lopez et al. 1997, Xiong et al. 1999 Ushijima et al. 1997


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Table 2.2. (continued) Methylated DNA binding column and denaturing gradient gel electrophoresis Methylated CpG island amplification Epigenomics microarray Two-color oligo array Identification of CpG Island exhibiting altered methylation patterns (ICEAMP) Amplification of inter-methylated sites (AIMS) Methylation- specific oligo array (MSO) Methylation amplification DNA Chip (MAD) Methylation-sensitive digital karyotyping NotI Cloning of deleted sequences (NotI CODE) Demethylation/expression analysis Different methylation hybridization (DMH) Similarly/differentially methylated regions (SMR/DMR) cloning Size fractination profiling Methylated-CpG island recovery assay (MIRA)

Masahiko et al. 1999 Toyota et al. 1999a, b Adorjan et al. 2002 Balog et al. 2002 Brock et al. 2001 Frigola et al. 2002 Gitan et al. 2002 Hatada et al. 2002 Hu et al. 2005 Li et al. 2002 Suzuki et al. 2002 Shi et al. 2002 Strichman-Almashanu et al. 2002 Tompa et al. 2002 Rauch and Pfeifer 2005

usually detect and quantify different types of modified bases. Some methods from this class are useful in the initial research to determine which species possess certain base modification and to which extent; the other methods from this group are useful for quick rough measures of ongoing hypermethylation in certain tissues. The second category consists of sequence-specific techniques, reliable for methylation analysis of already known genes and sequences (Thomassin et al. 1999). The most precise are based on modification by bisulfite, hydrazine, or permanganate, to accurately measure the level of methylation at given CG sites in target areas of genes. The third category would be the methods that provide information on methylation profiles; they are used for discovery of new methylcytosine hot spots—future biomarkers—without requiring prior knowledge of the DNA sequence. Recently a computational prediction progmam called HDFINDER was constructed and tested by Das et al. (2006) for predicting the methylation landscape of all 22 autosomal DNA, with 86% accuracy. Detection of chromatin modifications can be accomplished by chromatin immunoprecipitation (ChIP) and the ChIP on chip assay (Orlando 2000; Horak and Snyder 2002; Lippman et al. 2004; Gendrel et al. 2005). The ChIP assay is a powerful tool to identify genome-wide associations between histone proteins and their target genes. The key feature of the assay is that it makes use of an antibody that recognizes the protein of interest, not only free in solution but also when assembled into chromatin. The

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ChIP assay involves two steps: (1) in vivo crosslinking of whole cells that fixes protein-protein and protein-DNA interactions; and (2) immunoprecipitation of protein-DNA complexes with specific antibodies. The ChIP assay has been widely used for mapping the location of modified histones in the genome. Using chromatin immunoprecipitation with antibodies against specific histone modifications followed by PCR amplification of specific sequences, it is possible to detect the chromatin states of specific genes. This assay was used by Ko¨hler et al. (2003b) to identify PHERES 1 (PHE1) in Arabidopsis as the potential target gene of the MEA-FIE Polycomb group protein complex using antibodies against MEA and FIE proteins and PCR amplifications of different regions of the PHE1 gene after the immunoprecipitations. A highly promising recent technical advance has been the development of the so-called ChIP on chip assay. In this assay, chromatin immunoprecipitations are performed with antibodies against specific histone modifications, such as dimethyl H3K9 or dimethyl H3K4, indicators of heterochromatic or euchromatic chromatin regions, respectively. All of the DNA fragments recovered from the precipitate are then amplified. This DNA is enriched for sequences recovered from chromatin immunoprecipitation with antibodies raised against H3K9 or H3K4 modifications. The amplified DNA is then labeled and annealed to DNA microarray slides containing genomic regions of interest called genomic tiling microarrays (Lippman et al. 2004; Gendrel et al. 2005). In this way it is possible to study entire regions of a genome and map precise locations that are enriched for particular histone modifications. This techinique was used successfully to map H3K9 and H3K4 histone methylations across a 1.5Mb region in Arabidopsis (Lippman et al. 2004; Gendrel et al. 2005). Finally, RNAi identification of micro-RNAs has been accomplished with biochemical and in silico approaches. For the first ‘‘wet’’ approach, various biochemical methods were used in order to identify miRNAs in plants; however, only a small number of miRNAs could be isolated. The basic method of identifying miRNA genes has been to isolate total RNA, separate the small-RNA fraction (i.e., RNAs of about20 to 25nt long) on polyacrylamide gels, ligate modified 3’adaptors, reverse transcribe, clone, and sequence these small-molecular-weight RNAs (Elbasir et al. 2001). One drawback of this method is that it is biased for RNAs that are relatively abundant (Jones-Rhoades and Bartel 2004). As an alternative, in silico identification of miRNAs has been used widely in the past few years. Because in plants miRNAs have nearly perfect complementarity with their target sequences, it has been easy to develop computational approaches in order to identify miRNAs molecules in


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plants. Based on the conservation of short sequences between the genomes of Arabidopsis and rice, a search for conserved hairpins and target sequences has been possible. In this way, 91 potential miRNas were detected in Arabidopsis and in rice (Bonnet et al. 2004; JonesRhoades and Bartel 2004). Arabidopsis and rice miRNAs seem to be highly conserved, pointing to the critical role they play in various aspects in plant development. The plant miRNAs that had been identified in the past targeted primarily transcription factors operating at various stages in plant development. Introduction of mutations in miRNA sequences have led to striking developmental abnormalities (Kidner and Martienssen 2005). Currently 117 miRNAs from Arabidopsis, 178 from rice, 97 from maize, 16 from Medicago truncatula, 22 from soybean, 213 from poplar, 16 from sugarcane, and 72 from sorghum have been reported in the miRNA Registry (Griffiths-Jones 2004). Furthermore, a new computational tool for the identification of novel miRNAs and their targets in plants, named MIRO, was developed recently and will soon be available on the Internet (Ahren et al. unp. results). MIRO is based on evolutionary conservation and internal genome conservation and uses inversion analysis in order to predict hairpins. MiRNA target identification is performed with the MIRANDA algorithm (Enright et al. 2003). Finally a pattern-based method for the identification of microRNA binding sites and their corresponding heteroduplexes has been reported by Miranda et al. (2006). III. PLANT BIOLOGICAL PHENOMENA INVOLVING EPIGENETIC MECHANISMS A. Epigenetic Control of Transposable Elements and Repeats TEs were discovered and studied as the source of variegated maize phenotypes by Barbara McClintock and Peter Peterson, two illustrious pioneers of twentieth-century genetics. Earlier geneticists had studied variegation, which is obvious in maize seeds and many other plant parts. Alleles were classified as demonstrating somatic variation in contrast to the stable colored and colorless alleles. Parallel detailed analysis by Marcus Rhoades demonstrated the first case of transactivation of gene expression; specifically, the response of the a1-dt allele to a dominant Dotted factor elsewhere in the genome. McClintock’s work established that mobile DNA elements could be responsible for the in cis component of mutability, a discovery that surprised everyone (Fedoroff 1999; Walbot 2002). McClintock and Peterson showed that active forms of these elements were required in trans to mobilize

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derivative elements Ds and dSpm, respectively. More important, they demonstrated that alleles that acquired a transposon insertion fell under the control of the mobile DNA system. For that reason, McClintock named the transposons controlling elements, despite the fact that mobility was the most surprising property of the transposons mapped and analyzed. McClintock speculated that the controlling elements might be important in molding host gene expression patterns, an insight for which there is growing molecular evidence. It is also equally true that diverse host processes control TEs. TEs and their hosts have co-evolved tolerance: TEs are not completely silenced in the host, and the host is not killed by TEs. In the 50 years since multiple types of TEs have been discovered, the two basic categories have been identified: retrotransposons and DNA transposons. Retroelements insert through the production of an RNA copy of an existing TE; this RNA transcript is copied by reverse transcriptase into DNA, which inserts into a new chromosomal location. Retrotransposons can proliferate and insert, but they do not excise; as a consequence, retrotransposon-induced mutations have a stable, usually null, phenotype. In contrast, the ‘‘classic’’ DNA elements such as Ac/Ds and Spm/dSpm move as pieces of DNA. These elements are organized into families of autonomous and nonautonomous elements. Walbot (2002) provides data for the different families of TE and their percentages in the maize genome. Indicative that the methylation patterns of DNA may change during development is the fact that the RNA-silencing patterns can vary temporally during development. For example, work with two maize TEs Suppressor-mutators (Spm) and Robertson’s Mutator (Mu) has shown that silencing can increase during shoot development. siRNAs are important for the establishment of transposon silencing by DNA methylation. Plants that have active Spm or Mu transposons at germination display a gradual decrease in transposon activity and an increase in transposon methylation along the primary shoot of the plant. It was further shown that developmentally regulated methylation and silencing are paralleled by a gradual reduction in the polyadenylation of Muderived RNA and by an increase in the nuclear retention of these RNAs, resulting in fewer mature transcripts. The developmental regulation of Spm and Mu transposons was demonstrated most clearly in studies with the pale green mutant hcf106 and the lesion mutant Les28, whose mutant phenotypes are caused by the activity of Mu. Upon inactivation of Mu, the phenotypes of these mutants are suppressed, and sectors of wildtype tissue can be seen. These sectors increase in size and number in subsequent lateral organs as the plant develops. As a result, the


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juvenile leaves tend to have the greatest transposon activity, the adult leaves and ears (containing the female gametophyte) an intermediate activity, and the pollen the lowest activity. These changes in epigenetic states, although reversible under the appropriate conditions, are both mitotically and meiotically heritable. Because the pollen has a lower transposon activity than tissues generated during earlier developmental stages, it has a relatively high chance of passing on an inactive transposon state to its progeny. TEs and REs constitute a large fraction of the DNA in some species of animals, such as mice and humans, or plants, such as maize and wheat. There are three main views about the role of TEs and REs in the genetic material of these organisms. They have been considered as genome parasites or as ‘‘junk DNA’’ causing genetic diseases and evolving as retroviruses (Bird 2002). Another view argues that they are maintained in big numbers because of the mechanisms that make large, redundant genomes possible (Fedoroff 2002). Finally since there is evidence in support of each model, most probably synthesis of both is the correct one (Becker and Lonnig 2001; Bestor 2003). TEs were parasitic in nature at their origin, and host genome developed secure control system over their overamplification. Host genome found in those elements an excellent tool to generate variability and maintain its own integrity. For that reason, although TEs can be present in millions in a genome, only a very small fraction of them is normally active. In life-threatening situations, TEs and REs could be activated, increasing the mutation rate and restructuring the genome (Wessler 1996; Takeda et al. 1999). DNA methylation was the first process to be associated with inactivation of TEs and REs. Later evidence has confirmed the necessity of this process (Brutnell and Dellaporta 1994; Fedoroff et al. 1995; Hirochika et al. 2000; Gendrel et al. 2002; Kato et al. 2003). TEs inserted in or near transcription units can alter gene transcription both quantitatively and qualitatively. Transposon excisions create tremendous allelic diversity by introducing deletions and insertions into genes. Thus, it is true that hosts developed multiple EIS to effectively and stringently control their activity and transposition particularly prior to events leading to meiosis. In plants, somatic cell diversity may itself be a selected feature. Phenotypic variation in the soma can be very beneficial to plants in avoiding herbivory and pathogen attack; one branch that expresses novel chemicals may survive intact while other branches are defoliated (Walbot 1996). Consequently, maintenance of a low level of TE activity in somatic tissues may be selected for based on somatic survival and the fact that flowers arise from somatic tissues. The flowers on the hypothetical branch of novel phenotype may set more seed

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because photosynthesis locally can support better fruit development. As a consequence, progeny of these fruit could inherit a novel allele created by TE activity. Somatic selection within plants requires entertaining the idea that the various apical meristems likely to produce flowers are initially identical siblings that diverge as a result of mutation and epigenetic events. According to Walbot (2002), epigenetic regulation of TEs can create epialleles of host genes exhibiting novel quantitative regulation. Maize and other plants are thought to tolerate TE activity because the host can epigenetically silence transposons and also because haploid gametic selection reduces the impact of deleterious mutations (Walbot 1985). The utility of TEs in creating allelic diversity within the plant soma is proposed as an explanation for their retention in the genome. However, it is clear that the plant host is dominant to the TEs since their majority is transcriptionally and transpositionally inactive. There must have been an effective selection for host control on them during evolution. This might also suggest that multiple controlling mechanisms are operating to repress them and explain the high frequency of transgene silencing since transgene incorporation at aberrant sites resembles transposon insertion. Periodic transposon activation involves only a small part of the TEs and a few individuals in a population. Despite the potential beneficial role of TE-mediated diversification of gene expression, it seems that host suppression of all TE activities is the major outcome of the relationship between transposons and their host plant (Walbot 2002; Medstrand et al. 2005; Vitte and Panaud 2005). The importance of studying and understanding the role of TEs and REs as source of new important variants in plant breeding stems from additional recent findings where a TE family, the helitron, cause genomic sequence insertions in maize by an unknown mechanism. These elements can create multiple partial copies of genes or pseudogenes (Morgante et al. 2005). Such helitron actions definitely contribute to the lack of gene colinearity observed in modern maize inbreds (Lal and Hannanah 2005). The study of the helitron family of TEs with members specific to B73 and Mo17 maize inbred lines revealed also an active helitron that, significantly, produces an alternatively spliced and chimeric transcript joining together genic segments of different chromosomal origin contained within the helitron (Lai et al. 2005). This transcript potentially encodes up to four open reading frames. Thus, transcribed helitrons containing multiple gene fragments may occasionally give rise to new genes with novel biochemical functions by a combinatorial assembly of exons. In this way helitrons not only constantly reshape the genomic organization of maize and profoundly affect its genetic diversity, but also may be involved in the evolution of gene function (Brunner et al. 2005).


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Conclusively, comprehension of genome function could be better achieved by realizing that repetitive elements are responsible on many occasions for organization and architecture of the genome (Morgante 2006). B. Epigenetic Mechanisms and Stress Throughout their life span, plants continually encounter some kind of stress imposed by their environment. Due to their sessile nature, their ability to survive is based on their ability to withstand the consequences of stress and rapidly adapt to such stimuli without relocating themselves to a more favorable environment. For this reason plants evolved sophisticated physiological mechanisms to cope with adversity, either being prepared to face a stressful condition by having accumulated protective metabolites or responding it by deployment of a specific defense. It is well established that the cells contain metabolites, many of which represent preloaded defenses as, for example, antioxidant molecules and antimicrobial compounds. Also, both biotic and abiotic challenges initiate specific signaling cascades, which lead to fast responses for deployment of specific defense mechanisms and/or changing their asic architecture during development in response to such stimuli. How plants integrate endogenous developmental programs with environmental signals and then determine cell fates rapidly enough to ensure survival is not known, but recent experiments have revealed that epigenetic mechanisms play important roles (Costa and Shaw 2006). Another emerging aspect of stress tolerance and adaptation involves not only the physiological and developmental responses just described, but also genomic responses. Rapid changes in plant genome size have been documented historically without understanding a mechanism that could achieve these rapid size changes (Walbot and Cullis 1985). These genomic changes could be made in response to stress derived from genome changes that have adverse effects on the genome itself (genomic stress), or their cause and origin may be outside of the genome (e.g., environmental). McClintock (1984) introduced the concept of ‘‘genomic stress,’’ which can be used to distinguish between stress responses at the physiological level and ‘‘programmed responses to threats that are initiated within the genome itself’’ that lead to genomic modifications. These threats may encompass chromosome rearrangements, fragment loss and gain, and transposition of mobile elements, all of which are genetic (since they affect the primary DNA sequence), follow Mendelian inheritance, and are correlated with modifications of the transcriptional activity of many genes. Interestingly, such genomic structural changes may also be induced by environmental factors of either biotic or abiotic

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nature. There is evidence for transposon changes in barley cultivars where retrotransposon contribution is different at an examined chromosome location or in the genome as a whole. A study by Kalendar et al. (2000) has correlated the copy number and distribution of the Bare1 elements of barley with specific environmental conditions, suggesting that dynamic changes in copy number can occur quickly and be stabilized in contiguous populations. Furthermore, brief low doses of UV-B radiation are sufficient to activate quiescent DNA transposons such as Mu elements of maize (Walbot 1999). Perhars more revealing for the role of epigenetic phenomena in the transgeneration memory of stress in plants are the results obtained recently by Molinier et al. (2006). They have shown that Arabidopsis plants resulted in high somatic homologous recombination after a UV-C or bacterial flaggelin transgene stress and, more important, these increased levels of somatic homologous recombination persist in the subsequent generation even when the stresses were removed. According to McClintock (1984), genomic ‘‘responses, now known to occur in many organisms, are significant for appreciating how a genome may reorganize itself when faced with a difficulty for which it is unprepared.’’ It is evident that these responses to a changing environment have also an epigenetic component (Richards 2006). Alteration of DNA methylation and histone methylation and acetylation may result in response to stress. As mentioned, methylation in plants may be meiotically heritable over many generations and reset the chromatin landscape, causing genome-wide effects. Thus, stress can cause not only physiological but also genomic responses, which in many cases involve or are of epigenetic nature. The paragraphs that follow discuss recent data regarding epigenetic components of stress that are relevant to stress tolerance in plants. The focus is on examples of genomic stress derived from interspecific hybridization and polyploidization, tissue culture, and environmental stress of biotic and abiotic origin. These proved to be major forms of stress able to cause epigenetic genome-wide alterations. The implications of these in the improvement of plant tolerance or defense to various stresses are dealt in Section V. Polyploidization may encompass duplication of a genome (autopolyploid) or may arise from the fusion of two or more different genomes (allopolyploidy). Polyploidy is an important cause of plant evolution and speciation, and might be more common in plants than in animals, because plants produce powerful spindle inhibitors, such as nicotine and colchicine as a defense against herbivory. Furthermore, plants have few mechanisms to control their temperature; thus, subjection to heat and cold shocks might also promote polyploidy by inhibiting


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spindle formation (Cronk 2001). Many plants are obvious polyploids, as judged from chromosome number and chromosome behavior. Although Arabidopsis, with its five chromosomes, is not an obvious polyploid, whole-genome sequencing reveals that 60% of its genome is segmentally duplicated, for which a polyploidization event is the most likely cause. Bowers et al. (2003) proposed that Arabidopsis, like maize, is ancient tetraploid, and further putative polyploidy events are discernible in its genome. Similar conclusions have been obtained for poplar after the completion of its genome sequence where a polyploidy was revealed. All land plants and many algae will probably have polyploid events in their ancestry. A change of ploidy number is a severe stress for the genome and is associated with changes in the patterns of gene expression and altered phenotypes. In autopolyploids, it is expected that an increase of the number of loci could be related with increase in gene expression. This has been observed, for example in maize, where gene expression for several loci increased as ploidy increased (Guo and Birchler 1994), suggesting that gene expression and copy number are directly related in autopolyploids. However, epigenetic silencing can also occur in autopolyploids, as observed in Arabidopsis (Mittelsten Scheid et al. 1996). Later these investigators reported that interaction of active and inactive epialleles of a transgene in tetraploid Arabidopsis lines resulted in heritable gene silencing persisting after segregation from the inactivating allele, in a manner resembling paramutation. Further analysis indicated that hypermethylation was associated with silencing of the previously active allele and suggested that a functional chromatin remodeling activity was required for maintaining the silenced state (Mittelsten Scheid et al. 2003; Chen 2007). In allopolyploids, balanced expression of two or more genomes in one cell requires extensive coordination of gene expression. This is accomplished in early generations of allopolyploid formation, and established allopolyploids display phenotypic and genotypic stability. However, allopolyploids recently produced artificially by scientists lack the stability of their naturally established ancient counterparts. Reestablishment of a balance in gene expression between the parental genomes is achieved by silencing of many genes that sometimes correlates with hypermethylation of the silenced loci (Lee and Chen 2001; Madlung et al. 2002). But in other cases, both up-regulation and down-regulation in gene expression have been observed. Some changes are specific to one of the parental genomes (He et al. 2003), and gene silencing is correlated with increased DNA methylation at the affected loci (Shaked et al. 2001). These changes in DNA methylation and gene expression using synthetic allopolyploids have also been studied in Arabidopsis (Madlung

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et al. 2002), wheat (Kashkush et al. 2002, 2003), and cotton (Adams et al. 2004) among other plant species. Many expression alterations have been documented including up- and down-regulation of genes that can arise by the onset of polyploidization or in the course of subsequent generations. A subset of these has been found to have epigenetic causes (Rapp and Wendel 2005). All these alterations in gene expression may account for novel aberrant phenotypes that appear when a change in ploidy number is used for generating new genotypes in plant breeding (Wendel et al. 2002; Osborn et al. 2003). Allopolyploid plants can be considered a special type of hybrids where two (or more) homologous genomes are fixed in one nucleus, maintaining their integrity through sexual reproduction. Therefore, the advantage of hybrid vigor and heterosis can be fixed by polyploidization, in contrast to the wide recombination of the parental genomes in segregating progeny of diploid hybrids, which is one of the reasons for inbreeding depression (Fasoula and Fasoula 1997b). There is evidence that allopolyploidization and hybridization may cause transposon activation (Madlung and Comai 2004). Epigenetic changes in methylation and transcription were reported in rice following alien DNA introgression from Zizania latifolia, a related but differentiated species (Liu et al. 2004). The affected sequences included low-copy cellular genes, TEs and REs, suggesting that the alterations likely reflected a general and genome-wide phenomenon. In artificial allopolyploids formed recently between Arabidopsis thaliana and Arabidopsis arenosa, there was transcriptional activation of a repeat unit with similarity to transposons (Comai et al. 2000). Retrotransposon activation was also observed in rice hybrids (Liu and Wendel 2000) and in polyploid wheat (Kashkush et al. 2003). Some genes adjacent to transposon LTRs in wheat had also altered expression patterns, supporting the hypothesis that hybridization could activate previously silent genes altering heterochromatic regions. Direct evidence for hybridization-induced transposition via demethylation of elements, however, has not been reported so far (Madlung and Comai 2004). By extension, diploid hybrids may also be considered as a special case of allopolyploids, where haploid genomes that may retain significant disparity at various allelic chromosomal locations have to cooperate in a single nucleus and could display the range of atypical gene expression and epigenetic regulation patterns observed in early generations of real allopolyploid plants. Indeed, significant differences have been identified recently between inbred lines of maize with different origins, where large parts of DNA present in one line at a chromosomal position are missing the other and vice versa (Lee et al. 2002; Brunner et al. 2005;


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Buckler et al. 2006; Messing and Dooner 2006). This might account for incompatibilities between certain inbred lines and partly affect inbreeding depression observed in segregating populations of F1 hybrids. There are also data that link tissue culture conditions with stress due to chemical and physical causes, such as wounding, exposure to hormones in the tissue culture medium, and pathogen infection (Madlung and Comai 2004). Somaclonal variation, a frequent result of tissue culture, has been attributed to changes in DNA methylation pattern, activation of transposable elements or retrotransposons, and chromosome remodeling (Kubis et al. 2003). Both hypomethylation and hypermethylation of endogenous single-copy and repeated-gene loci as well as transgene sequences have been reported to occur in calli (Olhoft and Phillips 1999; Jaligot et al. 2000; Kaeppler et al. 2000; Koukalova et al. 2005). There appears to be mounting evidence for an important role of an epigenetically regulated network of defenses operating in plants during virus infections (see Section III.E). But is there a role of epigenetic mechanisms in infections by other pathogens, such as fungi and bacteria? Data on this issue are scarce, although indirect evidence may provide some clues. Pathogen recognition in plants is almost concomitant with production of reactive oxygen species (ROS) known as oxidative burst, which results from the action of a family of respiratory burst oxidase homolog (Rboh) proteins that encode the key enzymatic subunit of the plant NADPH oxidase (Torres and Dangl 2005). In addition to their well-established signaling roles in plant defense responses, ROS produced during the oxidative burst impose a severe stress termed oxidative stress and can damage a variety of biomolecules including DNA itself. This should evoke DNA repair mechanisms that may affect epigenetic patterns and genomic remodeling. Pathogens are not the only elicitors of oxidative stress in plants. Abiotic stress factors can also lead to an increase of ROS and possible epigenetic effects of ROS overload in the cell. Cassells and Curry (2001) reviewed the effects of oxidative damage in eukaryotic cells and cited examples of altered DNA methylation, changes in chromosome number from polyploidy to aneuploidy, chromosome strand breakage, chromosome rearrangements, and DNA base deletions and substitutions. Such changes could explain, at least in part, the range of variability found in plant cells, which is revealed as somaclonal variation in tissue culture. One of the most common oxygen radical–induced base alterations in DNA is formation of the oxygen radical adduct 8-hydroxyguanosine (8-oxoG) (Cerda and Weitzman 1997). Since there is no known enzymatic mechanism capable of producing 8-oxoG, the only route of its formation in the cell is through oxidative modification of guanine. The oxidation of

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guanine to 8-oxoG converts the N7 position of guanine from a hydrogen bond acceptor into a hydrogen bond donor and replaces the 8-hydrogen with an oxygen atom. Replacement of guanine with 8-oxoG alters methylation of adjacent cytosines. The methyl groups of both thymine and 5mC are also susceptible to oxidation. The oxidation of 5mC can generate 5-hydroxymethylcytosine (HmC), 5-formylcytosine, and 5-carboxycytosine. Each modification could potentially interfere with the recognition of the methyl-CpG dinucleotide by methylation binding proteins (see Section II.A). The replacement of a 5mC residue with HmC or the replacement of guanine by 8-oxoG reversed the increase in binding affinity afforded by a 5mC residue (Valinluck et al. 2004). Therefore, oxidation of 5mC to HmC or guanine to 8-oxoG was shown to inhibit MBPs binding, which could affect chromatin structure and result in potentially heritable epigenetic alterations (Zemach and Grafi 2007). In summary, plant stress responses, although programmed and inducible, may be not enough under certain conditions to ensure survival. These extreme and unpredictable challenges could lead to epigenetic reprogramming of major defense pathways that might provide additional means for their protection and perhaps generation of new variation in their progeny, increasing their chances of survival under the harsh conditions imposed. Plants take advantage of all the three epigenetic mechanisms and combine genetic and epigenetic solutions for enhanced stress tolerance and adaptation. C. Paramutation in Plants Paramutation is an allele-dependent transfer of epigenetic information that results in the heritable silencing of one allele by another (Chandler and Stam 2004). In plants, the term paramutation was coined by Brink (1956) to describe a heritable change in gene function directed by its allele (Kermicle 1996; Martienssen 1996; Richards 1997). The expression of the one allele, referred to as paramutable, changes following paramutation, thus violating Mendel’s first law. The allele that induces the change is called paramutagenic. Once the expression of the paramutable allele has been modified, it is called paramutated, which is designated by an apostrophe after the gene name, for example, R-r’. Alleles that do not participate in paramutation are called neutral alleles. When the paramutable/paramutagenic heterozygote is crossed to allow segregation of the two alleles, virtually 100% of the paramutable alleles transmitted display a decrease in expression. This decreased expression level (the paramutant phenotype) persists through many generations. Since there is no change in DNA sequence, but rather there are changes


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in DNA methylation or chromatin structures, paramutation is classified as a classical epigenetic phenomenon, and the paramutable and paramutant alleles are called epialleles. The paramutation phenomenon seems to be associated with a small number of alleles. However, the characteristic traits of paramutant alleles, including high-frequency penetrance and in some cases reversibility of traits, make this phenomenon interesting for breeders. The unknown epigenetic mechanism of interaction, where paramutagenic alleles influence homologous paramutable alleles in trans, leads to questions of how one allele influences another, how big the impact of that mechanism is in overall gene regulation, and why such a mechanism would evolve. One answer may be that paramutation is a fast way to adapt easily to a changing environment. The first report on paramutation phenomenon was in Pisum sativum (Bateson and Pellew 1915), soon followed by similar observations in other plants like snapdragon (Krebbers et al. 1987) and petunia (Van Houwelingen 1999). The most extensive research has been done in maize (Stam and Mittelsten Scheid 2005). Furthermore, similarity has been observed between paramutation of the R locus of maize and the behavior of A1 allele in certain transgenic petunia plants (Meyer 1996). Other transgenic loci behave like paramutants (Qin and Von Arnim 2002; Mittelsten Scheid et al. 2003), suggesting a link between paramutation and other epigenetic regulation of gene expression. Thereby paramutation can be redefined as trans-inactivation between homologous sequences that leads to a high frequency of heritable changes in the gene expression of one of the sequences (Stam and Mittelsten Scheid 2005). The extensive research on this phenomenon has revealed great specificity in behavior of paramutant alleles or great variation between paramutagenic loci in the amount of stability and penetrance (Chandler and Stam 2004). For example, the maize booster1 (b1) gene has been shown to be fully penetrant and highly stable while most other alleles do not show complete penetrance. Maize b1 gene, which expresses a basic helixloop-helix transcription factor, is required for activation of anthocyanin pathway in plant. The paramutable form, B-I, loses the heritable color phenotype when in a hetrezygotic condition with paramutagenic B’, the colorless form of allele. There are alleles neutral to paramutation and alleles that act as stabilizers for paramutable alleles, such as the b1 gene; but on the contrary, the heterzygosity of another paramutagenic allele, Pl’, with a neutral allele leads to loss of paramutagenic strength. Despite the penetrance of the new trait—for example, 100% for the b1 gene—not all paramutants are so efficient in establishing this new epigenetic state, nor are they so stable. Recombination mapping has defined a 6 kb region

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located 100 kb upstream of b1 promoter that included seven tandem repeats of an 853 bp sequence that is otherwise unique in the genome, whereas neutral alleles only have one copy of this sequence (Stam et al. 2002). DNA sequences are identical in B-I and B’, indicating that epigenetic mechanisms mediate the stable transcriptional silencing that is associated with b1 paramutation. The variable transcriptional silencing of the locus is brought about by differentially heterochromatization of the tandem repeats upstream of the locus causing greater DNaseI hypersensitivity in B’ (silenced) relative to B-I (the transcriptionally active allele), where repeats are hypermethylated and have an open chromatin structure. In newly formed B’ alleles, chromatin structure was intermediate between B-I and B’. There are four maize mutants that abolish paramutation: required to maintain repression loci, rmr1, rmr2 and rmr6 and one named mediator of paramutation1 (mop1) (Hollick et al. 2005). Recently Alleman et al. (2006) reported the cloning of mop1, an RNAdependent RNA polymerase gene most similar to the RNA-dependent RNA-polymerase (RDRP) described for siRNA production and gene silencing that is again strongly indicative for the critical role played by epigenetic mechanisms in manifestation of paramutations. The same group also reported that transcriptionally silenced transgenes in maize are activated by mutations defective in the paramutation epigenetic mechanism (McGinnis et al. 2006), indicating again that the two phenomena are epigenetically related. There are two major theories proposing two different mechanisms for how homologous sequences can interact in trans, bringing about paramutation. One proposed mechanism involves siRNA to mediate chromatin changes. Repeats that would give rise to siRNA are involved in several paramutation phenomena (Walker and Panavas 2001; Stam and Mittelsten Scheid 2005) but not all (Qin and Von Arnim 2002). The second proposed mechanism involves pairing between homologous sequences where paramutagenic locus is proposed to transfer its own transcriptionally inactive state onto the paramutable counterpart via pairing of homologous sequences (Chandler and Stam 2004; Grant-Downton and Dickinson 2004). A most complex model has emerged where the position of the gene and epigenetic memory could direct genes in organizer and transcription factory (Chakalova et al. 2005). D. Parental Imprinting Parental imprinting refers to the process whereby only one of parental alleles, either the maternal or the paternal one, is active in an offspring


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(De Chiara et al. 1991). An epigenetic modification, which must be mitotically stable, inactivates one of the parental alleles. Therefore, we have the parent-of-origin expression effect where one allele is inherited in a silent state and is not expressed; the other allele is inherited in an active state and consequently is expressed. Gene imprinting has been found both in animals and plants. The first imprinted gene to be reported in mouse was the insulin–like growth factor-2 (Igf-2) by De Chiara et al. (1991). Since then analysis of human and mouse chromosomes has revealed a large number of imprinted genes in these organisms (Kelsey et al. 1999). In mouse, nearly all imprinted genes are associated with differential DNA methylation of maternal and paternal alleles (Brannan and Bartolomei 1999; Feil and Khosla 1999). One of the best-characterized regions of the mouse genome contains a cluster of imprinted genes including Igf-2, H19, and Snrpm. Inspection of this region revealed the presence of differentially methylated regions in or near the imprinted genes. It was recently shown that in mammals, DNA methylation of large (up to 100 kb) specific intergenic regions, named imprinting control centers (ICR), regulate the expression of imprinted genes (Delaval and Feil 2004). For the Igf2 gene, it was also shown that the Igf2 paternal allele is silenced by a mechanism involving DNA methylation, an sRNA and the PcG gene Eed. Conversely, during female gametogenesis, methylation of the maternal Igf2 does not occur, and as a consequence the maternal allele is expressed (Delaval and Feil 2004). In plants, mutations that have either sporophytic or gametophytic parent-of-origin effects have been reported recently in Arabidopsis. One class of genes found to be maternally expressed is the FIS genes that have been mentioned (see Section II.B), such as MEA, FIE, FIS2, which play an essential role in seed development. The study of the MEA/FIE/ FIS2 complex has demonstrated that parental imprinting plays a central role in the transcriptional regulation of the FIS-class genes (Baroux et al. 2002). It was also mentioned that the target of the MEA gene is the gene PHERES1 (PHE1), a MADS box type I gene (Ko¨hler et al. 2003b). PHE1 is predominately expressed from the paternal allele during seed development whereas the maternal allele is also expressed but maternal transcript levels are much reduced (Ko¨hler et al. 2005). Moreover, it was shown that PHE1 is only expressed after fertilization. This was the first report of a paternally imprinted gene in plants. A DNA glycosylase, DEMETER (DME), is responsible for the activation of the maternal allele MEA before fertilization (Choi et al. 2002). Four met1 (mehtyltransferase1) mutant alleles were isolated in an effort to identify suppressors of dme (Xiao et al. 2003). It was suggested that MET1 is required to maintain cytosine methylation of the MEA promoter

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and that it acts antagonistically to DME. Parallel studies with FIS2 parental imprinting (Jullien et al. 2006) are also revealing for the essential role of DNA methylation for parental imprinting. This work shows that maintenance of FIS2 imprinting depends on DNA methylation whereas loss of DNA methylation does not affect MEA imprinting. Recent reports have shown that MEA imprinting is under self-regulation. This is achieved by DME-mediated specific demethylation (activation) of the maternal MEA allele (Gehring et al. 2006) and by maternal MEA-mediated histone methylation (silencing) of the paternal MEA allele (Baroux et al. 2006; Jullien et al. 2006). It seems that the other components of the PcG complex are involved in MEA autoregulation as fis2, fie, and msi1 mutants express the paternal MEA allele. MEA seems to be silenced during the vegetative and male gametogenesis phases of the life cycle of the plant by PcG complexes. In the endosperm, MEA expressed from the maternal allele maintains the silencing of the paternal MEA allele. In this way MEA ensures complete maternal control of MEA expression from both parental alleles. Another gene, FWA, which encodes a homeodomain transcription factor, has been also reported to be parentally imprinted in Arabidopsis (Kinoshita et al. 2004). The maintenance of endosperm-specific and parent-of-origin–specific FWA expression depends on MET1. The 5’ region of the FWA contains direct repeat sequences that are hypermethylated in all tissues except the tissues that FWA maternal transcript is present (i.e., the female gametophyte and the developing endosperm where the FWA promoter is hypomethylated). Furthermore, in the fwa-1 Arabidopsis mutant, the 5’ region of the FWA gene is hypomethylated, and this is associated with ectopic expression of FWA in vegetative tissues (Soppe et al. 2000). These studies indicate that unlike mammalian imprinting, the imprinting of MEA and FWA in plants is not established by allelespecific de novo methylation but rather by maternal-specific removal of methylation, which is dependent on the activity of the DME DNA glycosylase in the maternal gametophyte. This demethylation leads to specific activation of imprinted genes in the maternal gametophyte. Since the endosperm degenerates during seed maturation or germination, it does not contribute to the genetic or epigenetic information of the next generation. Therefore, the epigenetic state of the endosperm does not need to be reprogrammed, which means that the demethylated and thus activated genes need not be silenced again. An additional aspect of the regulation of the FWA gene imprinting came to light recently. Analysis of the 5’ region of the FWA gene revealed that it contained retrotransposon-derived tandem repeats that correspond to siRNAs (Lippman et al. 2004). In the same study a


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genome-wide analysis was performed by ChIP in Arabidopsis in order to define heterochromatic and euchromatic states of chromatin along large regions of the genome. It was shown that heterochromatin is composed of transposons and their related repeats, and they are controlled by the chromatin remodeling factor DDM1. Some of these transposon sequences correspond to siRNAs, suggesting that siRNAs may direct DDM1 to modify chromatin. Considering these results, it is possible that the regulation of FWA and other imprinted genes could be governed by transposon-derived sequences and siRNA-dependent DDM1 or MET1 activity. Many genes with phenotypes and mRNA accumulation patterns similar to MEA are not completely or selectively imprinted in the seed. These include genes that mediate seed phenotypes due to a requirement for expression in the gametophytes (Yadegari et al. 2000; Xiao et al. 2003). Thus, before differential expression can be interpreted as the result of imprinting, differential transcription in the zygote or endosperm must be demonstrated. Different proposals that are not mutually exclusive in every case of the genes studied have been made to explain imprinting. These proposals include the purifying selection hypothesis already described, which supports the view that any mechanism, imprinting included, leading to haploid phase and allele exposure to selection, could have a purifying selection efficiency, decreasing the mutational load, particularly from TEs and REs activities (Walbot 2002; 2004). The parental conflict proposition also presents an attractive mathematical model proposing that imprinting is driven by conflicts of interest between the mother and father over resource allocation. Imprinting arises when half-sibling embryos from multiple-pollen parents compete with each other on the same mother. Imprinting of growth regulators can serve the interest of polygamous mothers via equal growth of all their progeny, whereas the interests of competing fathers are best served by preferential treatment of progeny carrying their own genes, at the expense of maternal half-sibs (Wilkins and Haig 2003). The failure of endosperm to cellularize and the runaway cell proliferation in mutants of paternally silent genes, such as MEA and the FIS-class genes, could result in increased resource demand in these seeds and therefore are consistent with the parental conflict hypothesis. The same could be true for effects of reciprocality on interploidy crosses (see Section V.F). But the conflict model is incompatible with the differential expression data (Wilkins and Haig 2003), which is a widespread phenomenon during seed development (Dilkes and Comai 2004). On the contrary, a differential dosage sensitivity hypothesis in

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which the dose of a gene product determines the phenotype is compatible with the wide occurrence of differential expression during seed development (Dilkes and Comai 2004). The dosage hypothesis relies on a dosage sensitivity described by Birchler et al. (2001) according to which it arises at regulatory pathways because the ‘‘stoichiometric relationship of the components of regulatory complexes affects target gene expression.’’ In other words, genes causing dosage effects are expected to encode the subunits of macromolecular complexes, and a decrease in one component affects the function and assembly of the whole complex (Vietia 2002). The balance hypothesis applies well to the dosage-sensitivity interpretation of parent-of-origin effects in interploidy crosses (Dilkes and Comai 2004). E. Viral-Induced Silencing PTGS can be induced in plants by viral vectors harboring specific genes through the virus-induced gene silencing (VIGS) (Dalmay 2005). Plant viruses have different morphologies, genetic structures, vectors, and host range. Their genome may be single stranded (ss) or double stranded (ds) DNA and ss or dsRNA. However, over 90% of plant viruses have ssRNA genomes that are replicated by a virus-encoded RdRP to produce both sense and antisense (termed plus-strand and minus-strand), and thus accumulate a traceable amount of dsRNA within the plant. When a plant is infected with unmodified viruses, the VIGS mechanism is specifically targeted against the viral genome. However, with virus vectors carrying a copy of an insert derived from host genes, the process can be additionally targeted against any native or homologous transcripts (Lu et al. 2003). Many groups have used VIGS in order to study the function of a variety of plant genes (Baulcombe 2005). Moreover, VIGS has facilitated the study of resistance gene pathways (see Section V.F). The VIGS technology was applied in tomato for silencing the constitutive triple response 1 and 2 (CTR1 and 2) genes that negatively regulate ethylene responses, by introducing the Arabidopsis CTR1,2 (Liu Y. et al. 2002). Although there is no experimental evidence so far, it is generally assumed that 85% nucleotide identity would be the lowest limit for triggering the VIGS mechanism (Benedito et al. 2004). Moreover, conserved boxes of gene families can potentially trigger silencing of other members concomitantly (Ratcliff et al. 2001). Overexpression of the petunia flowering gene (PFG) induces cosuppression of PFG itself and of flowering binding protein 26 (FBP26), which shares high overall sequence homology with PFG (74%), particularly within the MADS box (88%) (Immink et al. 1999). Consequently, it can


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be hypothesized that the sequence homologies found in conserved boxes of gene families are more important for multiple gene silencing than are their overall homologies for VIGS mechanism. Recent findings by Yang and his colleagues (2007) showed that virus-induced gene silencing of NbBTF3 that encodes a Nicotiana benthamiana homolog of human BTF3 triggered both leaf yellowing and abnormal leaf morphology but no modification in the development of the plant. In summary, the VIGS system can be used in reverse genetics without requiring plant transformation, thereby allowing the study of multiple genes. Additionally, the procedure is rapid—the VIGS phenotype develops within one or two weeks instead of months or even years when the traditional methods that require transformation procedures are used. Most important, the target mRNA is not silenced until the virus vector infects the plant. It is possible therefore to suppress essential genes for host cell growth and development (Peele et al. 2001). It is important, however, to take into consideration that, until now, this system has been efficiently used in only a few plant species, such as Nicotiana benthamiana, tomato, and barley. Nevertheless, the increasingly developing novel vectors will augment the number of species that respond efficiently to VIGS. In order to protect themselves from degradation, viruses have evolved RNAi inhibitors, which allow them to overcome plant RNAi action and successfully infect the plant. This has been demonstrated with the identification of the potyviral Helper component-Proteinase (HcPro) (Anandalakshmi et al. 1998; Brigneti et al. 1998; Kasschau and Carrington 1998) and the 2b protein of CMV (Brigneti et al. 1998) that suppress PTGS targeting different components in the silencing mechanism. Strong suppressors could prolong and increase virus accumulation, whereas a weak suppressor could not allow a high level of virus accumulation. Lakatos and his colleagues (2004) demonstrated that a viral protein of tombusviruses (p19) inhibits RNA silencing in vivo by strongly binding siRNA in virus-infected cells. When the silencing of the RNAi mechanism was suppressed by p19 in tobacco plants, the expression of various transgenes in transient expression assays was increased 50-fold (Voinnet et al. 2003). Recent findings by Mlotshwa et al. (2005) demonstrate that developmental defects in the viral silencing suppressor P1/Hc-PRO result from the aberrant Dicer activity, suggesting a possible role of Dicers in development, independent of target RNA degradation, and proposing a possible mechanism of surpassing the viral suppression of RNA silencing. New light was shed recently on the mechanism underlying induction and suppression of RNA silencing in antiviral defense by Deleris et al. (2006). These authors showed that DCL4 and DCL2 exhibit specific hierarchical activities in

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antiviral silencing. DCL4 is the one to exert its antiviral effect first by producing 21-nt siRNAs, which recruit an antiviral RISC complex. DCL2 also forms 22-nt siRNAs with antiviral activity, but only when DCL4 is inhibited by a viral suppressor. These findings will assist in elucidating the mechanism of the poorly understood antiviral defense process in plants. F. Transgene Silencing The spread of techniques that allow introduction and functional expression of foreign genes in plant cells enabled the production of transgenic plants with improved insect and disease resistance, superior quality of seeds and fruits, and increased tolerance to extreme environmental conditions. Vaccines against serious human diseases have also been developed using transgenic plants (Herrera-Estrella et al. 2005). However, the frequent use of gene transfer methods has revealed that many transgenes do not express as expected. Early findings by Matzke et al. (1989) demonstrated that transformation can induce epigenetic phenomena. The copy number of a transgene that integrates into the genome is unpredictable, as is the integration site. Thus, one or multiple copies can integrate at one or multiple loci. Transgene silencing could take place through TGS or at a PTGS. Transgene-induced silencing phenomena in plants could be observed either in cis, when it refers to the silencing of transgene expression integrated at a single locus, or in trans, when it refers to the silencing effect of one locus on another. Transcriptional cis-inactivation can result from the insertion of multiple, rearranged copies of a transgene at a single locus that is located in or next to heterochromatin regions. Therefore, methylation may spread from adjacent sequences into the transgene, leading in this way to silencing (Vaucheret et al. 1998). Moreover, transcriptional cis-inactivation may occur when closely linked copies of transgene can attract TGS inspection systems like methylation or heterochromatin-forming proteins although they integrate at a hypomethylated locus. Such RIGS correlate with increased methylation and changes in chromatin configuration (Assaad et al. 1993; Ye and Signer 1996). Cis-inactivation can also take place in some instances when single copy of a transgene is inserted at a hypomethylated locus. This phenomenon can be due to the difference in sequence composition between the transgene and the integration region (i.e., when a transgene derived from a monocotyledonous plant was introduced into a dicotyledonous plant), leading to methylation of sequences, which the plant recognizes as foreign (Elomaa et al. 1995). This form of TGS transgene inactivation also involves increased methylation, suggesting


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again that hypermethylation and chromatin condensation are general characteristics associated with transcriptional silencing. TGS transinactivation can result from the effect of one transgene on another (i.e., an active copy of a transgene can become repressed by another silenced homologous gene, either linked or not,) and can acquire the capacity of inactivating other copies in subsequent crosses (Meyer et al. 1993). Trans-inactivation can affect any transgene that is expressed under the control of the same promoter regardless of the coding region. Sequences as short as 90 bp are sufficient for mediating silencing (Vaucheret 1993). Previous studies proposed that this type of inactivation requires interaction of the sequencing locus with the target sequence by direct DNA-DNA pairing between the loci, resulting in a transfer of the silenced state from one locus to another, or that aberrant promoter transcripts can cause RNA-directed DNA silencing (Mette et al. 1999). Recent findings suggested that siRNAs, produced by a miRNA-directed RdRP, could mediate methylation of genomic DNA (Ronemus and Martienssen 2005). PTGS is occurring when RNA does not accumulate even though transcription occurs. As opposed to TGS, which is meiotically heritable, PTGS is reset after meiosis (Vaucheret 1993; Park et al. 1996). Another difference is that methylation of the promoter region typically results in TGS transgene silencing whereas coding region methylation results in PTGS transgene silencing (Iyer et al. 2000). For many years TGS transgene inactivation was linked to a modification of the epigenetic state of the transgene while the PTGS transgene inactivation was related to RNA degradation. However, this distinction is under consideration after recent findings that suggest the involvement of RNA molecules in the induction of transgene methylation and TGS events such as chromatin modification (Matzke et al. 2004; Depicker et al. 2005). What can be assumed from studies in plants is that the presence of inverted repeats and multiple copies of transgenes is often associated with PTGS. It is believed that ectopic DNA-DNA or DNA-RNA pairing or transcription of inverted repeat structures and simultaneous expression of both sense and antisense transgenes induce the initiation of RNA silencing either by the formation of aberrant RNA transcripts that activate silencing or by the accumulation of normal transcripts that ‘‘exceed’’ a threshold level (Lindbo et al. 1993; Muskens et al. 2000; De Buck et al. 2001). For RNA silencing events, several transgene characteristics can trigger the induction of RNA silencing. The composition of the transgene (promoter selection, promoter strength, transgene stability) determines whether a single-copy transgene will trigger RNA silencing. The possibility of silencing seems higher in homozygous lines for the transgene.

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Moreover, transformed plants with multiple transgene copies have a higher possibility of being silenced at a posttranscriptional level, implying that there is a higher likelihood of production of threshold levels of transgene transcripts generating RdRP-mediated primary silencing signals and for inverted-repeat arrangements. Several studies from various groups on transgene-induced cosuppression in Nicotiana tabacum and Arabidopsis provided the first evidence that PTGS might play a role in phase change (De Carvalho et al. 1992; Elmayan and Vaucheret 1996; Elmayan et al. 1998). The silencing of transgenes and any endogenous gene by its cognate siRNA, caused by the overexpression of a sense transcript, is known as the phenomenon of cosuppression. Transgenes expressed under the control of the 35S promoter in young seedlings were often downregulated in successive leaves until little or no transcript remained. The level of gene expression determined the degree of suppression: Whereas homozygous transgene transcription was inhibited, hemizygous transgene transcription continued to be active (De Carvalho et al. 1992; Elmayan and Vaucheret 1996; Elmayan et al. 1998). Similar to the inheritance of Pl-Blotched, silencing seems to be reset at every generation, as judged by the fact that the progeny and parents shared the same developmentally regulated transgene-silencing pattern. Finally environmental effects may cause transgene silencing; several reports demonstrated that transgenes were silenced when they were grown in the field but not in the greenhouse (Brandle et al. 1995). RNA silencing in plants can produce a systemic signal that triggers degradation of homologous RNA at long distances through the plant. The factors involved in such amplification and transmission of the signal are under study via grafting experiments that aim to unravel the mechanisms (Baulcombe 2005; Voinnet 2005b). All these data clearly suggest that transgene silencing still remains a problem to be solved. The phenomenon seems more persistent in plants, probably due to the capacity of plants to fight against invasive DNA and put up with genome duplication. IV. EPIGENETIC MECHANISMS AND PLANT DEVELOPMENT A. Plant Development Despite the over a billion-year dichotomy between plants and animals, there are many common elements in their developmental strategies. The complete genome sequences of animals and plants indicated that, grossly speaking, similar numbers of genes are required for building


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these multicellular higher organisms. The two groups share the main developmental strategies, such as cell-cycle, cell-cell signaling, polarity, modularity, as well as basic genetic and epigenetic mechanism of gene and genome control such as DNA methylation, PcG, ATX complexes, and RNAi(Meyerowitz 2002). But there are also fundamental differences in the life cycles of plants and animals. All plants exhibit at least one form of apical meristem consisting of one or more cells that are functionally analogous to metazoan stem cells because they are histogenetic (i.e., able to generate specialized tissues). Plants, however, differ from animals in that the plant apical meristem has the additional capability of generating organs like leaves and stem, inflorenscences or flowers throughout the life of the plant, whereas the number and form of metazoan organs are embryonically determined. Development continues throughout the life of the plant and proceeds by the often indeterminate, repeated iteration of modules (leaves, roots, and stems). By contrast, animals have a single developmental trajectory, which is completed at maturity and ends with a fixed number of organs. The iteration of these modules is controlled by the signals that plants exchange with the environment. This continuous feedback between development and the external environment is not seen in animals, which, in general, have the power of changing their environment thanks to their capacity to move and their behavior. The presence of a thick cell wall is also a distinguishing feature of plants. The elaborate cell wall of plants not only makes plants sessile, but also prevents cell movement in plant development. Animal cells can slide past each other to take up programmed developmental positions. By contrast, fate information during development in plants is probably controlled more by cell-cell signaling than by developmental history (cell lineage). As a result, there is no distinction between germ line and soma in plants: the development of germ cells is determined by cell position and not cell lineage (Cronk 2001; Gilbert 2003a). Plants are also characterized by the alternation of generations, a life cycle in which haploid and diploid generations alternate with each other. Very different developmental trajectories, which are linked to ploidy level, are thus contained in the same genome. The specific developmental paths undertaken by haploid spores and diploid zygotes indicate that radically different gene-expression patterns are possible— a phenomenon that might be linked to changes in DNA methylation and epigenetic changes in general. Over evolutionary time from bryophytes, to ferns and angiosperms, there has been a steady reduction in diversity of structures during the haploid phase (Gilbert 2003b). During the course of land plant evolution, heterochronic control mechanisms

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perhaps shifted the starting point of the diploid phase earlier and earlier in development at the expense of the earlier haploid organism (Cronk 2001). Gametophytes in flowering plants, for example, are nonphotosynthetic and contain few cell types. Finally, the double fertilization event during sexual reproduction described previously for the production of both embryo and the nutritive tissue endosperm are unique features of flowering plants (Walbot and Evans 2003). Land plants originated monophyletically from freshwater green algae belonging most probably in the charophycean group. Thus some characteristics of land plants, such as cellulosic cell wall, multicellular body, cytokinetic phragmoplast, plasmodesmata, donated to plants from algae. The origin of a well-defined sporophytic apical stem cell and a system for its proliferation correlated with capacity for organ production and branching occurred sometime between the divergence of modern bryophytes and vascular plant lineages. Roots and their meristem and a multilayered tunica-corpus shoot apical meristem arose later (Graham et al. 2000). It is important to understand these fundamentally unique aspects of plant development, the mechanisms involved, and their consequences. For example, as it was stressed most of DNA transposon-based diversity is generated in somatic tissues; this means that most of the potentially deleterious new mutations and chromosome breaks occur in somatic cells. It is interesting to consider that somatic diversity may itself be a selected feature. Phenotypic variation in the soma can be very beneficial to plants in avoiding herbivory and pathogen attack; one branch that expresses novel chemicals may survive intact while other branches are defoliated (Walbot 1996). Consequently, maintenance of a low level of transposon activity in somatic tissues may be selected for based on somatic survival and the fact that flowers arise from somatic tissues. The flowers on the hypothetical branch of novel phenotype may set more seed because photosynthesis locally can support better fruit development. As a consequence, progeny of these fruit could inherit a novel allele created by transposon activity as well as by the active transposable element system. Somatic selection within plants requires entertaining the idea that the various apical meristems likely to produce flowers are initially identical clones that may diverge as a result of genetic and epigenetic events. Stringent regulation of events preceding meiosis and in the resulting haploid cells could readily be selected based on survivorship in the progeny. In plants, according to Walbot and Evans (2003) and Gu et al. (2003), the gametophytic stage presents an opportunity for natural selection on the haploid genome—as in other haploid organisms, recessive deleterious alleles that would be masked in diploid organisms are removed from the


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population. In fact, many of the parent-of-origin effects enumerated by Dilkes and Comai (2004), such as differential expression of genes in the female and male gametophyte, the gametes’ delayed expression of genes from one of the other parent in the early zygote development, and differential expression of parental genes in the developing seed, could have a purifying role of a haploid selection scheme, among other roles, such as dosage regulation. Early data for the significant general role played by DNA methylation during plant development came from examining the effects of genomewide demethylation in Arabidopsis. As mentioned in Section II.AII.A, transformation with an antisense construct of MET1 resulted in plants with abnormal phenotypes. These included reduced stature, abnormal root morphology, altered leaf size and shape, abnormal floral development, and altered flowering time. The same was later proven to be true for mutations of histone-modifying enzyme genes, such as the Arabidopsis BRAHMA, whose silencing results in reduced fertility, curly leaves, homeotic transformations during flower development, and photoperiod-independent early flowering (Farrona et al. 2004). Finally, mutations of genes in the sRNA pathways have similar effects, as exemplified by an allelic series of AGO1 mutations that displayed a complete loss of adaxial/abaxial polarity, ectopic ovule formation, and inversion of polarity in the epidermal cells (Kidner and Martienssen 2005). The aim of this section is not to describe each developmental step and organ formation. The reader can find detailed information of these topics in excellent recent reviews (Cronk 2001; Walbot and Evans 2003), books (Cronk 2002), or textbooks (Gilbert 2003a). In the following paragraphs an analysis will be undertaken of certain critical plant developmental events or formation of specific organs with emphasis on the particular role played by each EIS. B. Shoot and Leaf Formation and Development Flowering plants go through two major developmental life stages: an embryonic stage composed of two phases (early embryonic and late embryonic) followed by a postembryonic stage consisting of a juvenile, adult, and reproductive phase. During their vegetative life (juvenile and adult), plants are composed of three basic organs: shoot, leaves, and roots. Inflorescences and flowers with floral organs such as sepals, petals, stamens, and carpels are the structures formed after the plant makes its transition from vegetative to reproductive phase. Therefore, it is essential to understand shoot and leaf development in order to comprehend full aerial plant morphogenesis.

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Shoot meristems are groups of indeterminate cells, which act in a coordinated manner to form the aerial organs. The shoot apical meristem (SAM) initiates during embryogenesis and is a collection of stem cells that reside at the tip of each shoot and are maintained throughout the life of the plant. In Arabidopsis the SAM is comprised of several hundred cells that are divided into different functional domains: the central zone (CZ) located at the tip of the meristems composed of slowly dividing stem cells; the peripheral zone (PZ) surrounding the CZ, which is composed of more rapidly dividing cells from which leaf and flower primordia will be formed; and the rib meristem (RM) zone located below the CZ from which cells of the stem form. Axillary meristems initiate from the peripheral zone of the SAM at the axil of a subtending leaf (Steeves and Sussex 1989; Meyerowitz 1997; Sussex and Kerk 2001). The fate of an axillary meristem is regulated by developmental and environmental cues. It may develop as a vegetative branch or as an inflorescence resulting in flowers and seeds. During reproductive development, axillary meristems play a crucial role in the establishment of different inflorescence structures that ultimately lead to the formation of flowers. An axillary meristem may also remain dormant throughout the life of the plant. This flexibility as to fate determination of axillary meristems provides plants with the necessary plasticity that allows them to respond to environmental signals and also to distinguish functions within the shoot. The SAM often suppresses development of axillary branches; this phenomenon is termed apical dominance. The first step in shoot branching is the establishment of axillary meristems. It is regulated by a series of transcription factors belonging in the GRAS-, MYB-, and bHLH- families of transcription factors. The organization of axillary meristems probably involves communication between the presumptive axillary meristem cells and the vascular system, and is governed by the REVULOTA subfamily of HD-ZIP transcription factors, REV, PHB, and PHV. It was recently shown that the second step in shoot branching, lateral bud outgrowth, is under the control of MAX (more axillary growth) and RMS (ramosus) proteins (Schmitz and Theres 2005). These proteins seem to produce, perceive, and transduce a signal of yet unknown identity that inhibits lateral bud outgrowth. Although its relation to MAX genes is not yet clear, the first gene suppressing lateral bud outgrowth to be identified was the teosinte branched 1 (TB1) gene in maize (Doebley et al. 1997; Wang et al. 1999; Hubbard et al. 2002). TB1 homologs were later identified in rice, tomato, and other crop plants. TB1 is one of the main genes responsible for changes associated with the domestication of maize from its ancestor, teosinte. Teosinte is typically highly tillered with long axillary


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branches developing from the majority of its nodes. Conversely, domesticated maize, which arose from overexpression of TB1, exhibits increased apical dominance, as demonstrated by the suppression of axillary bud outgrowth (Doebley et al. 1997). TB1 is a member of the TCP family of transcription factors such as barren stalk (BA) or ramosa (RA) (lateral inflorescence bud outgrowth) (Gallavotti et al. 2004; Vollbrecht et al. 2005), which were identified in maize and other plants and shown to control lateral bud outgrowth. The group of R. Martienssen elegantly demonstrated that all of these genes are heterochronic genes, which delay or prolong a developmental stage at the expense of neighboring (previous or following) developmental stages (Vollbrecht et al. 2005). According to Cronk (2002), heterochronic changes are employed by higher organisms more easily than heterotopic changes. This may be due to the fact that a single genetic or epigenetic change in the promoter area of a gene coding for a developmentally important transcription factor could have significant phenotypic changes that could be assessed and selected. Interestingly, all these genes code for important developmental transcription factors, and the changes—for instance, in the case of TB1 in maize leading to its higher, thus prolonged expression—are located upstream in its promoter (>41 kb upstream of tb1), as it was demonstrated by Doebley and coworkers (Clark et al. 2006). A great deal of knowledge has been obtained concerning the transition of plants from the adult to the reproductive phase (floral transition). However, the transition from juvenile to adult phase has not been studied as intensively. Juvenile and adult vegetative phases are best distinguished in woody plants, such as Hedera helix, where they were first described, but they are also obvious in herbaceous plants, such as maize and Arabidopsis (Poethig 2003). In Arabidopsis, during the juvenile phase leaves develop round with smooth margins, a low blade-to-petiole ratio and no abaxial trichomes. Conversely, leaves of the adult phase are characterized by an ovate shape, serrate margins, relatively short petiole, downward-curling edges, and abaxial trichomes (Willmann and Poethig 2005). The processes of juvenile-to-adult and adult-to-reproductive transition in various species are controlled by hormones such as gibberellic acid (GA), phytochrome B, and vernalization (Willmann and Poethig 2005). Most likely, these two transitions are coordinated by these and other unkown factors. A number of genes coding for specific miRNAs as well as genes coding for their metabolism are involved in the transition from juvenile to adult phases, indicative of the significant role played by EIS in controlling plant development (Kidner and Martienssen 2005;

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Willmann and Poethig 2005). Changes in the timing of function of these miRNAs exert heterochronic control in the timing of developmental phase changes (Willmann and Poethig 2005). Leaf primordia, similar to shoots, initiate from flanks of the SAM. Leaf development requires a balance between the proliferation of meristem cells and a commitment to the formation of leaf primordia. Expansion of the leaf blade requires the establishment of adaxial-abaxial polarity. Adaxial-abaxial polarity refers to the two opposing faces of a leaf blade, which have distinct cell types with different biological functions (Hudson 2000; McConnell et al. 2001). Early leaf primordia removed from the SAM could only form a small radial leaf without adaxialabaxial differentiation. This suggested that a meristem-derived signal directs adjacent regions of leaf primordia to become adaxial and regions farthest from the meristem to become abaxial (Bowman et al. 2002). Leaf shape and size are regulated by the expression in the leaf primordia of certain genes such as CLAVATA1, CLAVATA3, WUSCHEL, KNOTTED1, and PHANTASTICA. Most likely these genes regulate the establishment of hormonal gradients and transport. Finally, establishment of leaf form in the later stages of development involves coordination between different processes, such as establishment of hormonal gradients, release of biophysical constraints, cell division, and cell differentiation (Kessler and Sinha 2004). Leaf development requires the establishment of proximodistal, mediolateral, and adaxial-abaxial polarities. Several genes have been identified in Arabidopsis that play a role in the formation of the adaxialabaxial polarity, and epigenetic regulation of some of these has been demonstrated. Class III homeodomain/leucine zipper genes, PHABULOSA (PHB), PHAVULOTA (PHV), and REVULOTA (REV) genes are expressed in a polar fashion (adaxially localized) in leaf primordia and are required for adaxial cell fate (McConnell et al. 2001; Emery et al. 2003). In addition, members of the YABBI and KANADI gene families are required for determining abaxial cell fate (Eshed et al. 2001; Kerstetter et al. 2001). Moreover, it has been shown that the ASYMMETRIC LEAVES1genes (AS1 and AS2) are also involved in establishing leaf polarity by determining leaf adaxial identity, and they were found to positively regulate the PHB gene (Xu et al. 2002, 2003). Two miRNAs from the Arabidopsis miR165 and miR166 families contain complementary sites for the PHB, PHV, and REV transcripts, and they direct their cleavage in vitro. The miRNA 165/166 complemetary site is conserved between Arabidopsis and maize. Disruption of the miRNA 165/166 complementary sites leads to the presence of mutant transcripts in the abaxial side and formation of adaxialized leaves. In maize,


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mutations in the rolled leaf 1 (rlf1), a member of the class III homeodomain/leucine zipper genes, leads to an upward curling of the leaf blade caused by adaxialization or partial reversal of leaf polarity. The miRNA166 represses rolled leaf1 and in this way mediates maize leaf polarity (Juarez et al. 2004) by restricting expression of the homeodomain/leucine zipper genes in the adaxial site. In a study also in Arabidopsis, it was shown that the RDR6 gene acting together with the AS1 and AS2 genes also regulate leaf development. The rdr6 single mutant plants displayed minor altered phenotypes, whereas the rdr6 as1 and rdr6 as2 double mutants exhibited dramatically altered phenotypes with severe defects in the leaf adaxial-abaxial polarity and normal leaf morphology (Li et al. 2005). The double mutants exhibited ectopic expression of a class I KNOX gene BREVIPEDICELLUS (BP), which is down-regulated during leaf primordia initiation. They also displayed dramatically elevated levels of the miRNAs 165/166 and decreased transcript levels of the PHB and REV transcripts. These results indicated that the Arabidopsis RDR6-associated epigenetic pathway synergistically acting with the genes AS1 and AS2 represses the expression of BP and miRNAs 165/166 and is required for proper leaf development. Cell division processes also govern proper leaf development. Studies of mutant Antirrhinum plants had assigned a role for proper leaf development to the CINCINNATA (CIN) gene. Plants lacking CIN exhibited leaves with a crinkly appearance whereas wild- type plants had flat leaves (Nath et al. 2003). CIN codes for a TCP transcription factor protein (Cubas et al. 1999). TCP transcription factors are involved in regulating the growth of plant organs by arresting cell divisions in specific meristematic zones. It was suggested that in cin mutants, there is a delay in cell division arrest in the developing leaf, leading to accumulation of excess cells and a crinkly phenotype. Studies in Arabidopsis based on a genetic screen identified a mutant named jaw-D whose phenotype resembled the cin mutant in that the leaves had uneven shape and curvature. By analyzing global expression profiles, it was demonstrated that the expression of four TCP genes was reduced in the jaw-D mutant (Palatnik et al. 2003). In addition, a new miRNA, miR-JAW, was identified with sequence similarity to a well-conserved region of the four TCP genes. By mutagenesis and transformation experiments, this region was demonstrated to be a true target of the miR-JAW microRNA that guided the cleavage of the corresponding TCP transcripts. Abnormal expression and distribution of the TCP mRNAs during leaf cell differentiation lead to abnormal leaf development. In summarary, sRNAs associated with EIS have been proven to be major regulators of genes involved in the vegetative phase of plant development.

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C. Flowering The transition from vegetative growth to flowering is controlled epigenetically primarily by the PcG protein complex described, as well as by certain other chromatin remodeling factors. Although the MEA/FIE/ FIS2 PcG complex controlling seed development is the best studied in Arabidopsis, the first PcG gene characterized was Curly Leaf (CLF), and it was shown to be involved in flower morphogenesis. The CLF protein is similar to E(Z) and contains the typical SET domain found in methyltransferases. The clf mutants have a phenotype that is similar to transgenic plants with constitutive expression of the homeotic MADS-box gene AGAMOUS (AG) (Goodrich et al. 1997). These mutants have small and curly leaves, flower earlier, and show partial homeotic transformation of sepals and petals into carpels and stamens, respectively. Further research has shown that CLF is responsible for maintaining AGAMOUS in a repression state (Goodrich et al. 1997). EMF2 is another polycomb gene that was shown to be involved in floral induction (Yoshida et al. 2001). Mutant emf2 does not form any rosette leaves but creates small inflorescences whose lateral buds produce only flowers but not additional inflorescences. The emf2 also shows ectopic expression of AG. The EMF2 belongs to the same family with FIS2 of Arabidopsis and SU(Z)12 of drosophila. It is likely that FIE, CLF, and EMF2 are all part of a complex similar to the PcG complex for FIE, MEDEA, and FIS2 that is formed during seed development (Grossniklaus et al. 1998) (Fig.3). This hypothesis can also be based on the fact that the emf2 and clf mutants show similar phenotypes: early flowering and curly leaves. Furthermore, a partially complemented fie mutant starts flowering in the seedling stage, as is the case for emf2. Finally, as there are no other genes identical to FIE in the Arabidopsis genome, FIE is probably part of various PcG complexes that control different developmental processes in plants. This is also indicated by the fact that only heterozygous fie mutants are viable whereas other fis mutants can be made homozygous (Spillane et al. 2000). One of the most interesting aspects of PcG-based epigenetically regulated processes is the involvement of PcG in the regulation of the vernalization response. Vernalization is the process by which exposure to long periods of cold promotes flowering in plants. Vernalization ensures that flowering does not take place in the fall but in the favorable conditions of the spring. The term vernalization originates from the Latin word vernus, which means ‘‘spring.’’ Plants usually achieve a vernalized state only after a long-term exposure to cold winter conditions. Previous studies had shown that the site of cold perception is the meristem, and during


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vernalization the meristem becomes competent for flowering (Sung and Amasino 2004a). Once meristems have been exposed to low temperatures for a sufficiently long period, they ‘‘remember’’ that they have been vernalized, and this memory is inherited through cell divisions. After vernalization, plants do not necessarily initiate flowering but become competent to do so (Sung and Amasino 2005). A major determinant in flowering and vernalization is the FLOWERING LOCUS C (FLC) MADS-box gene. It has been shown that in Arabidopsis, the acceleration of flowering by prolonged exposure to cold is associated with the down-regulation of the FLC gene, which usually prevents flowering when it is upregulated. FLC is a MADS-box transcription factor that acts as a repressor of flowering by inhibiting the activation of a set of genes required for the transition of the apical meristem from the vegetative phase to the reproductive phase (Michaels and Amasino 1999). Following prolonged exposure to cold, the FLC transcript levels are down-regulated, and they remain low during subsequent development. This observation leads to the concept that this cellular ‘‘memory’’ for the cold vernalization response must have an epigenetic basis. Indeed, genetic screens in Arabidopsis identified mutants in which the vernalization response was impaired. In this way three genes were identified, VERNILIZATION 2 (VRN2), VERNALIZATION1 (VRN1), and VERNALIZATION INSENSITIVE 3 (VIN3). VRN2 encodes a homolog of the drosophila Suppressor of Zeste 12 (Su(z)12), a member of the PcG transcriptional repressor (Gendall et al. 2001). VRN1 encodes a plant specific DNA-binding protein (Levy et al. 2002). VRN1 and VRN2 were shown to be required for the maintenance of FLC repression during subsequent development following prolonged cold exposure. VIN3 encodes a PHD plant homeodomain finger protein (Sung and Amasino 2004b). PHD finger proteins are often associated with protein complexes involved in chromatin remodeling (Aasland et al. 1995; Gozani et al. 2003). To investigate whether histone modifications were involved in the vernalization-dependent regulation of FLC, ChIP experiments were performed (Bastow et al. 2004). It was demonstrated that vernalization results in histone methylation in discrete domains within the FLC MADS-box gene. In particular, there was increased dimethylation of H3K27, which is indicative of a heterochromatic repressed chromatin state. Dimethylation of H3K27 was lost only in the vrn2 mutant whereas dimethylation of H3K9 was absent from both vrn1 and vrn2, indicating that VRN1 functions downstream of VRN2. In another study, the acetylation of H3K9 and H3K14 was examined in the chromatin environment of FLC (Sung and Amasino 2004b). A region of intron I and a region upstream of the transcriptional initiation site

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exhibited decreased acetylation during vernalization, an indication of inactivation. This decrease was maintained after transfer to warm growing conditions. In contrast, in the vin3 mutant, the vernalizationmediated changes in FLC acetylation did not occur. In the vin3 mutant, the vernalization-mediated increase in dimethylation of H3K9 does not take place as is the case for the vrn1 and vrn2 mutants (Sung and Amasino 2004b). Furthermore, it was shown that VIN3 is transiently expressed upon cold conditions both in the wild type and in the vrn1 and vrn2 mutants. Taken together, these results lead the researchers to propose a model for the epigenetic basis of vernalization also in Arabidopsis. Exposure to prolonged cold induces the expression of VIN3 that results in deacetylation of the FLC locus. This leads to histone methylation and the formation of mitotically stable heterochromatin at the FLC locus by a process involving VRN1 and VRN2. In summary, a series of epigenetic chromatin modifications in the FLC locus leads to the epigenetic memory of winter cold, known as memory of winter. After the transition from vegetative to reproductive meristems, the appearance of flowering plants is determined by the forms of flowerbearing branch systems, known as inflorescences, and the overall structure of the plant. Inflorescence architecture comprises the stereotypical number and arrangement of floral branches that characterize each species of flowering plant (Weberling 1989). The presence or absence of long branches, for example, dictates the capacity for flower and seed production and largely crop yield. Thus, inflorescence architecture reflects reproductive meristems activity, arrangements, and numbers. And the duration of activity of this reproductive meristems correlates with branch length (Sussex and Kerk 2001; Vollbrecht et al. 2005). Developmental decisions such as meristem allocation, fate, and timing (Sussex and Kerk 2001) are thus observed as quantitative variation in the number of main axis inflorescence meristems established, the number of elongated axillary inflorescences, or the timing of shoot bolting. Identification of genes that lead to variation in quantitative aspects of inflorescence morphology can thus provide insights into developmental pathways that lead to diversity in plant reproductive shoot architectures. Plant reproductive ecology is determined, in part, by the architecture of the inflorescence shoot (Schoen and Dubec 1990; Fishbein and Venable 1996; Diggle 1999). Inflorescence architectures display a wide range of diversity among plant species (Coen and Nugent 1994) and are critical determinants of interspecies differences in plant morphology and life history. Significant architectural changes in inflorescence took place during domestication and breeding of many crops. In the last few years, geneticists have identified and isolated several genes


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that regulate the development of the inflorescence in Arabidopsis (Shannon and Meeks-Wagner 1991, 1993; Bowman et al. 1992; Bradley et al. 1997; Koornneef et al. 1998; Levy and Dean 1998; Schmitz and Theres 1999). In Arabidopsis, genes like TFL1 (Shannon and Meeks-Wagner 1991, 1993) were cloned, characterized, and studied. In grasses, genes like ra1, which imposes short branch identity as branch meristems are initiated, have been cloned and characterized (Vollbrecht et al. 2005). The gene ramosa1 encodes a transcription factor that appears to be absent in rice, is heterochronically expressed in sorghum, and may have played an important role in maize domestication and grass evolution (Bommert et al. 2005). Characterization of the maize gene ramosa2 has shown that it encodes a protein with the highly conserved domain lateral organ boundary, which is essential for determining the fate of stem cells in branch meristems of maize (Bortiri et al. 2006). An extensive genome-wide study on quantitative trait loci (QTLs) exerting major effects on quantitive differences in Arabidopsis inflorescence was conducted by Ungerer et al. (2002). Flower patterning is also controlled by various epigenetic regulators. The crucial role of DNA methylation in flower patterning has been demonstrated by examining the effects of genome-wide demethylation in Arabidopsis. Transformation with an antisense construct of MET1 resulted in plants with abnormal flower phenotypes (Finnegan and Dennis 1993; Finnegan et al. 1996; Ronemus et al. 1996). Homeotic transformation of floral organs in these plants resembled the phenotypes of superman (sup) and superman/agamous (sup/ag) double mutants (Finnegan et al. 1996). In addition, the leaves of the MET1 antisense plants exhibited ectopic expression of the two floral homeotic genes APETALA3 (AP3) and AGAMOUS (AG). Examination of the methylation state of the SUPERMAN and AGAMOUS genes in MET1 antisense plants revealed that the genes were hypermethylated, and this hypermethylation correlated with the absence of SUPERMAN and AGAMOUS transcripts in floral buds, respectively (Finnegan 2001). Flower shape has been shown to be determined by the action of two closely related genes that belong to the family of TCP transcription factors, CYCLOIDEA (CYC) and DICHOTOMA (DICH). Expression of both genes is required to produce the typical asymmetric flower morphology in Antirrhinum and Linaria. Null mutations in the CYC and DICH genes resulted in the classic peloric mutant phenotype, originally described by Linnaeus 250 years ago. Peloric is characterized by a change from bilateral to radial flower symmetry both in Antirrhinum and Linaria (Cubas et al. 1999; Luo et al. 1999). Cubas et al. (1999)

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demonstrated that in the Linaria mutant, the CYC gene is extensively methylated and transcriptionally silenced, and this epigenetically controlled phenotype has been stable for more than 250 years. Another large family of plant-specific transcription factors has been described recently, the NAC family, which takes its name from the first studied members of the family, NAM, ATAF, and CUC2. Both NAC and TCP transcription factors have been implicated in the establishment of organ boundaries. The NAC transcription factors comprise a superfamily of more than 100 members in Arabidopsis and nearly as many in rice (Olsen et al. 2005). Mutations in the NAC genes lead to severe perturbations in embryonic, floral, and vegetative development. The first NAC mutant to be characterized was the nam (no apical meristem) mutant in petunia (Souer et al. 1996). The nam mutant lacks a shoot apical meristem and dies at the seedling stage. It exhibits fusion of cotyledons, and occasionally plants developed from escape shoots display abnormal flowers. Another NAC mutant, the cuc2 mutant (for cup-shaped cotyledons) was later characterized in Arabidopsis (Aida et al. 1997). Cuc1 and cuc2 double mutants display severe defects in the separation of cotyledons, sepals, and stamens and in proper shoot apical meristem formation (Aida et al. 1997; Takada et al. 2001). A critical role has been assigned to miRNA 164 in the regulation of CUC1 and CUC2 (Mallory et al. 2004). The miRNA 164 complementary sites were detected in several NAC genes including CUC1 and CUC2, and miRNA-directed cleavage of the mRNA of those genes was validated experimentally. Arabidopsis plants transformed with a miRNA164a- and 164b-resistant version of CUC1 displayed dramatic effects in cotyledon, leaf, and flower formation. Furthermore, miRNA164c has been also shown to be involved in the early extra petal1 (eep1) mutant in Arabidopsis, which is characterized by extra petals in early-arising flowers. MiR164c controls petal number by regulating the transcript accumulation of CUC1 and CUC2 (Baker et al. 2005). Finally, another miRNA, miR172, has been shown to target the AP2 (APETALA 2) transcription factor and is associated with proper flowering timing and floral organ formation (Aukerman and Sakai 2003). In summary, all three EIS are major determinants of transition to flowering, flower patterning, inflorescence and flower numbers (thus seed numbers), and flower organ formation and growth. D. Seed Development Flowering plants and conifers bear seeds. Seeds contain the embryo and the endosperm, which provides nutrients and sustains embryo devel-


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opment and germination. The endosperm stores reserves, such as starch, fatty acids, and proteins. The endosperm of cereal crops (rice, maize, wheat, barley) represents 60% of the world’s food and feed supply. Consequently, understanding and manipulating endosperm development for increasing seed yield is of outmost interest for agriculture. In flowering plants, the seed is formed through the process of double fertilization. Fertilization of the egg cell by a sperm cell from the male gametophyte generates the diploid embryo from which the organs, tissues, and meristems of the plant will be generated. Fertilization of the adjacent central cell by a second sperm cell forms a triploid endosperm, in angiosperms, which supports embryo growth and development by producing storage proteins, lipids, and starch (Brown et al. 1999). In dicots, such as Arabidopsis, the endosperm is consumed by the embryo during seed maturation, whereas in monocots, such as cereals, the endosperm persists after embryo development is completed and constitutes the major portion of the mature kernel (Gehring et al. 2004; Olsen 2004). Epigenetic regulation has been shown to play a crucial role in proper seed development. Proper embryo and endosperm development depends on the maternal allele of a group of genes encoding the PcG transcription regulators. The identification of PcG genes such as MEA, FIE and FIS2, already described, indicated that the endosperm shows defects in proliferation and polarity when these genes are mutated. In addition, embryos in mea and fis2 mutants rarely reach the heart stage, and there is no embryo development in fie mutants (Ko¨hler et al. 2002). In Arabidopsis, MSI 1 protein is part of the MEA/FIE complex and interacts directly with FIE (Ko¨hler et al. 2003a). The msi mutants also show similar phenotypic behavior to that of the other fis mutants, which adds more evidence for its contribution to proper seed development. However, the exact role of this complex in gene silencing has not been completely elucidated. Microarray analysis in Arabidopsis detected two potential targets of the MEA/FIE PcG complex, namely the genes PHERES1 (PHE1) and MEIDOS, as their transcript levels were shown to be increased in mea and fie mutants (Ko¨hler et al. 2003b). PHE1 encodes a MADS-box type I transcription factor, and MEIDOS has not been cloned yet. ChIP analysis showed that the MEA/FIE complex strongly associates with the promoter of PHE1, indicating that PHE1 is a direct target of the MEA/FIE PcG (Ko¨hler et al. 2003b). PHE1 is transiently expressed in embryo and endosperm after fertilization, and it remains highly expressed until the embryo aborts in fis mutants. Reduced levels of PHE1 expression

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in mea mutants result in rescue of mea seed abortion. These observations suggested that PHE1 has an important role in seed development and that mea mutant seed abortion is due to deregulation of its expression. PHE1 is also parentally imprinted, as it is only the paternal allele that is expressed (Ko¨hler et al. 2005). Homologs of the E(Z) and FIE genes have been recently identified in maize (Springer et al. 2002), but no mutants are available to analyze gene function. Three E(Z) genes have been isolated: MEZ1, MEZ2 and MEZ3. Phylogenetic analysis showed that these are orthologs of the EZA1 gene in Arabidopsis (for which no phenotype is yet available) and very distantly related to MEA. MEA homologs have not been detected in the rice EST or genomic sequence databases. This may suggest that monocots have another E(Z) homolog that performs the function of MEA and may reflect differences in the regulation of seed development between monocots and dicots (Springer et al. 2002). Asymmetric expressions of genes in the zygote and the endosperm interfere with seed development. This asymmetry in gene expression is associated with structural differences in the nuclear genomes of the gametes. Plant sperm have highly condensed chromatin, as compared with the egg and central cell (Mogensen 1982; Scholten et al. 2002). Thus, there is a potential imbalance of regulatory factors contributed by the two parents to the products of fertilization (Dilkes and Comai 2004). The pollen parent contributes a small compact nucleus, whereas the female parent contributes an active and decondensed genome, much cytoplasm, and many RNAs. It follows that the female parent might also contribute factors necessary for remodeling and unpackaging sperm chromatin, and development might impose a strict timing requirement on this phase. Interestingly, the zygote and endosperm develop at different rates: The zygote develops slowly whereas the endosperm rushes into a series of cell divisions, engaging in four rounds of mitosis before the embryo divides once (Boisnard-Lorig et al. 2001; Brown et al. 2003). Perhaps, as a consequence of this mechanism, widespread suppression of many paternal genes occurs during early seed development (Vielle-Calzada et al. 2000; Weijers et al. 2001; Guo et al. 2003). As development progresses, this suppression are partially relaxed, resulting in maternally skewed biallelic expression in the endosperm (Weijers et al. 2001; Guo et al. 2003). Transgenes follow a pattern similar to that of endogenous genes (Vielle-Calzada et al. 2000; Baroux et al. 2001; Weijers et al. 2001), displaying either absolute imprinting and differential expression or a reduction in expression from alleles contributed from the pollen parent. Differential expression of paternal alleles has also been demonstrated in maize endosperm, although twice


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as many mRNAs are preferentially expressed from the maternal allele (Guo et al. 2003). The dosage sensitivity hypothesis described in Section III.D implies that a dosage-sensitive phenotype affected by any of these gene products, imprinted or not, would result in parent-of-origin effects and could also explain the dosage sensitivity of endosperm development observed during interspecific hybridization. Asymmetric parental contributions during sexual reproduction have long been known to cause problems. Dissimilar paternal and maternal types may be created by different copy numbers of heterochromatic elements, which further could create an impediment to hybridization. If, for example, a critical level of repressor is needed to suppress a locus, a maternal deficiency of repressor would result in locus activation, similar to hybrid dysgenesis in animals. The more rapid the developmental pace, the sooner remodeling must be complete and the greater the reliance on preexisting resources rather than the de novo synthesis of required factors. That is, early entry of the fertilized central cell into proliferative cell cycles might underlie endosperm sensitivity (Boisnard-Lorig et al. 2001; Dilkes and Comai 2004; Olsen 2004). This section has examined different critical plant developmental steps, the formation of different organs, and growth parameters in light of the important role played by the three EIS. All these developmental events (e.g., flowering time, early- or late-spring flowering, and early- or late-leaf senescence) or organ formation and growth (e.g., the number of inflorescences, the determined or indetermined nature of inflorescence, the number of flowers or fertile flowers, the number of seeds and their size, which is actually the size of endosperm or embryo or both in different seed types) are major targets of many plant breeding programs. They are also for many cases major determinants and components of yield and yield stability. Thus, it is important to understand them in molecular, genetic, and epigenetic terms in order to comprehend and enhance plant breeding efficiency in both conventional and modern plant breeding.

V. IMPLICATIONS IN PLANT BREEDING A. Genetic and Epigenetic Variation Phenotypic variation is traditionally parsed into components that are directed by genetic and environmental variation. The line between these two components is currently blurred by inherited epigenetic variation, which is potentially sensitive to environmental inputs.

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A unifying theme in biology is that the characteristics displayed by organisms are controlled—ultimately—by the nucleotide sequence of their genome. Another cornerstone of modern biology is that inherited information that is transmitted on the chromosomes changes only at random, without direction from the environment toward particular phenotypic outcomes. These elements of our current biological thinking are being tested by recent work in the field of epigenetics. The chromatin and DNA methylation-based mechanisms and the involvement of RNAi mediate a semi-independent epigenetic inheritance system at the interface between genetic control and the environment (Richards 2006). Epigenetic states in plants, once established, can be inherited through the transmission of epigenetic alleles (epialleles) over many generations (Kakutani 2002). These heritable epigenetic alleles can be considered as a new source of polymorphism and may produce novel phenotypes. An example of the stability and heritability of alleles and epigenetic variation is found in the morphological variant of toadflax Linaria vulgaris described 250 years ago by Linnaeus. Ironically, this variant, which has played such a significant role in the history of botany, turned out to be neither a new species (as Linnaeus thought) nor a mutation (as de Vries and others thought), but a fairly stable epimutation due to an alteration in DNA methylation of the Lcyc gene. It is not clear what caused the methylation change in the first place, but once formed, it seems to have been transmitted, more or less steadily (although there is residual instability), for many generations. Such epigenetic variants could have significant implications in plant breeding. Heritable phenotypic variation within populations is the basis for selection and breeding. The genetic causes of phenotypic variation are attributable to mutations that create allelic variation and recombination that alters the genetic structure in which alleles are expressed, offering new backgrounds for epistatic and pleiotropic allelic interactions. In addition to mutations that create the genetic variation underlying phenotypic traits, epimutations produce a new source of variation for selection. Most important, epigenetic alleles can result from a genome response to stressful environments and may enable plants to tolerate stress (Tsaftaris and Polidoros 2000; Finnegan 2001; Sherman and Talbert 2002; Steward et al. 2002). The two mechanisms generating polymorphism (mutations and epimutations) have been compared by Tsaftaris and Polidoros (2000). Epialleles could emerge at high frequency in a single generation, far exceeding the rate of mutational events that give rise to new alleles. Their reversion rate is far higher and will interfere in heritability estimation. Their emergence is highly affected by plant growth conditions while random mutational


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events are largely considered independent of growth conditions. Furthermore, DNA methylation, in particular, can give rise to more permanent mutant alleles at a locus by its mutational role. However, mutations rarely lead to new epialleles (when, by chance, critical C-residues in methylation sites are eliminated or generated). Assessing the importance of methylated epialleles in plant breeding requires the determination of: (1) the extent of variation in methylation patterns among individuals within the selection population; (2) the degree to which methylation patterns affect phenotypes; and (3) the extent to which methylation variants linked to superior phenotypes are stably inherited. These are challenging tasks, but the technical potential exists now for genome-wide assessment of methylation pattern and chromatin structure differences between individuals (Zhang et al. 2006). A better understanding of the role and significance of this new source of polymorphism in plants will be achieved as more data accumulate for the role of DNA methylation and other EIS in plant evolution, domestication, and breeding. Research on the regulation of activity of transposable elements just described strongly support the involvement of EIS in generating new genetic variation. Extended inactivation of eukaryotic transposable elements is accomplished through chromatin modifications. Reactivation may occur when the epigenetic mark is lost, which may result to transposition and disruption of gene loci causing genetic mutation, even though it has an epigenetic origin. The possible activation of an inactive element depends on several factors, all of which have been shown to affect chromatin structure including methylation levels. Among these factors is position in the plant, parental origin, presence or absence of other active elements, developmental stage, and influence of the environment (Tsaftaris and Polidoros 2000; and Section III). All these represent possible opportunities for generation of new genetic variation that is heritable since TEs may stably integrate in the regulatory area of a specific locus and exert their effect for many generations. Plant tissue culture that was frequently used as another source of variation (somaclonal variation) in breeding programs is also found to involve EIS as causes of epigenetic variation. Tissue culture has many applications, including micropropagation, elimination of viruses by meristem-tip culture, production of doubled haploids by anther culture, the use of cell cultures for the production of secondary products, and more recently as a source of target cells in Agrobacterium-mediated gene transfer. In these applications it is of paramount importance that the plants derived from culture are true to type. However, uncontrolled instability can occur when plant cells are cultured in vitro because single

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cells or tissue explants dedifferentiate from the developmental state in the explanted tissue to form callus and then redifferentiate into new tissue types. Tissue culture inflicts a type of severe stress that includes wounding and genome restructuring to promote developmental changes and reprogramming. This reprogramming of the genome causes phenotypic variability known as somaclonal variation. Dedifferentiation of the cultured cells has been accompanied with changes in rRNA gene methylation in some plant systems (Anderson et al. 1990; Vyskot et al. 1993), but not in others (Avivi et al. 2004; Komarova et al. 2004). The initial reduction of CG and CNG methylation in both intergenic and genic regions of the rDNA cistron in fully dedifferentiated callus was followed by the establishment of stable epigenetic patterns that were maintained throughout prolonged culture. However, regenerated plants and their progeny showed partial and complete remethylation of the ribosomal units (Koukalova et al. 2005). Although these data suggest a role of tissue culture–induced stress in the epigenetic patterns of chromatin structure, a functional relation between methylation and gene expression has been documented by Mitsuhara et al. (2002); Fojtova et al. (2003); and Avivi et al. (2004). Madlung and Comai (2004) discussed the effects of tissue culture stress on genome structure and concluded it is likely that tissue culture compromises the epigenetic homeostasis of plant genomes and can result in secondary genomic effects. Last but not least, an important source of epigenetic variation emerges from the regulation of gene expression by means of RNAi. As it is described, this epigenetic mechanism can inhibit the expression of an otherwise normal allele and make it seen as a null mutation. For example, Todd and Vodkin (1996) have shown that the soybean color phenotypes are likely the outcome of mutations affecting different enzymes of the anthocyanin and proanthocyanidin pathways. The I locus corresponds to a 27-kb-long chalcone synthase gene cluster that exhibits a unique tissue-specific gene silencing mechanism in the seed coats mediated by short-interfering RNA (Todd and Vodkin 1996; Tuteja et al. 2004). Parallel studies with the flavonoid pigment pathway in maize also found that the dominant inhibitory chalcone synthase allele C2-Idf (inhibitor diffuse) acts via an endogenous silencing mechanism (Della Vedona et al. 2005). C2-Idf is a stable dominant mutation of the chalcone synthase gene, c2, which encodes the first dedicated enzyme in the flavonoid biosynthetic pathway of maize. Homozygous C2-Idf plants with two defective alleles show no pigmentation. This allele also inhibits expression of functional C2 alleles in heterozygotes, producing a less pigmented condition instead of the normal deeply pigmented phenotype. The gene structure of the C2-Idf haplotype differs substan-


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tially from that of the normal c2 gene in that three copies are present. Two of these are located in close proximity to each other in a head-tohead orientation, and the third is closely linked. In nuclei of C2-Idf/C2 heterozygotes c2 transcription occurs, but mRNA is destructed by a siRNA coded by the C2-Idf allele. Perhaps this is the first indicative result of a new mechanism for explaining alleles frequently observed in nature, exhibiting negative dominance. The first commercially used cultivar involving RNAi was the rice mutant line Low Glutelin Content-1 (LGC-1; Kusaba et al. 2003). LGC1 rice has low-protein content and is useful for patients whose diet requires low protein intake, such as those who suffer from kidney disease. Glutelin is the major storage protein in rice, and LGC-1 is again a dominant mutation in the glutelin gene. The negative mutant allele produces a hairpin RNA from an inverted repeat of the glutelin gene sequence. Via an RNAi mechanism it leads to lower glutelin content in the rice. The LGC mutant was isolated in the 1970s, and the lowglutenin mutant character appears to have been stable for over three generations. This suggests that the suppression of gene expression by natural hairpin RNA-induced RNAi may be inherited in a stable manner. The siRNA example with the rice LCG glutenin gene family, in addition to negative dominance of a defective allele in a normal one, is a revealing example of negative epistasis. The glutenin gene family consists of more than eight members, all of which are suppressed by one allele. These data suggest a new approach as to how the expression of a family of homologous genes could be down-regulated, for breeding purposes, using transgenic technology (see also Section V.G). B. Improving Plant Stress Tolerance The abundant evidence demonstrating that epigenetic changes occur as a direct consequence of different stresses has been described in the previous sections. Some of these stresses are internal genomic stresses involving, for example,polyploidization, interspecific hybridization, movement of transposable elements, and so on have already been mentioned. Polyploidization, for example, was identified in Section III.C as a genomic stress factor causing epigenetic changes. Most of the major crops are polyploids, and even if they behave as diploids, many of them have experienced polyploidization events. Allopolyploid plants can be considered as a special type of hybrids where two or more homeologous genomes are fixed in one nucleus maintaining their integrity through sexual reproduction. Therefore, the advantage of hybrid vigor and heterosis can be fixed by polyploidization (Fasoulas 1993). In contrast,

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diploid hybrids undergo wide recombination of the parental genomes in segregating progeny. Hindered recombination between the parental genomes in allopolyploids could be unfavorable on an evolutionary time scale. But this disadvantage would be not dramatic in self-fertilized or asexually propagated species, which are genetically uniform. The widespread distribution of allopolyploid hybrids in such species implies that the benefits of permanent fixation of heterosis through polyploidization overwhelm any disadvantage imposed by limiting the recombination of the parental genomes. How a polyploid plant genome ‘‘senses’’ the stress of polyploidy and how this leads to epigenetic changes is not understood. Quantitative changes of different transcripts due to gene dosage changes in the pollypdoid could be one possible mechanism inducing the mechanisms of epigenetic changes, but this requires further research. External environmental conditions imposing biotic and abiotic stresses during plant growth also are proven to induce epigenetic changes in plants. Pathogen attack, for example, is a severely stressful event, and plants have developed an array of defense mechanisms to cope with its effects. These defenses, particularly against viruses, include epigenetic components like RNA silencing, which also leads to genome methylation, as described in detail in Section III.E. High-temperature stress also leads to epigenetic modifications. Exposure of transgenic Petunia plants to high temperature increased the 5mC levels of the 35S CaMV promoter region of a maize A1 transgene construct (Meyer et al. 1992). This treatment resulted in the change of flower color conferred by decreased activity of the A1 gene. Other types of stress have also been reported to affect epigenetic states. In Bryonia dioica, mechanical stress caused DNA methylation levels to drop from 25% to nearly undetectable in less than an hour, and remained at that level for at least three hours (Galaud et al. 1993). In Stellaria longipes, demethylation of ramets coinciding with initiation of rapid stem elongation was observed in plants growing for four days under long-day warm conditions and depended on the relative ratio of Red/Far Red light. The degree of methylation was a crucial factor in controlling the stem elongation response in different ecotypes, since prairie ecotype plants grown in Murashige and Skoog media supplemented with 5-azacytidine (5-AzaC) required greater doses of 5-AzaC, and thus lower methylation levels, than the alpine ecotype plants in order to promote maximal stem elongation (Tatra et al. 2000). Water deficit is another type of stress that has been reported to result in epigenetic modifications. Hypermethylation of tobacco heterochromatin was observed in response to osmotic stress (Kovarik et al. 1997), while hypermethylation


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at specific chromatin sites of pea root tips was observed in response to water deficit (Labra et al. 2002). Environmental stress conditions, such as drought, salinity, high light, or heavy metals, cause a rapid and excessive accumulation of ROS in plant cells. Superoxide dismutatses represent the first line of defense against superoxide accumulation by rapidly converting superoxide to H2O2 and molecular oxygen. A fundamental insight into the regulatory role of microRNAs in stress responses of defense genes was provided by the identification of miR389 as a repressor of superoxide dismutases CSD1 and CSD2 in Arabidopsis. Expression of miR398 was downregulated by oxidative stress. This down-regulation proved important for the posttranscriptional induction of CSD1 and CSD2 expression under oxidative stress conditions. Furthermore, it was shown that relieving miRNA-directed suppression by overexpression of a miR398resistant version of CSD2 leads to great improvement of plant resistance to oxidative stress conditions such as high light, heavy metal, and methyl viologen (Sunkar et al. 2006). It is conceivable that other gene targets of miR389 should also be relieved from suppression under oxidative stress. It is important to note that relieving microRNA-guided suppression of defense gene expression might prove to be an effective new approach to improving plant productivity under stress. Flax exhibits phenotypic and genomic changes associated with environmental factors. In a stressful environment, flax can bear progeny called genotrophs that exhibit stable phenotypic changes associated with highly specific DNA changes at multiple loci. Although the parents remain phenotypically plastic when grown in different environments, the altered phenotypes of the genotrophs are stable. A site-specific insertion sequence (LS-1) was identified in the genotrophs that is also found in natural populations of flax (Chen et al. 2005). An intact LS-1 is not present in the genome of the progenitor flax line. Flax genotrophs support the idea that beyond genetic, a yet uncharacterized mechanism of inheritance might be operating to shape a dynamic plant genome, providing multiple alternatives for chromosomal changes that may confer better adaptation in response to various challenges. How plants ‘‘sense’’ all the above abiotic stresses and how this leads to the epigenetic changes described is not very well known. The accumulaton of oxygen radicals, discruptive for different macromolecules including DNA, under stress conditions of growth is one mechanism already mentioned. Repair efforts on behalf of the plant genome could lead to genetic and/or epigenetic changes in the damaged area. A recently described example involving developmental changes of plant epigermal pattering under nutritional (Pi and Fe)

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stress is also indicative of the role of environmental conditions on plant growth (Guimil and Dunand 2006). Under conditions of limiting iron and phosphate, Arabidopsis forms many more and longer root hairs (Lopez-Bucio et al. 2003; Muller and Schmidt 2004). However, this is an adaptive response to increase the absorptive surface area in contact with the rhizosphere in order to maximize water and nutrient uptake. How these environmental signals are translated into changes in epidermal cell fate is not understood. Recent experiments have revealed that epigenetic regulation has an important role in the determination of cell fate in the root epidermis (Costa and Shaw 2006). Furthermore, results obtained after treatment of germinating Arabidopsis seedlings with trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs), promoted hair cell formation and growth at non–hair cell positions (Xu et al 2005). Application of TSA resulted in the hyperacetylation of core histones H3 and H4 at the CAPRICE, GLABRA2, and WER, a nuclear-localized R3 loci. And mutants of HDAC family members lead to the same phenotype. All these facts strongly support the role of epigenetic mechanisms as mediators of environmental condition effects on changes in the genome. Breeding plants for stress tolerance, low input demands, better exploitation of limited resources for growth, and stability of performance in general is a major goal for many breeding programs involving different crops. And, despite the success in a number of cases, very little is known about: (a) the underlying genetic or epigenetic mechanisms responsible for the tolerant genotype obtained, and (b) which are the appropriate conditions during selection for the most efficient breeding for stress-tolerant varieties. A better understanding of these mechanisms will help breeders to fulfill the goal of breeding for stress tolerance in a more efficient way. C. Epigenetic Mechanisms, Yield, and Heterosis A lasting mystery in biology, from the days of Charles Darwin, is how hybrids display superior growth and fertility over their parents (Darwin 1876). Heterosis, or hybrid vigor, refers to the phenotypic superiority of a hybrid over its parents with respect to traits such as growth rate and reproductive success and plays significant role in evolution. Hybrid vigor, particularly for yield, was rediscovered in maize breeding almost 100 years ago and has subsequently been found to occur in many crop species (Duvick 2001). The importance of heterosis in plant breeding and in agriculture is evident from the dramatic increases in yield over the past 50 years, following the influx of hybrids to crop production


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(Duvick 2001). Maize has provided the genetic tools to study heterosis in plant breeding because parental inbreds have been artificially selected for maximum combining ability, and the creation of structured genetic populations enables more accurate quantitative phenotyping than in natural populations. Indeed, much of our genetic knowledge of heterosis comes from classical genetic studies on maize, mainly examining the participation of dominance versus overdominance and their epistatic interactions in the manifestation of heterosis. Despite decades of research, it is widely assumed that the genetic components of heterosis are still obscure (Lippman and Zamir 2007). Recent efforts on mapping and identification of QTLs controlling different phenotypes, including heterosis, addressed the classical models by breaking down heterosis into Mendelian factors and addressing their modes of inheritance, showed that both dominance and overdominance and epistasis have a varying role in heterosis (see Lippman and Zamir 2007 for a recent review). Working with tomato Semel et al. (2006) created populations of isogenic lines (ILs) that control epistasis (allowing dominance or overdominance effects to be studied) and were able to identify a clear overdominant tomato QTL for yield and fitness. ILs are now available for other crops, such as rice, and similar studies on QTLs and heterosis are expected (Tian et al 2006). In a study comparing maize diploid and triploid hybrids of B73 and Mo17 lines (in order to separate the additive effects), Auger et al. (2005) also identified nonadditive gene expression, in addition to the additive gene dosage involved. In addition to commercially used diploid hybrids, such as maize and rice, many important crops, such as wheat, cotton, and canola, are allopolyploids (Wang et al. 2004). Both autopolyploids and allopolyploids can maintain high levels of heterozygosity through generations (Osborn et al. 2003), and hybrid vigor is positively correlated with polymorphism of the contributing genomes in the formation of autotetraploid potato and alfalfa (Mok and Peloquin 1975; Bingham et al. 1994). Theoretically, hybridization of inbreds in diploid species should not be as stressful as allopolyploidization, since it brings together in one nucleus two homologous haploid genomes. But hybrid breeding proved that combining ability of different inbred lines varies greatly, probably due to incompatibilities of haploid genomes even in the same species, which may result in imbalanced gene expression and genomic stress in the hybrid that, even though less severe, could still trigger similar responses as those reported in allopolyploids. Indeed, allelic variation of gene expression that was not due to parental effects has been observed in maize hybrids (Guo et al. 2004). Moreover, allele-specific responses to abiotic stress were recorded, suggesting a functional

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disparity of the parental alleles in the hybrid, which may have an impact in combining ability, stability of performance, and heterosis (Guo et al. 2004). Genomic imbalance in hybrids may not be due only to allelic variation of gene expression. It could also encompass disrupted colinearity of the haploid genomes. Although karyotypic polymorphisms in plants have been studied for many years (Levin 2002), the possibility of polymorphism in gene content and order within species has been largely neglected. Since local gene order differences can be observed between species, there must have been a point in time when they were polymorphic within species, and it is thus reasonable to suppose that some polymorphism in gene order is present within contemporary species as well (Vision 2005). Indeed, different maize inbred lines are polymorphic for the presence or absence of genic sequences at various allelic chromosomal locations due to gene movements caused by transposons (Lai et al. 2005). This polymorphism has been documented for example for the bz genomic regions of two North American maize lines that differ extensively in the organization and content of the intergenic retrotransposon clusters in the region, but also in the content of the genes themselves (Fu and Dooner 2002; Brunner et al. 2005; Buckler et al. 2006; Messing and Dooner 2006). It is currently not known how widespread this structural disturbance might be in other plants or animals. But thanks to the advancements with the human genome, the phenomenon of genic polymorphism even between human individuals has been identified in the human genome, indicating that genic polymorphism is by far more extensive than ever thought (Tuzun et al. 2005). The most convincing evidence for structural genic differences within plant species comes from disease-resistance polymorphisms that are known to result from the presence or absence of particular genes (Tian et al. 2002; Scherrer et al. 2005). At present, the molecular basis of heterosis remains elusive and efforts are ongoing to understand this important phenomenon in molecular terms. Since heterosis is a genome-wide phenomenon, Tsaftaris (1995) proposed that it involves mechanisms of changing globally gene expression. Biochemical and molecular investigations on heterosis indicate that indeed quantitative variation of gene expression may be important in vigor manifestation (Tsaftaris and Polidoros 2000) and molecular models based on classical genetic hypotheses proposed by Birchler et al. (2003). Variation of gene expression could result by a shift in gene regulation in hybrids and support the significance of regulatory mechanisms involved in the quantitative modulation of gene expression. Alternatively, Fu and Dooner (2002) suggested that in different


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maize lines, the presence or absence of individual genes that are members of gene families should cause quantitative (additive) effects rather than qualitative ones. Lines lacking different additive genes would complement one another and show hybrid vigor, while lines lacking mostly the same genes would not complement and, consequently, would not exhibit heterosis. As mentioned, Auger et al. (2005) demonstrated that a substantial number of genes are not expressed at the midparent level in hybrids. In mouse hybrids, complementation of alleles produced spatial and temporal expression patterns beyond the range observed in the parents (Cowles et al. 2002; Guo et al. 2004). Similar results of nonadditive gene expression in a hybrid were found using enhancer trap lines in Drosophila (Hammerle and Ferru 2003), analyzing zein expression in maize hybrid endosperms (Song and Messing 2003), and examining protein levels in maize root tips (Romagnoli et al. 1990; Leonardi et al. 1991). Adams et al. (2003) also found unequal contributions from the two genomes in newly synthesized allopolyploids of cotton. A growing body of data therefore indicates that when diverse genomes are brought together in intra- or interspecies hybrids, gene expression patterns will not be predicted by simply averaging the expression of the parental lines, but are a consequence of regulatory interactions producing novel effects on genes under hybrid conditions. Thus, regulatory genes will exhibit some measure of dosage dependence, whereas genes that encode metabolic functions will be less likely to show a dosage effect (Birchler et al. 2001). Most of the studies just mentioned tested relatively small numbers of genes, but with the new microarray-based technology available, global estimations of genomewide expression patterns have become possible (Auger et al. 2005; Stupar and Springer 2006; Swanson-Wagner et al. 2006) with conflicting results mainly due to inherited technical and analytical difficulties of microarray analysis. As stressed by Lippman and Zamir (2007), besides the technical difficulties with these approaches, a fundamental problem is that they cannot associate novel expression patterns in hybrids with any heterotic phenotypes. One way to address this would be to include inbred parents and hybrids that have increased heterosis, in addition to hybrids with low heterosis. This comparison would reveal whether distinct patterns of gene expression or novel activity among genes belonging to specific functional categories were associated with highly heterotic hybrids. This approach with smaller number of genes was followed by Tsaftaris and his colleagues (Tsaftaris and Kafka 1998; Tsaftaris and Polidoros 2000; Kovacevic et al. 2005; Tani et al. 2005), by comparing a common parental line generating a highly heterotic and a nonheterotic hybrid when crossed to two other parental lines and by

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examining this set of three parental inbreds and the two hybrids in different conditions of growth, since it is well known that the conditions of growth affect the manifestation of heterosis (Tsaftaris et al. 2005). Epigenetic mechanisms, such as RNAi, chromatin modifications, and particularly DNA methylation could be considered as genome-wide general regulatory mechanisms that globally might affect the expression of many genes that are important for the manifestation of heterosis (Tsaftaris et al. 1997, 1999, 2005; Tsaftaris and Kafka 1998; Phillips 1999). By examining parental transcript accumulation in maize hybrids using allele-specific RT-PCR analysis, Guo et al. (2004) found that 11 of the 15 genes studied showed significant differences between parental alleles, which can be a result of transcriptional or posttranscriptional regulations, such as mRNA degradation or cis-acting sequence polymophisms, which can cause allelic difference in transcriptional regulation. In rice, patterns of 5mC in an elite rice hybrid and its parental lines were detected by a methylation-sensitive amplification polymorphism technique (MSAP) (Xiong et al. 1999). There were three classes of patterns of 5mC according to differences in degree of methylation between the hybrid and the parental lines: (1) no difference in methylation status between the parents and hybrid; (2) an increase of methylation level in the hybrid compared to the parents; and (3) a decrease in hybrid methylation level. In the first case, the banding patterns appeared to follow simple Mendelian inheritance. In both of the latter cases, the banding patterns were not inherited in a Mendelian fashion. As described earlier, epigenetic changes in new polyploids might lead to gene repression or to expression of genes that were repressed in the diploid that is derepression (Osborn et al. 2003). These changes could affect phenotypes directly if they involve genes encoding enzymes or structural proteins. Moreover, epigenetic modifications of homolog genes in polyploids in response to environmental cues and developmental programs could be used as a means for adaptive selection and domestication because the best combination of gene expression patterns may be selected (Wang et al. 2004). In maize, DNA methylation patterns differ between tissues and developmental stages and are influenced by growth conditions (Banks et al. 1988; Rossi et al. 1997; Cocciolone et al. 2001; Sturaro and Viotti 2001; Steward et al. 2002). Results from several studies in maize hybrids and their parental inbred lines carried out in our lab by measuring global methylation using HPLC and local methylation of random sequences using coupled restriction enzyme digestion–rapid amplification (CRED-RA) indicated that: (1) hybrids are, in general, less methylated than their parental inbreds; (2) heterotic hybrids are less methylated


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than related nonheterotic hybrids; (3) old, low-yielding inbreds are more methylated; (4) most modern inbreds, especially those selected for high and stable yield under low-density planting in the absence of competition (Fasoula and Fasoula 1997a, b), have lower percentage of methylation in comparison with old progenitor lines (Tsaftaris et al. 1997, 1999, 2001; Tsaftaris and Polidoros 2000; Tani et al. 2005). These data are in agreement with results from other studies revealing an impact of the environmental conditions on DNA methylation. Temperature changes, for instance, altered the activity and methylation state of the transposon Tam3 in Antirrhinum (Hashida et al. 2003). Moreover, when maize seedlings were exposed to cold stress, a genome-wide demethylation occurred in root tissues, suggesting that DNA methylation functions as a common switch of gene expression and that naturally induced changes in DNA methylation may result in heritable epigenetic modification of gene expression (Rossi et al. 1997). However, other studies revealed methylation increases in the DNA of pea root tips exposed to water deficit, which specifically, for the second cytosine of the CCGG target sequence assayed by the MSAP technique, accounted for about 40% of total sites investigated (Labra et al. 2002). Planting density also affected the methylation state of the Ac element in maize (Tsaftaris and Kafka 1998). Results obtained for three consecutive years revealed that demethylation (activation) of a methylated Ac element was significantly more frequent in plants grown under spread than dense planting. In subsequent research using Restriction Landmark Genomic Scanning (RLGS), which is a method capable of screening in a single assay the methylation status of more than 1,000 genes, we determined the polymorphism of methylation patterns in maize inbred lines and hybrids, and examined the effects of high-density growth on genome methylation in a maize hybrid selected for its stable performance (Kovacevic et al. 2005). Methylation levels varied up to 20% between different sib inbred lines. A slight increase of methylation was recorded in a stable hybrid growing under high-density stress (Kovacevic et al. 2005), which is in agreement with our previous reports indicating that hybrids are more resistant than inbreds to site-specific methylation changes under stress. This increase was due to both demethylation and new methylation of DNA fragments in the F1 hybrid, supporting the hypothesis that methylation can be released or repatterned when inbred lines are crossed to generate hybrids. These results show that part of the methylation inheritance is not Mendelian, which indicates that novel regulatory circuits may be formed in the hybrid to account for the quantitative variation in gene expression observed in many studies

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(Romagnoli et al. 1990; Leonardi et al. 1991; Damerval et al. 1994; Tsaftaris and Kafka 1998). It is possible that stability of performance is related with increased resistance to changes in methylation patterns under stress, which could explain the differences observed between inbreds and hybrids. Guo et al. (2004) claims that hybrids in general originated from different breeding programs for specific combining ability can surpass their inbred parents in different level, timing, or duration of gene expression, and in their response to developmental and environmental signals. Overall, these data point to a possible involvement of methylation in manifestation of hybrid vigor in conventional hybrids and encourage further study of the role of epigenetic inheritance in heterosis. Resistance of hybrids in induced genome methylation alterations under different stresses could be at the core of high and, perhaps more important, stable hybrid yield, especially if critical cytosine residues (e.g., regulatory genes, promoter regions of protein-coding genes) are preferably involved. Evidence has been provided that developmental changes of DNA methylation and chromatin structure at, or close to, the promoter region of a gene are responsible for epigenetic regulation of expression (Hoekenga et al. 2000). Other regions of the genome (e.g., heterochromatic DNA) remain highly methylated permanently, throughout plant growth. This could explain why slight changes of total methylation are concomitant with significant changes in gene expression. The need for robustness that stability of performance on one side, and developmental plasticity and environmental interaction on the other give, suggests that plants would intensively employ epigenetic regulatory strategies that can give heritable, often reversible, changes in their genetic information without immediate altering of their primary nucleotide sequence. In summary, results from these studies support the hypothesis that hybrids perform better than inbred lines as they resist alterations in methylation under stress. Epigenetic changes like DNA methylation are also factors affecting hybrid vigor. D. Changes in Plant Development and Architecture Yield and seed yield in particular is a plant characteristic that is difficult to study and understand at the genetic or molecular level. Genetic or molecular genetic methodologies have advanced our knowledge in many different aspects of plant biology, contributed marginally in the understanding of yield. For example, loss-of-function mutants are very informative for establishing the involvement of a gene in a specific biological process; however, in the case of yield, loss of function would


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be manifested as a deficiency in growth or a reduction in fruit and seed production, phenotypes that are obviously shared by many essential genes and not diagnostic of a specific role in yield. Gain of function could be manifested by a yield improvement. Yet the relative yield increase in such a case often will not surpass the resolution limit of standard greenhouse or growth chamber. Far more accurate than currently available phenotyping technologies are required for field evaluation of yield. Plant growth and the contribution of plant growth to harvestable yield are continuous processes, features that make the dissection of these processes quite difficult (Van Camp 2005). Despite these limitations, the successes enhancing yield, particularly seed yield, that have been achieved in recent years are encouraging. In a number of cases, many and diverse approaches that have been successful in enhancing yield involve genes controlling plant architecture and development. The great potential of developmentally important genes that could largely enhance seed yield is illustrated by the green revolution genes, which are based on the improvement of plant architecture; the introduction of semi-dwarf cultivars doubled the crop yield in wheat and rice due to increases in harvest index and the improved environment (by adding water and fertilizer among main inputs) (Khush 1999). The mutations that are responsible for the short stature in wheat and rice have now been identified (Sakamoto and Matsuoka 2004), and both relate to the plant hormone gibberellin. The role of gibberellin in plant dwarfism and the nature of the green revolution genes has recently been reviewed (Sakamoto et al. 2004). The same review also deals with the recent identification of two genes, MONOCULM1 (Li et al. 2003) and the rice ortholog of TEOSINTE BRANCHED1 (Takeda et al. 2003), which control the formation of tillers in rice. In spite of the fact that yield is considered a multifactorial trait, the integration of various developmental and physiological processes provides mounting evidence that yield can be increased by genetic modification of single genes affecting yield components, such as plant height and number of tillers. Similar results were obtained in tomato with genes controlling fruit yield (Fridman et al. 2004; Semel et al. 2006). Many other yield-enhancement genes remain elusive, thus feeding a long-standing debate as to whether such genes actually exist. In the past few years significant progress has been made toward the elucidation of plant genes that, as single variables, are able to improve yield. The debate therefore seems to evolve in favor of the proponents of yieldenhancement genes. Increasing yield through manipulations of single or few genes using conventional or modern biotechnology methods has been reviewed by

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Sakamoto and Matsuoka (2004), Beemster et al. (2005), and Van Camp (2005). Some representative examples that show the involvement of EIS mechanisms in controlling plant development and architecture, which influences yield components, will be discussed. The different sRNAs, and their described associated changes to chromatin structure controlling leaf shape, size, and senescence exert significant role in yield since leaves greatly affect yield (Ku et al. 2001; Lieman-Hurwitz et al. 2003). The stay green gene is perhaps one of the best examples of leaf senescence effect on seed yield (Ying et al. 2000; Valentinuz and Tollenaar 2004). Single genes such as TB1, RA and others that change dramatically the shoot architecture making maize look so different from its wild progenitor teosinte also have a significant affect on yield. The same is true for genes controlling inflorescence and flower formation since they control the sink capacity, another major component of yield (Giroux et al. 1996;Regierer et al. 2002; Smidansky et al. 2002, 2003). AP2, best known for its role in the regulation of flower meristem and flower organ identity, also plays an important role in determining seed size, seed weight, and the accumulation of seed oil and protein (Jofuku et al. 2005). It is well established that AP2 is epigenetically regulated by miRNA (Chen 2004). In addition, the gene apetala2 was found to regulate the activity of the stem cell niche in Arabidopsis shoot meristem (Wrschum et al. 2006). Results from conventional plant breeding underscore the fact that tillering, profligacy, number of florets, and number of seeds per plant are major determinants of yield and stability of yield under different conditions (Fasoula and Fasoula 1997a, b, 2000; Andrade et al. 1999; Vega et al. 2001; Tollenaar and Lee 2002; Fasoula and Tollenaar 2005). Van Camp (2005) introduced in rice a SYTa gene involved in chromatin remodeling (SanzMollinero 2004) and enhanced seed size and seed yield, suggesting the possibility of yield enhancement by changes in the architecture of the seed through EIS mechanisms. Plant development and developmental genetics was left out of plant breeding thinking in the past. But the data presented in this chapter provide ample support for the role of genetic and epigenetic mechanisms operating mainly in critical regulatory areas of the genome to generate adaptive phenotypic change. The examples described and many others, such as the TB1 and VGT1 loci (Clark et al. 2006; Salvi et al. 2002), provide strong evidence for the role of regulatory elements operating during development and providing instructions as to when, where, and for how long different tool genes will be used in the transforming a genotype to its phenotype. It is time to introduce plant development into plant breeding thinking.


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E. Manipulating Parental Imprinting There has been increased interest in recent years to better understand the role of epigenetic mechanisms that control parental imprinting in seed and particularly endosperm development in plants of high economic value. It had been shown from interploidy cross experiments that the parental genome ratio in Arabidopsis seeds is responsible for proper seed development, viability, and size (Scott et al. 1998). Altering the relative contribution of the maternal and paternal genome caused changes in the cell cycle and the differentiation of the endosperm. Even more striking was the observation that the size of the mature seed in Arabidopsis was dramatically affected when the parental genome dosage was altered (Scott et al. 1998). Seeds from 4x 2x (the seed parent is mentioned first) crosses were lighter than seeds produced by diploids, despite containing more genomes, whereas seeds from 2x 4x crosses are heavier than seeds produced by tetraploids. In general, seeds where the paternal contribution was doubled had accelerated mitosis, delayed endosperm cellularization, and larger-than-normal size when mature. Conversely, double dose of the maternal genome led to reduced endosperm mitosis and seeds smaller than the normal size. The balance of the two parental genomes is related to parental imprinting and thereby to the differential expression of the maternal and paternal genes. Similar interploidy crosses experiments in maize showed that disturbing the parental genome equilibrium resulted in abnormal endosperm development and seed abortion (Lin 1984). As in Arabidopsis, a 2:1 ratio of maternal to paternal genomes in the endosperm was shown to be crucial for normal maize kernel formation. In addition, like Arabidopsis, allele-specific expression was reported in maize for the genes ZmFIE1, NRP1 (no apical meristem-related protein), MEG1 (maternally expressed gene), and Mez1, one of the three E(z) maize homologs (Danilevskaya et al. 2003; Guo et al. 2003; GutierrezMarcos et al. 2004; Haun et al. 2007). All three genes are maternally active during early seed development. NRP1 and MEG1 are not expressed in the female gamete, which suggests that they are regulated by parental imprinting. There are two ZmFIE maize genes, which are homologous to Arabidopsis FIE. ZmFIE1 is maternally expressed at six days postfertilization, and maternal transcription continues throughout seed development in the endosperm. The second maize FIE gene (ZmFIE2), is expressed before fertilization and biallelically expressed in embryo, endosperm, and vegetative tissues (Danilevskaya et al. 2003). The function of FIE genes in maize is currently unknown as no fie mutants have been reported in maize so far. It is possible that FIE in

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maize also plays a role in endosperm development. Mez1 displays a monoallelic expression pattern of the maternal allele throughout endosperm development, which is probably due to differential methylation of the Mez1 promoter in the maternal and the paternal alleles (Haun et al., 2007). In Arabidopsis the FIS-class genes (MEA, FIE, and FIS2) are regulated by parental imprinting (see Section III.D). Only the maternal allele is expressed during the early stages of seed development; it plays an important role in regulating endosperm development (Gehring et al. 2004; Grossniklaus 2005). Thus, further research in the function and epigenetic control by parental imprinting of FIS genes in crops could lead to a better understanding of seed development and provide a new tool for crop improvement. This is particularly important for cereals whose endosperm is the main part of seed, making seed size or seed yields the major component of crop yield. Manipulation of parental imprinting could eventually lead to the development of larger seeds in cereals that are cultivated for their endosperm. Better understanding of parental imprinting could also lead to better understanding and exploitation of the much-desired phenomenon of apomixis (Savidan 2000; Koltunow and Grossniklaus 2003; Bicknell and Koltunow 2004; Spillane et al. 2004). Apomixis, defined as asexual reproduction through seeds, is the production of fertile plant progeny without double fertilization and thus identical to the mother plant (Savidan 2000). About 1,000 apomictic plant species are currently known that can reproduce asexually to form seeds that are clones of the seed parent. However, apomixis is not found frequently in crop plants and conventional breeding has not been successful, so far, in transferring apomixis. The phenotype of fis mutants resembles that of apomixis in apomictic plants as a large number of apomictic species exhibit autonomous endosperm proliferation (Spielman et al. 2001; Lohe and Chaudhury 2002; Gehring et al. 2004; Groosniklaus 2005). Mutations in MEA, FIE, or FIS2 all lead to initiation of endosperm development without fertilization, and in mea, fie, and fis2 mutants, there is no embryo development in the absence of fertilization. In these mutants, embryos develop abnormally after fertilization and are eventually aborted. Therefore, the FIS-class genes may play a crucial role in regulating endosperm and embryo development. Studying such genes in apomictic plants may reveal whether they might also be involved in apomixis. One of the major difficulties in making use of apomixis in cereal crops is the strict requirement for a 2:1 ratio of maternal-topaternal genomes in the endosperm tissue. Deviations from the 2:1 ratio in many plants and most cereal crops leads to seed abortion (Brichler


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1993), a phenomenon that is related to parental imprinting and the dosage sensitivity hypothesis described also in seed development. Indicative of this relation are data proving that maize, when used as the seed parent, can make fertile hybrids with the apomictic relative Tripsacum dactyloides. The highest fertility results when using diploid maize and tetraploid Tripsacum (Kindiger and Beckett 1992). A study of the functions of FIS homologs in apomictic plants could provide new insights into the process of apomixis. Moreover, manipulation of the genomic imprinting mechanisms associated with seed development may solve the current problems of generating apomixis in economically important crop plants (Grossniklaus 2005).

F. Improving Plant Resistance to Viruses and Other Parasites Suppression of PTGS is a widespread property and probably a necessary adaptation of RNA and DNA viruses of plants (Voinnet et al. 1999). This is perhaps the most convincing argument that PTGS acts as an antiviral mechanism in plants. Transgenic resistance against viruses based on the PTGS mechanism easily fulfills the current high demands for biosafety. Indeed, PTGS-based resistance does not involve the transgenic production of functional viral genes or proteins, nor does it lead to the presence of transgenic RNA (Goldbach et al. 2003). An important drawback of this approach in biotechnology is the high level of sequence specificity required for RNA degradation. Thus, viruses that contain more than 10% nucleotide discrepancy cannot be subjected to RNA degradation. Another drawback until recently was the size of the transgene. It should be more than 300 base pairs, in order to trigger efficient RNA silencing. There is experimental evidence using genetically engineered miRNA that a 21-nucleotide sequence was sufficient to trigger complete silencing in transgenic plants (Voinnet 2002). However, the major technical limitation for technologies based on RNA silencing is that many important plant crop species are difficult or impossible to transform. Moreover, public concerns over the potential ecological impact of virus-resistant transgenic plants have significantly limited their use so far (Fermin et al. 2004). Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antiviral defense mechanism. Haque and his colleagues (2007) have produced transgenic Nicotiana benthamiana plants that expressed the coat protein gene (CP) of sweet potato feathery mottle virus, and they have shown that RNA silencing spreads in the 5’–3’ direction, but not in the 3’–5’ direction, along the transgene mRNA.

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Until now, most of the applications of VIGS have been in the plant virologists’ model plant, N. benthamiana. Nevertheless, new vectors are used with the ability to support VIGS in Arabidopsis (Dalmay et al. 2000) and tomato (Liu Y. et al. 2002). VIGS technology was used very recently for characterization of genes associated with local and systemic resistance in barley and other cereals (Hein et al. 2005). The main concern for the application of VIGS is the use of genetically modified plant pathogens; thus, all experiments must be carried out in strict control. The development of RNA silencing as an effective and environmentally safer approach is a long-term goal to control plant virus diseases. Although considerable progress has been made in understanding this mechanism, much still remains to be discovered (Tenllado et al. 2004). G. The Use of RNAi for Crop Improvement Improving the nutritional value of plants can be achieved either by classical breeding based on selection of the natural or induced genetic variation or by generating transgenic plants that carry the desired agronomic traits. Transgenic technologies have advantages over classical breeding because the genotypic alterations, which will lead to superior phenotypes, can be specifically designed and introduced to the plant in a highly regulated manner as to abundance and spatial expression. Even though traditional gene knockout approaches have been used in the past to suppress expression of certain genes in plants, RNAi technology provides a way to reduce the product of a gene in a much more efficient and regulated fashion. Most important, RNAi is extremely useful when families of numerous copies of gene homologs need to be down-regulated rather than a single locus. RNAi technology has been widely used in the past as a tool for analyzing gene function in plants. A common way to achieve RNA interference is to use a transgene that produces hairpin RNA (hp RNA) with a ds RNA region (Waterhouse and Helliwell 2003). Double-stranded RNA had been demonstrated in the past to be a good method to trigger gene silencing in plants. However, RNAi that has been induced by hairpin RNA has proven to be much more efficient than conventional gene silencing methods, which relied on introducing antisense RNA into the plant (Chuang and Meyerowitz 2000). In a hairpin RNA-producing vector, the target gene is cloned as an inverted repeat that contains a spacer of unrelated sequence. This is important for the stability of the construct in E. coli after cloning. A strong promoter, such as the 35S CaMV promoter for dicots or the maize ubiquitin 1 promoter for monocots, drives the inverted repeat. The efficiency of


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silencing increases dramatically (almost 100% of the transformed plants show gene silencing) when the spacer fragment is an intron (Helliwell and Waterhouse 2003). For genome-wide analysis of gene function, where the silencing of large numbers of genes of unknown sequences is desired, a novel method has been developed named SHUTR (Silencing by Heterologous 3’-UTRs) (Brummell et al. 2003), which relies on the construction of a vector containing an inverted repeat of the 3’-untranslated region (3’UTR) of an heterologous gene and a strong promoter. Gene sequences are incorporated 5’ upstream of the 3’UTR of the heterologous gene as to provide a dsRNA region at the 3’end of the transcript. This method was tried out using the the 3’-UTR region of the nopaline synthase (nos) gene from Agrobacterium tumefanciens, and it was shown to induce highly efficient silencing of a polygalacturonase transgene in tomato and two transcription factor genes in Arabidopsis (Brummell et al. 2003). Constructs containing tissue-specific promoters have also been used successfully in guiding RNA silencing. For example seed-specific genes have been effectively silenced using the napin and lectin promoters (Smith et al. 2000; Stoutjesdijk et al. 2002). Similarly, very efficient organ-specific silencing has been achieved both in Arabidopsis and Brassica using MADS-box gene promoters to transform petals into sepaloid structures (Byzova et al. 2004). A long list of genes, ranging from transcription factors to biosynthetic enzyme encoding genes, has been knocked down using hairpin RNA technology in order for their function to be analyzed (www.pi.csiro.au/RNAi/different gene_eg.htm). Examples include the Bes1 gene, which codes for a transcription factor involved in regulation of brassinosteroid gene expression in Arabidopsis (Yin et al. 2005), and the male fertility gene Ms45 and several other anther-expressed genes whose transcriptional silencing by RNAi resulted in male sterile plants in maize (Cigan et al. 2005). RNAi is particularly useful for silencing of genes in polyploids or genes that belong to multigene families. Traditional gene knockout and conventional breeding cannot be used for the repression of a multigene family by the accumulation of mutations for each member of the family, particularly when the members are tightly linked. For example, in the naturally occurring low-glutelin RNAi mutant in rice LCG-1, the glutelin family consists of at least eight members, five of which are clustered in a particular chromosomal location. However, reduction of the levels of glutelin was achieved from a single RNAi locus and not from mutations for each of the members of the glutelin family (Kusaba et al. 2003; Kusaba 2004). RNAi technology has also been used as a tool for crop improvment. The primary proteins of the maize endosperm, the so- called zein pro-

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teins, are classified in four subfamilies, the a-b-g-d zeins (Ueda and Messing 1993). The a-zeins account for 70% of the total zein content, and this nutritional protein class is responsible for the overall low content of seed lysine, a significant amino acid in human and animal diet. The a-zeins belong to a multigene family whose members are clustered in several chromosomal sites (Hunter et al. 2002). In the past, the opaque-2 (o2) maize mutant had been isolated, which displays significantly reduced levels of the 22kd a-zein protein in the endosperm (Mertz et al. 1964; Schmidt et al. 1987). Analysis of this mutant showed that the mutation was the result of differential inhibition of the transcription of the 22kd a-zein transcript (Kodrzycki et al. 1989). Unfortunately, the O2 gene, which encodes a basic leucine zipper transcriptional factor, controls the expression of not only the 22kd a-zein protein, thus the o2 mutant although high in lysine has undesirable agronomic traits such as low seed quality and yield (Tang and Galili 2004). Segal et al. (2003) described the production of dominant O2 maize variant in which selective repression of zein protein synthesis occurred independently of the O2 gene, which was achieved through the use of RNAi constructs derived from the sequence of the 22kd a-zein. In this mutant, the 22kd azein proteins were specifically reduced, whereas accumulation of the other zein proteins remained unaffected. Most important, the altered trait was stably inherited and the mutant maize plants produced normal seeds with high levels of lysine-rich proteins. Other efforts to increase the lysine content in plants focused on the gene that codes for dihydrodipicolinate synthase (DHPS), the first enzyme specifically committed to lysine biosynthesis. Lysine synthesis is strongly regulated by lysine-mediated feedback inhibition of the activity of DHPS but also by genes regulating lysine catabolism, Lysketoglutarate reductase (LKR), and saccharopine dehydrogenase (SDH) (Galili 1995; Galili et al. 2001). Studies with an Arabidopsis AtLKR/ SDH knockout mutant, which was designed to express a bacterial lysine-insensitive DHPS specifically in seeds, showed that the mutant accumulated high levels of lysine in the seeds but the growth of seedlings from these seeds was dramatically decreased (Zhu and Galili 2003). However, in a subsequent study, coexpression of the bacterial DHPS and a AtLKR/SDH RNAi construct both under the control of a seed-specific promoter led to increased lysine content in seed and improved seedling growth (Zhu and Galili 2004), which again supports the advantages of RNAi-mediated technology for altering gene expression in plants. RNAi technology has been successfully applied to reduce caffeine content in Coffea arabica. Decaffeinated coffee is of major interest for


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the coffee industry because the stimulatory effects of caffeine cause health problems to sensitive individuals (high blood pressure, palpitations, insomnia). Ogita et al. (2003) constructed transgenic coffee plants where the expression of one of the caffeine-synthesizing enzymes was specifically repressed by an RNAi-based technique, and caffeine content was reduced by 50 to 70%. Another application of RNAi technology has been the modification of the fatty acid composition of cotton oil. Two key enzymes in the metabolism of fatty acids are the stearoyl-acyl-carrier protein D9-desaturase and oleoyl-phosphatidylcholine v6-desaturase (Liu Q. et al. 2002). RNAi mediated by a hairpin RNA was used in cotton in order to reduce the expression of the genes encoding these two enzymes. The expression of the two desaturases was successfully down-regulated, and the resulting cotton plants produced high levels of oleic and stearic cottonseed oils, which are essential fatty acids for maintaining a healthy heart condition. In another study RNAi technology was utilized to increase the carotenoid and flavonoid content in tomato (Davaluri et al. 2005). Tomato is the principal dietary source of lycopene, b-carotene, and flavonoids, all of which are phytochemicals that benefit human health. Lycopene and flavonoids are powerful antioxidants that have been associated with reduced risk of heart disease and certain types of cancer. b-carotene is the precursor of vitamin A. Past attempts to regulate genes in the biosynthetic pathways of carotenoids and flavonoids resulted in the increase of the content of either one phytochemical but never of both. The authors demonstrated that suppression of a photomorphogenesis regulatory gene, DET-1, using RNAi techniques resulted in tomato fruits containing higher levels of both carotenoids and flavonoids while other fruit quality indicators remained unaltered. RNAi silencing operates as an ancient self-defense mechanism against foreign invaders, such as viruses and transposons, present in a broad range of eukaryotic organisms. In plants, RNAi serves as an antiviral system. Successful viral infection requires suppression by the virus of gene silencing that was induced by the host plant. During viral invasion, most plant RNA viruses form a dsRNA intermediate, which is cleaved to generate siRNAs that target viral RNA. In order to protect itself from degradation, the virus has evolved RNAi inhibitors, which allow the virus to overcome plant RNAi action and infect the plant successfully. Recently it was demonstrated that a viral protein of tombusviruses (p19) inhibits RNA silencing in vivo by strongly binding siRNA in virus-infected cells (Lakatos et al. 2004). When RNAi silencing was suppressed by p19 in tobacco plants, the expression of various

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transgenes in transient expression assay was increased 50-fold (Voinnet et al. 2003). Thus, RNAi silencing suppression could be potentially used as an effective tool for the overproduction of desired proteins in plants. Kru¨tzfeldt et al. (2005) have achieved the chemical engineering of oligonucleotides, termed antagomirs, which can efficiently silence miRNAs in vivo. Antagomirs are single-stranded RNA analogs complementary to miRNAs, which have been chemically modified for stability and cholesterol-conjugated for delivery. Intravenous administration of antagomirs against certain miRNAs resulted in a significant decrease of the respective miRNAs in mice. Because miRNAs play a crucial role in the regulation of gene expression in human disease, this could prove to be a therapeutic technology with enormous potential. Such technology might be applied in plants. For example, it may be possible to deliver such antagomir constructs in the plant liquid-stage endosperm (a multinucleate structure that lacks a cell membrane) in order to manipulate seed development. H. Securing Stability of Transgenes Although it is not yet possible to completely rule out transgene silencing, it can be avoided by following several strategies. First, it is advisable to screen transgenic lines for the insertion of single-copy transgenes into hypomethylated genomic regions. It is wise therefore to exclude transgenic lines that have transgene rearrangements and are candidates for silencing (Depicker et al. 2005). This can be accomplished by analyzing the genomic regions adjacent to the transgene integration site and discarding plants with transgenes that are inserted into hypermethylated genomic regions (Meyers 1998). Moreover, because plant genomes are mosaics of isochores, the transgene should match the isochore composition of the host organism. In order to minimize the impact of the sequence composition of the transgene integration site, matrix attachment regions (MARs), which are DNA elements that bind specifically to the nuclear matrix in vitro (Allen et al. 2000), can be positioned on either side of a transgene and may help to reduce variance in expression levels (Vain et al. 1999; Halweg et al. 2005). Another approach is the integration of the transgene into a fixed chromosomal site-by-site–specific integration (Albert et al. 1995). It is important to exclude any plasmid or phage vector in the transgene constructs as they might be recognized as foreign and serve as targets for TGS or PTGS (Iyer et al. 2000). Other factors that should be taken into account are the transformation vectors and the choice of transformation


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technique. Multiple uses of the same promoters should be avoided, as they can be subjected to TGS (Vaucheret 1993). Multiple uses of identical 3’ regions for the generation of a transgenic plant also should be avoided as they can also be targets for TGS or PTGS (De Neve et al. 1999). Silencing has probably evolved as a defense mechanism against parasites and viruses and not as a mechanism to inactivate transgene expression. Therefore, it is possible that a methodical study of these mechanisms will help us to overcome silencing. Plant viruses have evolved their own counterdefenses by virus-encoded silencing suppressors, showing that silence can be broken (Qu and Morris 2005).

VI. OUTLOOK Frequently, when unexpected phenomena are observed, they first are ignored, then timidly explored, and only later published and debated with firm conviction in a more coherent framework. The third phase of research associated with epigenetic mechanisms operating in plant development and evolution provides new insights into such an important matter. The discovery of sRNAs has introduced a new paradigm of gene function and regulation and uncovered the once-hidden role of function and regulation of non–protein-coding areas of genomic DNA. In addition, these small RNA species provided a link to interconnect the three EIS (DNA methylation, histone modifications, and RNAi) into an interrelated triangle of interactive mechanisms. Perhaps the described stability of the peloria epiallele for more than 250 years is indicative of the similarities in the stability of some epigenetic and genetic changes (Cubas et al. 1999). The example of lowglutelin variant in rice due to RNA silencing of multiple members of the glutelin gene family and its stability in this case for 25 years (Kusaba et al. 2003) is also indicative for the role of EIS and siRNA in particular in creating useful variation for selection. The genetic alternative for simultaneous knocking out a gene family would require the rather unlikely concurrent mutation of all the individual members of the family (Kusaba 2004). The same holds true for genes with multiple copies in polyploid crops (Lawrence and Pikaard 2003). The low-glutelin rice variant and its study indicates the opportunities offered by the existence of such epigenotypes, which have been selected in these breeding efforts, as a valuable source of material for molecular biologists studing EIS. Epigenetic mechanisms are currently proven as major mediators of a genomic effect of the environment. What is emerging, with strong

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experimental support, is that environmental changes in addition to the induction of the known physiological response on behalf of the sessile plant are playing a major restructuring role on plant genomes. These data are revolutionary with implications not only in plant breeding but also in plant biology and evolution in general. Developmental genetic and epigenetic variation could be of particular importance for plants. Most structures of a mature plant are formed after embryogenesis by the reiterative action of meristems. By delaying the bulk of growth and differentiation until after embryogenesis, the sessile plants can fashion their form to the environment, allowing development to substitute for behavior to some extent. The structures produced by the apical meristems change through developmental time. The varying epigenetic stages of TEs in somatic cells, for example, is indicative of the extent of this somatic cell variation, of the power of EIS in generating variation, and of the possibilities that emerge. The pattern of plant development allows substantive opportunities for embryogenic cells to vary, and of course such variant cells can subsequently give rise to varying gametes. Epigenetic and genetic systems are involved in the development of individual plants; thus they could be a source of significant and useful variation and could play a vital role in plant breeding. During a large part of the twentieth century, plant breeding was basically considered an artificial selection scheme applied to a genetically segregating material. Darwin’s theory of natural selection, introduced in 1859, proposed a mechanism of evolution at the organism level. As Darwin had no acceptable theory of genetics, natural selection foundered and was bitterly opposed in the early twentieth century (Cronk 2001). However, the rediscovery of Mendelian rules of inheritance by Correns, De Vries, and von Tschermak allowed natural selection to shift to the gene level. R.A. Fisher brilliantly reestablished natural selection as the dominant evolutionary mechanism by showing mathematically its potency in acting on changing allele frequencies in a population. The paradigm of natural selection acting at the gene level led to the ‘‘modern synthesis’’ and has lasted to the present day. The shift from Darwin’s organism and species selection, to the frequencies of alleles in populations in the neo-Darwinian theory, transferred the emphasis from individuals to populations in both evolution by natural selection and plant breeding by artificial selection. Thus development and developmental genetics were left out of this thinking (Cronk 2001; Robert 2004). However, the recent explosion of the genomic revolution and the availability and comparison of sequences have shifted the emphasis from the neo-Darwinian alleles to single nucleotide


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differences and other changes emphasizing the significance of the effect of nucleotide changes in the cis-regulatory elements of transcription factors and other regulatory genes. Such changes, leading to altered specificity of DNA-protein binding chromatin modification, to production or new targets of sRNAs, may in turn lead to heterotopic and heterochronic changes in the expression domains of key regulators. Data presented in this review provide ample support for the role of genetic and epigenetic mechanisms operating in these critical areas of the genome driving adaptive changes in the phenotype. As the coding sequences of developmental genes are highly conserved, they are unlikely to be sources of organismal diversity. Studies in maize (Doebley et al. 1997; Wang et al. 2005), rice (Spielmeyer et al. 2002; Li et al. 2003; Li et al. 2006), and tomato (Cong et al. 2002; Liu J. et al. 2002; Van der Knaap et al. 2004) indicate that genetic and epigenetic polymorphisms in the controlling areas operating during development provide instructions as to when, where, and how the ‘‘tool’’ genes will be used, which should be the stimulus not only for further work on similar developmental and genetic studies in other crops but also for the making of individual plant development the target of artificial selection breeding (Fasoula and Fasoula 2002). The newly established field of Evo-Devo (Carroll 2005) that brought development to evolutionary natural selection should now be extended to studies of plant domestication (DomestDevo) and plant breeding (Bred-Devo). VII. ACKNOWLEDGMENTS The authors thank Drs. A. Argyriou and T. Farmaki (INA-CERTH) for their critical review of this manuscript and the three anonymous reviewers as well as the series editor, Dr. Jules Janick, for their contibutions in substantially improving the text. We apologize to our colleagues whose results could not be included and discussed in the review for space reasons. Work in our laboratories in Greece was supported by the Ministries of Development and Education. VIII. LITERATURE CITED Aasland, R., T.J. Gibson, and A.F. Stewart. 1995. The PHD finger: Implications for chromatin-mediated transcriptional regulation. Trends Biochem. Sci. 20:56–59. Achwal, C.W., P. Ganguly, and H.S. Chandra. 1984. Estimation of the amount of 5-methylcytosine in Drosophyla melanogaster DNA by amplified ELISA and photoacustic spectroscopy. EMBO J. 3:263–266.

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3 Enhancing Crop Gene Pools with Beneficial Traits Using Wild Relatives Sangam L. Dwivedi and Hari D. Upadhyaya International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) Patancheru PO 502324 A.P. India H. Thomas Stalker North Carolina State University, PO Box 7620 Raleigh, North Carolina 27695 USA Matthew W. Blair Centro Internacional De Agricultura Tropical (CIAT) A.A. 6713 Cali, Colombia David J. Bertioli Universidade Catolica De Brası´lia Pos Graduacao Campus II Sgan 916, DF CEP 70.790-160 Brası´lia, Brazil Stephan Nielen PBI, EMBRAPA Recursos Geneticos E Biotecnologia Parque Estaçaõ Ecologica Norte Final AV W5 Norte Brası´lia-DF 70770-900, Brazil Rodomiro Ortiz Director, Resource Mobilization CIMMYT Apdo. Postal 6-641 06600 Mexico, D.F., Mexico I. INTRODUCTION II. GENETIC RESOURCES FROM WILD RELATIVES III. BARRIERS AND APPROACHES TO INTERSPECIFIC GENE TRANSFER A. Prefertilization Barriers to Hybridization B. Postfertilization Barriers to Hybridization IV. BENEFICIAL TRAITS FROM WILD RELATIVES CONTRIBUTING TO CROP GENE POOLS Plant Breeding Reviews, Volume 30. Edited by Jules Janick Copyright © 2008 John Wiley & Sons, Inc.

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VI. VII. VIII. IX.

S. L. DWIVEDI A. Resistance to Biotic Stresses B. Tolerance to Abiotic Stresses C. Cytoplasmic Male Sterility D. Yield, Nutritional Quality, and Adaptation Traits BIOTECHNOLOGICAL APPROACHES TO ENHANCE UTILIZATION OF WILD RELATIVES IN CROP IMPROVEMENT A. Chromosome-Mediated Alien Gene Transfer B. Using Cloned Genes from Wild Relatives to Produce Transgenics C. Developing Transgenics with Large-Scale Transfer of Exogenous DNA from Distant Relatives D. Marker-Aided Introgression E. Resynthesizing the Crop Progenitors to Capture Variability Lost During Crop Evolution and Domestication F. Developing Exotic Genetic Libraries OUTCOMES OF WILD RELATIVES USE IN GENETIC ENHANCEMENT OF CROPS FUTURE OUTLOOK ACKNOWLEDGMENTS LITERATURE CITED

ABBREVIATIONS AFLP

Amplified fragment length polymorphism

BC

Backcross

BLAST

Basic local alignment search tool

cDNA

Complementary deoxyribonucleic acid

CGIAR CIAT

Consultative Group on International Agricultural Research Centro Internacional de Agricultura Tropical

CIMMYT

International Maize and Wheat Improvement Center

CMS

Cytoplasmic male sterility

CSSLs

Chromosomal segment substitution lines

DNA

Deoxyribonucleic aicd

EMBRAPA

Empresa Brasileira de Pesquisa Agropecuaria

EBN

Endosperm balance number

FAO GCP

Food and Agriculture Organization Generation Challenge Programme

GP

Gene pool

ICRISAT

International Crops Research Institute for the Semi-Arid Tropics

MAAL

Monosomic alien addition lines

PGR

Plant genetic resources

QTL

Quantitative trait loci

rDNA

Recombinant deoxyribonucleic aicd

RFLP RILs

Restriction fragment length polymorphism Recombinant inbred lines

SNP

Single-nucleotide polymorphism

SSRs

Simple sequence repeats

3. ENHANCING CROP GENE POOLS

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I. INTRODUCTION The Russian geneticist and plant explorer Nikolai Ivanovich Vavilov is known for his pioneering research on the phyto-geographic basis of plant breeding and the world centers of crop origin, the laws of homologous series in the inheritance of variability, and the study of immunity of plants from infectious diseases (Vavilov 1951). Vavilov also understood the role of wild species in the genetic enhancement of crops, as shown by his reports on the use of plant genetic resources in crop breeding. For example, after analyzing the interspecific constitution of bread wheat, Vavilov (1951) referred to the potential use of genera such as Aegilops, Secale, Haynodia, and Agropyrum in wheat improvement. A crop wild relative is a wild plant taxon that has an indirect use derived from its relatively close genetic relationship to a crop (Maxted et al. 2006). An understanding of the taxonomic and evolutionary relationships between cultigens and their wild relatives is prerequisite for the exploitation of wild species in crop improvement (Hawkes 1977). Harlan and de Wet (1971) proposed the gene pool (GP) concept to define relationships among crop plants and related taxa that could be useful to plant breeders and geneticists wishing to make use of these resources in crop improvement. They assigned various genetic resources to the three GPs based on ease of hybridization: primary (GP-1), secondary (GP-2), and tertiary gene pool (GP-3). GP-1 includes germplasm that can be easily hybridized, for example, cultivars, land races, and elite germplasm. GP-2 consists of species that can be crossed with GP-1 without major problems of F1 infertility. Gene transfer from species in GP-2 to cultivars is possible, but may be difficult to accomplish. GP-3 is the outer limit of potential genetic resources associated with cultivated species and hybrids between GP-1 and GP-3 species almost always require in vitro techniques for F1 rescue due to lethality and physiological abnormality of complete sterility. In these cases, bridging species are often needed to effect gene transfer from the tertiary gene pool to the cultivated crop, but this is usually a laborious process, and selection for the desired gene can be very difficult. It is important to note that despite the problems of inter-gene pool hybridization, the need for these types of crosses is great, given that the cultivated gene pools of many food crops are narrow and genetic improvement is stifled by a lack of variability. Therefore, there is a need to broaden the basis of crop gene pools in order to enhance their genetic potentials and meet the growing food demands of the developing world. The use of plant genetic resources (PGR) in crop improvement is one of the most sustainable methods to conserve valuable genetic resources

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for the future and simultaneously to increase agricultural production and food security. Haussmann et al. (2004) summarized issues related to use of PGR in crop improvement that include information resources on the Internet; documentation and evaluation of PGR; access to PGR, equitable sharing of profits, and material transfer agreements; impediments to the use of PGR in crop improvement; classical methods of using PGR in crop improvement; use of landraces in breeding for specific adaptation to stress environments; utility of molecular markers and genomic approaches for using PGR in crop improvement; and gene transfer. The greatest economic impacts of wild relatives in crop improvement to date have been in increasing disease and pest resistances in several crops (Harlan 1976; Stalker 1980; Goodman et al. 1987; Lenne´ and Wood 1991; Hoisington et al. 1999). The increases in annual crop yields from crop improvement since 1945 are worth approximately US $60 billion per year (USBC 1995). Assuming that contribution of genetic resources is responsible for 30% of yield increase and that much of this is from wide crosses with wild accessions, the introduction of new genes from wild relatives contributes per year approximately $20 billion toward increased crop yields in the United States and $115 billion worldwide (Pimentel et al. 1997). Wild relatives have also shown very high levels of resistance or tolerance to abiotic and biotic stresses in contrast to cultivated species. However, gene introgression from wild relatives to cultivars has been difficult due to cross incompatibility, hybrid sterility, and linkage drag (Stebbins 1958; Zeven et al. 1983). This review documents the contributions of wild relatives to broadening the gene pools emphasizing selected cereal (wheat, rice, maize, barley, oat, sorghum, and pearl millet) and legume (soybean, common bean, chickpea, pigeonpea, cowpea, and peanut [groundnut]) crops found in the Food and Agriculture Organization (FAO)–mandated collections of the Consultative Group on International Agricultural Research (CGIAR). The institutes of the CGIAR have the responsibility to collect, preserve, characterize, evaluate, and document the genetic resources of the cultivated and wild relatives of these crops. Additionally, they have genetic improvement programs for their utilization. These gene banks have provided genetic resources to researchers around the world for use in crop improvement. Among the cereal and legume accessions preserved in the CGIAR collections, 17.4% (65,200) of the total collection (375,200) belong to wild relatives. Although this review concentrates on cereals and legumes, additional examples will be given from other crop species where they may serve as models for the use of germplasm for enhancing productivity to meet growing food demands.


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II. GENETIC RESOURCES FROM WILD RELATIVES Novel germplasm can be used in breeding programs to create new crops and new uses for existing crops, to meet breeding objectives for sustainability of crop production, and to ensure the entire world’s people benefit from crop improvement through enhanced food security and quality (Heslop-Harrison 2002). The key to successful crop improvement is a continued supply of genetic variability and beneficial traits contained in this diversity. Most plant breeders/geneticists exploit variability only from the primary gene pool of a specific crop. However, variability for some traits of interest exists only in the secondary or tertiary gene pools. Novel germplasm that is available in species not currently used commercially can be expoited by domesticating them for direct use or introducing their genes into existing cultivars through conventional breeding, by use of in vitro technologies, or through use of genetic transformation. Molecular biology–based technologies such as marker-assisted selection now can assist in identifying and tracking allelic variants associated with beneficial traits and identifying desirable recombinant plants with the markers of interest. Alternately genes can be cloned from distantly related species or genera and introduced into existing crops. Indeed, gene technologies are now available to exploit species in the tertiary gene pool and even more distantly related taxa. As a result, the concept of gene pools is not static and shifts as new technologies become available to affect gene transfer. For example, a dominant gene for resistance to bacterial blight was cloned from a distantly related wild relative of rice (Song et al. 1995) and transferred into transgenic versions of commercial cultivars of rice, which then showed the same level of resistance as observed in the wild relative from which the gene was cloned (Zhai et al. 2001). Obviously, the potential for exploiting novel germplasm has greatly increased and will increase the potential of plant breeders to continue improving crops with high yields, better adaptation, and enhanced quality. As part of this new paradigm for using genetic resources, the Generation Challenge Programme (GCP) (www.generationcp.org) was established with the objective of unlocking the genetic diversity existing in a wide spectrum of germplasm collections in order to solve some of the world’s most serious agricultural and food security issues. This program supports global competitively funded projects to (1) exploit natural genetic variation by developing genomic resources and introgression lines for four AA genome rice relatives; (2) enhance genetic diversity in wild relatives of peanut with genomic and genetic tools; and (3) discover genes in wild relatives of rice for tolerance of saline and

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phosphorus-deficient soils to enhance and sustain productivity on marginal lands. It is expected that the novel germplasm and associated technologies developed in these projects will be useful to applied breeding for improved adaptation of these crops to conditions in developingcountry agriculture. Other examples of the exploitation of genetic resources from the secondary and tertiary gene pools (or even more distantly related species) and generation of novel cultivated germplasm exist for wheat, barley, and related small grains: Hordeum bulbosum, the only species belonging to secondary gene pool (Bothmer et al. 1995), contributing novel alleles for resistance to several leaf diseases to cultivated barley (H. vulgare) (Pickering and Johnston 2005); synthetic wheat with demonstrated potential to enhance grain yield and/or yield components (de Blanco et al. 2001); tritipyrum with high levels of salt tolerance (King et al. 1997b); and hexaploid tritordeum with high water-use and nitrogen-utilization efficiency and grain characteristics like bread wheat (Martin et al. 1999). However, brittle rachis in the hexaploid tritordeum is a serious limitation to threshing by mechanical harvester. To establish tritordeum as a new commercial cereal, Prieto et al. (2006) developed double disomic substitution lines, 2D(2Hch) and 3Hv(3Hch), that possess nonbrittle rachis, tenacious glumes, and compact spikes (a character highly desirable for the improvement of tritordeum threshability), and these lines can be used for introgression to wheat or tritordeum background. Similarly in maize, exploitation of wild relative Tripsacum has the potential for fixing hybrid vigor through apomixes found in this species and contributing abiotic and biotic stress resistances not present in maize (Hoisington et al. 1999).

III. BARRIERS AND APPROACHES TO INTERSPECIFIC GENE TRANSFER Barriers to interspecific hybridization are common among species. They can be caused by spatial or phonological differences in flowering, sexual incompatibility, and cytological abnormalities, or because they are genetically too distant to be compatible. Because plants that are being crossed are typically maintained in nurseries, sexual barriers are of most concern to the scientist. Reproductive barriers are differentiated by the time they occur, typically at pre- and postfertilization. Accessions within a species can be highly variable in their compatibility, and there are times when only a few accessions or cultivars within a species will serve as a parent in interspecific crosses, and often in only a unidirectional manner (Baum et al. 1992). Thus, it is important to


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attempt difficult crosses with a range of germplasm and in a range of environments. For example, hybrids with cultivated peanut and the related species Arachis glabrata must be made with the wild relative as a female parent and have only been successful in crosses made under natural sunlight to those in greenhouse. Interspecific crosses can also be enhanced or prevented by single genes (Fedak and Jui 1982; Laurie and Bennett 1989), and their effect on plant breeding is reviewed by Frankel and Galun (1977). It is beyond the scope of this review to provide in-depth discussion on the reproductive barriers and the technologies for circumventing these barriers that are published elsewhere (Liedl and Anderson 1993). A number of techniques have been used to either circumvent or overcome barriers to hybridization. A summary of these techniques is presented in two sections, depending on the timing of the barriers to hybridization, either before or after fertilization. A. Prefertilization Barriers to Hybridization Incompatability between the male and female parents can first occur in the stigma or stylar tissues before fertilization occurs. To overcome these incompatibilities, techniques can be employed, such as the use of (1) compatible pollen that is capable of germinating but with inactivated nuclei mixed with the pollen desired for crossing, or (2) mixtures of compatible and incompatible pollen with identification of hybrids versus selfs in the subsequent generation (Brown and Adiwilaga 1991). Other methods of overcoming stigma/style inhibitions include cutting part or all of the style tissue, thus removing the stigma and many of the inhibition factors that may be present (Wietsma et al. 1994), or heating styles before pollination to inactivate inhibition factors (Ascher and Peloquin 1968). Alternatively, chemical applications of gibberellins or auxins and cytokinins to reproductive tissues can result in faster pollen tube growth and fertilization or enhanced fruit development (Dionne 1958; Larter and Chaubey 1965; Kruse 1974; Alonso and Kimber 1980; Sastri and Moss 1982). Immunosuppressants applied to floral tissues have also been used to enhance hybridization in cereals (Baker et al. 1975; Mujeeb-Kazi and Rodriguez 1980) and in legumes (Baker et al. 1975). B. Postfertilization Barriers to Hybridization Causes of postmating barriers include polyploidy differences between species, chromosome loss or rearrangements, cytoplasmic incompatibilities, physiological seed dormancy, apomixes, and hybrid breakdown resulting from lethals or low plant vigor in the first or subsequent

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generations. At least as important as producing hybrids is genetic recombination between the species to transfer the desired genes into the cultivated species genome. This is a straightforward event in some interspecies crosses, especially when the species are at the same ploidy level and have common genomes, but it can be an extremely rare event in other cross combinations. In other cases, apomixes but not recombination occurs after generation of the interspecific hybrid. When apomixes are present in one of two species to be crossed, interspecific crosses are made with the apomictic species as the male parent to avoid this trait. A more difficult circumstance occurs when it becomes necessary to cross two apomictic species because sexual hybridization is not possible. Although a limited number of sexual progenies develop even in apomictic species, large numbers of recombinants are usually required for intragenomic chromosome pairing to occur or to break up linkage groups associated with lethality. After fertilization and gamete fusion and usually after a period of embryo growth, in vitro culture techniques are commonly used to recover young embryos from interspecific crosses before they abort (Williams et al. 1987). Ovule culture is more often used when embryo abortion occurs at a very early stage; alternately, sequential culturing of ovules and then embryos can be employed, as was done for interspecific hybrids of Trifolium (Przywara et al. 1989). In general, legumes are more difficult than grasses to manipulate in vitro, but in either case, culturing procedures are not trivial exercises. Another complexity occurs when cultivated and wild species are at different ploidy levels. This is important given that many cultivated species, including wheat, oat, sugarcane, peanut, cotton, and potato, are polyploids whereas related species for these crops are often at a different ploidy level. Genomic imbalances in hybrids between species with different chromosome numbers can be overcome by several approaches: First, direct crossing followed by chromosome doubling of the sterile hybrids can often restore fertility to the F1 generation. Subsequently, the colchicines-treated plants can be either backcrossed with the cultivated species (as the recurrent parent) or they can be selfed in an effort to generate spontaneous chromosome reduction to the ploidy level of the cultivar. An example of this is interspecific hybridization between peanut (2n ¼ 40) and A. cardenasii (2n ¼ 20), where the hybrids were initially triploid (2n ¼ 30), but were treated with colchicines to double the number of chromosomes to the hexaploid level, followed by selfpollination for multiple generations to derive tetraploid progenies (Company et al. 1982). Although time-consuming and unpredictable, the advantage of the selfing approach is the increased number of gen-


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erations in which recombination can occur between the chromosomes of different genomes. A second method of overcoming ploidy differences is to raise the chromosome number of the species at the lower ploidy level to that of the species at higher ploidy level, which is usually the cultivar prior to making pollinations between species. For cultivars that are autopolyploid, this has been highly successful, but in allopolyploid species, there remains a considerable amount of sterility in F1 hybrid or later generations. To partially circumvent this type of genomic sterility, bridging species at the lower ploidy level and chromosome doubling can be used to make the desired interspecific hybrid with the respective cultivated species (Simpson 1991). Some species can be manipulated to lower the chromosome number of the higher-ploidy species. For example, Voigt (1971) reported a diploid sexual plant in the tetraploid apomictic grass species Eragrostis curvula, in tobacco where tetraploid Nicotiana tabacum can be hybridized as females with N. afracana to generate maternally derived haploids (Burk et al. 1979), or by reduced parthenogenesis in species of Solanum (Peloquin and Ortiz 1992). Polyploids can then be resynthesized to equal the chromosome number of the higher-ploidy species. In wheat, techniques have been used for nearly a half century to induce translocations or create chromosome addition or substitution lines with these methods (Khush 1973). Although not a cereal, Saccharum is one of the easiest genera to manipulate, in large part because of its high ploidy level, and it has been possible to introgress partial or complete genomes from wild species into S. officinarum L. (2n ¼ 80), creating modern cultivars that vary in number but all of which have more than 100 chromosomes. Ploidy manipulations with haploids, 2n gametes, and use of wild species remain as impressive and exciting crop germplasm enhancement methods that have developed from cytogenetic research (Ortiz et al. 2006). Professor Stanley J. Peloquin (University of Wisconsin, Madison, USA) and his associates were able to develop new potato genotypes that combine high and stable yield with disease or pest resistance, which allow potato to be grown in areas previously unsuitable for this crop (Ortiz et al. 2005). The genetic enhancement of potato germplasm often involves the use of species that are the source of genetic diversity, haploids that provide a method for utilizing this diversity, and 2n gametes with the endosperm balance number (EBN) that provide an effective method for transmitting the diversity into cultivars. There are two main methods for ploidy manipulations in potato: unilateral sexual polyploidization (4x-n gametes 2x-2n gametes or vice versa) and bilateral sexual polyploidization (ensuing from crosses between 2x-2n gametes producing parents) (Ortiz 1998).

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For these breeding schemes, the diploid progenitors ensue from crosses between potato haploids and tuber-bearing diploid species. Maternal haploids are easily extracted through parthenogenesis from most tetraploid potato cultivars, and they can be crossed with diploid species. The locally adapted haploid-species hybrids are used for selection because they possess 2n gametes, acceptable tuber characteristics, and additional desired attributes (e.g., disease or pest resistance). Polyploids can then be resynthesized because most of the hybrids ensuing from sexual polyploidization in potato are tetraploids (due to a strong triploid block in potato). Other difficulties in transferring genes from wild to cultivated species are: (1) nonflowering of hybrids; (2) selective chromosome elimination of the donor parent; (3) hybrid breakdown due to hybrid lethality or low vigor; and (4) physiological seed dormancy of the hybrid (Baum et al. 1992). Unfortunately, options to circumvent these problems do not exist for crop improvement. Biotechnology is increasingly being utilized for interspecific hybridization efforts. Although marker-assisted selection is useful both for identifying and following genes of interest in wild species crosses (Tanksley and McCouch 1997), it is not useful for overcoming barriers per se. Transformation technologies, however, are useful for overcoming these barriers because in many cases a single gene is desired from the wild species parent and can be more precisely moved through genetic engineering rather than genetic crosses. This is true in the case of monogenic resistance to plant pathogens, as discussed in Section V.B. It is worth keeping in mind, however, the large amount of research involved in identification and cloning of specific genes from a wild relative, as well as their transformation into the genome of the cultivated species and selection for stable expression. As more genomes are fully characterized, gene cloning and transformation methodologies will become more useful and common techniques for gene introgression will be available. IV. BENEFICIAL TRAITS FROM WILD RELATIVES CONTRIBUTING TO CROP GENE POOLS A. Resistance to Biotic Stresses Pests and diseases are the major causes of low productivity in cultivated plants. For biotic stresses, resistance is either not available in cultivated species or is available only at very low levels. In contrast, very high levels of resistance to pests and diseases have been reported in many wild relatives of cultivated species, and in some case attempts to


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transfer this resistance have been successful (Table 3.1). Examples of successful gene transfer include resistance to bacterial blight (Xanthomonas oryzae pv. Oryzae), blast (Magnaporthe grisea), sheath blight (Rhizoctonia solani), brown plant hopper (Nilaparvata lugens), whitebacked plant hopper (Sogatella furcifera), yellow stem borer (Scirpophaga incertulas), and grassy stunt virus in rice; leaf rust (Puccinia triticina), yellow rust (also known as stripe rust) (Puccinia striiformis f. sp. tritici), stem rust (Puccinia graminis), karnal bunt (Tilletia indica), powdery mildew (Erysiphe graminis), common root rot (Cochliobolus sativus), fusarium head blight (Fusarium graminearum), hessian fly (Mayetiola destructor), green bug (Schizaphis graminum), root-knot nematode (Meloidogyne naasi), and soil-born mosaic and spindlestreak mosaic viruses in wheat; leaf blight (Bipolaris maydis) in maize; and powdery mildew (Bulmeria graminis f. sp. hordei), septoria speckled leaf blotch (Septoria passerinii), leaf rust (Puccinia hordei), scald (Rhynchosporium secalis), and barley mild mosaic and barley yellow mosaic viruses in barley. Unlike cereals, the contribution of wild relatives to legumes has been limited to a few pests and fungal or bacterial diseases that include resistance to nematodes (Heterodera spp.) in chickpea and soybean; to common bacterial blight (Xanthomonas axonopodis pv. phaseoli), fusarium root rot (Fusarium solani f. sp. phaseoli), white mold (Sclerotinia sclerotiorum), and bruchid (Zabrotes subfasciatus) in common bean; and to rust (Puccinia arachidis), early leaf spot (Cercospora arachidicola), late leaf spot (Phaseoisariopsis personata), nematodes (Meloidogyne spp.), southern corn rootworm (Diobrotica undecimpunctata howardi), corn earworm (Heliothis zea), Spodoptera (Spodoptera spp.), and velvetbean caterpillar (Anticarsia gemmatalis) in peanut (Dwivedi et al. 2005). B. Tolerance to Abiotic Stresses Drought, salinity, acidic soils, phosphorous deficiency, and variation in temperature are the major abiotic stresses contributing to the substantial loss of production in cereal and legume crops. Many wild relatives have shown high levels of tolerance to these stresses, and some genes conferring these stress tolerances have been successfully transferred into cultivated species (Table 3.2). Examples include tolerance to acidic soils and aluminum toxicity in rice; drought and salinity tolerance in wheat; and drought tolerance in barley. Interspecific progenies in chickpea have shown tolerance to drought and cold temperature. Unlike cereals, this is the only example of gene transfer for abiotic stress tolerance from wild to cultivated legumes.

190

Wild Relatives Contributing Genes for Resistance to Biotic Stresses

Toubia-Rahme et al. 2003

Xu and Kasha 1992; Pickering et al. 1995 von Korff et al. 2005

Pickering and Johnston 2005

Walther et al. 2000

Zhang et al. 2001

Ruge et al. 2003

Ruge-Wehling et al. 2006

Reference

Chickpea (Cicer arietinum) Cyst nematode Resistance to cyst nematodes (Heterodera ciceri and H. rosii) transferred from Malhotra et al. 2003 C. reticulatum and C. echinospermum to C. arietinum. Wilt, foot rot, and Inter-specific derivatives involving C. reticulatum showed resistance to wilt Singh et al. 2005 root rot (Fusarium oxysporum f. sp. ciceris), foot rot (Phacidiopycnis padwickii), and root rot (Fusarium spp. and Rhizoctonia solani) diseases.

Barley (Hordeum vulgare) BaMMV and BaYMV H. bulbosum contributed gene, RYM16Hb, conferring resistance against all European viruses of soil-borne virus complex [BaMMV, barley mild mosaic virus; BaYMV, barley yellow mosaic virus (BaMMV, BaYMV-1, 2)] in H. vulgare. H. bulbosum contributed gene, Rym14HB, for resistance to BaMMV and BaYMV in H. vulgare. Leaf rust Resistance to rust (Puccinia hordei) transferred from H. bulbosum to H. vulgare. Leaf rust and mosaic Resistance to all leaf rust and mosaic viruses transferred into winter viruses H. vulgare from H. bulbosum. Multiple resistance H. bulbosum contributed genes for resistance to leaf rust, powdery mildew (Bulmeria graminis f. sp. hordei), scald (Rhynchosporium secalis), septoria speckled leaf blotch (Septoria passerinii), and BaYMV/BaMMV in H. vulgare. Powdery mildew H. bulbosum contributed gene for resistance to powdery mildew in H. vulgare. Powdery mildew, H. vulgare ssp. spontaneum contributed genes for resistance to powdery leaf rust, and scald mildew, leaf rust, and scald in H. vulgare. Septoria speckled Resistance to SSLB (Septoria passerinii) transferred into barley from H. bulbosum. leaf blotch (SSLB)

Pest/Disease

Table 3.1. Wild relatives contributing resistance to pests and diseases in barley, chickpea, common bean, maize, oat, peanut, rice, soybean, and wheat.

191

A. macrostachya contributed gene for resistance to powdery mildew (Erysiphe graminis) in oat.

Herrmann 2006

Goodman et al. 1987

Singh 2001

Cardona and Kornegay 1989, 1999 Singh and Munoz 1999

Kornegay et al. 1993

Blast (Bl) and BB

Rice (Oryza sativa) Bacterial blight (BB)

A dominant gene for resistance to BB (Xanthomonas oryzae pv. Pryzae) transferred from O. longistaminata to O. sativa.Several introgression lines resistant to BB developed by crossing IR 56 with O. brachyantha. Resistance to Bl (Magnaporthe grisea) and BB transferred from O. minuta to O. sativa.

(continues)

Amante-Bordeos et al. 1992

Khush et al. 1990Brar et al. 1996

Peanut (Arachis hypogaea) Nematodes and insect Wild Arachis species contributed resistance to nematodes (Meloidogyne spp.), Stalker et al. 2002a; Stalker pests Spodoptera (S. frugiperda and S. litura), corn earworm (Heliothis zea), southern and Lynch 2002; Dwivedi corn rootworm (Diobrotica undecimpunctata howardi), velvetbean caterpillar et al. 2003; Mallikarjuna (Anticarsia gemmatalis), and Jassids (Empoasca ssp) to A. hypogaea. et al. 2004 Rust and leaf spots Wild Arachis species contributed resistance to rust (Puccinia arachidis), Stalker et al. 2002b; early leaf spot (Cercospora arachidicola), and late leaf spot (Phaeoisariopsis Dwivedi et al. 2003; personata) to A. hypogaea. Mallikarjuna et al. 2004

Oat (Avena sativa) Powdery mildew

Common Bean (Phaseolus vulgaris) Bruchid Monogenic arcelin-based weevil resistance against the bruchid (Zabrotes subfasciatus) transferred from wild common bean to CIAT breeding lines. Resistance to bruchid from wild bean populations from highlands of Mexico transferred to P. vulgaris. Common bacterial Resistance to CBB (Xanthomonas axonopodis pv. phaseoli) transferred from blight (CBB) tepary bean to P. vulgaris. CBB, fusarium root rot, Moderate resistance from P. coccineus to CBB, fusarium root rot and white mold (Fusarium solani f. sp. phaseoli), and white mold (Sclerotinia sclerotiorum) introgressed to dry/snap beans. Maize (Zea mays) Leaf blight Resistance to northern corn leaf blight (Bipolaris maydis) transferred from Tripsacum dactyloides to maize.

.

192 Resistance to cyst nematode (Heterodera glycines) introgressed from G. tomentella to G. max.

Resistance to three biotypes of BPH (Nilapavata lugens) transferred from O. officinalis to O. sativa. An introgression line resistant to BPH selected from BC2F4 population of O. australiensis O. sativa. A gene for resistance to GSV transferred from O. nivara to O. sativa. Tolerance to RSNV transferred into double haploid progenies involving Caiapo and O. rufipogon. Interspecific progenies, involving improved varieties and O. rufipogon, showed good tolerance to Rhizoctonia solani, Sarocladium oryzae, and Bipolaris oryzae. Resistance to tungro virus from O. rufipogon, to yellow stem borer (Scirpophaga incertulas) from O. longistaminata, and to sheath blight (Rhizoctonia solani) from O. minuta transferred to O. sativa. Resistance to WPH (Sogatella furcifera), BPH, and BB from O. officinalis transferred into elite breeding lines. Resistance to BPH and/or BB from O. australiensis transferred into elite breeding lines.

Wild Relatives Contributing Genes for Resistance to Biotic Stresses

Wheat (Triticum aestivum) Barley yellow dwarf virus Thinopyrum intermedium and Th. ponticum contributed genes for (BYDV) and/or wheat resistance to BYDV and WSMV to T. aestivum. streak mosaic virus (WSMV) Common root rot Aegilops ovata contributed resistance to common root rot (Cochliobolus sativus) in T. aestivum. Fusarium head blight Resistance to fusarium head blight (Fusarium graminearum) transferred from T. dicoccoides, T. timopheevi, T. monococcum, and Ae. speltoides to T. aestivum. Hessian fly Resistance to hessian fly (Mayetiola destructor) from Aegilops species (Ae. geniculata, Ae. triaristata, Ae. squarrosa, and Ae. ventricosa) transferred to T. aestivum/T. durum.

Soybean (Glycine max) Soybean cyst nematode

White-backed plant hopper (WPH), BPH, and BB

Tungro virus, yellow stem borer, and sheath blight

Grassy stunt virus (GSV) Rice stripe necrosis virus (RSNV) Soil-borne diseases

Brown plant hopper (BPH)

Pest/Disease

Table 3.1. (continued)

El Khlifi et al. 2004

Cai et al. 2005

Bailey et al. 1993

Sharma et al. 1995; Fedak et al. 2001; Jiang et al. 2005

Riggs et al. 1998

Multani et al. 1994

Jena and Khush 1990

Brar and Khush 1997

Khush et al. 1977 www.ciat.cgiar.org/riceweb/ pdfs/poster_alleles.pdf www.ciat.cgiar.org/riceweb/ pdfs/poster_alleles.pdf

Ishii et al. 1993

Jena and Khush 1990

Reference

193

Yellow rust (stripe rust), stem rust, and leaf rust

Yellow rust and leaf rust

Stem rust

Rust, WSMV, and WSSMV

Rust

Root-knot nematode

Powdery mildew

Leaf rust

Hessian fly, leaf rust, and soilborne-mosaic virus Karnal bunt

Hessian fly, green bug, and rust

Ae. squarrosa contributed resistance to hessian fly, green bug Gill and Raupp 1987 (Schizaphis graminum), and leaf rust (Puccinia triticina) to T. aestivum. Ae. squarrosa contributed genes for resistance to hessian fly, leaf rust, Cox et al. 1990 and soil-borne mosaic virus to T. aestivum. Resistance to karnal bunt (Tilletia indica) transferred from T. tauschii Villareal et al. 1995 to T. aestivum. Resistant to leaf rust race 5 transferred from Ae. speltoides to Dvora´k 1977 T. aestivum. Resistance to powdery mildew (Blumeria graminis) transferred from Ae. Spetsov et al. 1997 variabilis to T. aestivum. T. turgidum var dicoccoides contributed gene for resistance to Rong et al. 2000 powdery mildew, Pm 26, in T. aestivum. Resistance to powdery mildew transferred from T. urartu into Qiu et al. 2005 T. aestivum. Ae. variabilis contributed resistance to root-knot nematode Yu et al. 1990; Barloy et al. (Meloidogyne naasi) to T. aestivum. 2000 Lr32 from T. tauschii transferred to T. aestivum. Kerber 1987 Rust-resistant genes Lr41, Lr 42, and Lr 43 from T. tauschii introgressed Cox et al. 1994 to T. aestivum. Resistance to rust, WSMV (wheat soil-borne mosaic virus), and WSSMV Cox et al. 1995 (wheat spindle-streak mosaic virus) transferred from T. tauschii to T. aestivum. Resistance to stem rust (Puccinia graminis) transferred from T. turgidum McFadden 1930 L. ssp. dicoccum to T. aestivum. Resistance to stem rust transferred from T. monococcum to T. aestivum Kerber and Dyck 1973 via T. durum. T. turgidum and T. tauschii contributed genes for resistance to yellow Ma et al. 1995 rust in synthetic hexaploid wheat. Triticum spp. and Ae. speltoides contributed genes for resistance to Valkoun 2001 yellow rust and T. baeoticum and Ae. speltoides to leaf rust to T. durum. Resistance to yellow rust (Puccinia striiformis) from Ae. comosa; to stem Goodman et al. 1987 rust from Ae. speltoides, T. monococcum, and T. timopheevi; to leaf rust from A. squarrosa, A. umbellulata, and A. elongatum transferred to T. aestivum.

194

Wild Relatives Contributing Gene(s) for Abiotic Stress Tolerance

Amphidiploids from T. turgidum and Ae. Speltoides showed greater salinity tolerance than either parent.

Wheat (Triticum aestivum) Drought Drought tolerance from Agropyron elongatum incorporated to T. aestivum. Traits related to drought tolerance from T. urartu, T. baeoticum, Aegilops speltoides, and Ae. tauschii transferred to T. dicoccoides. T. tauschii contributed genes for traits related to drought tolerance in T. aestivum. Salinity Salt tolerance from Ae. cylindrica and Thinopyrum junceum transferred to T. aestivum.

Chickpea (Cicer arietinum) Drought and cold C. reticulatum and C. echinospemum contributed tolerance to drought and cold to C. arietinum. Rice (Oryza Sativa) Acidic sulphate soils Tolerance to acidic sulfate soils and toxicity to iron and aluminum transferred from and aluminium O. rufipogon (AA genome) to O. sativa. toxicity

Barley (Hordeum vulgare) Drought Some progenies involving H. vulgare ssp. spontaneum and H. vulgare ssp. agriocrithon with H. vulgare ssp. vulgare outyielded control variety by 13–22% under dry Mediterranean environments. Recombinant inbred lines (Arta x H. spontaneum 41–1) produced higher grain yield under drought-stress conditions. Tolerance to drought selected from the BC1F2 population (H. vulgare H. spontaneum). Progenies involving Barke and wild barley (HOR11508) produced greater yield under varying water availability. Drought and cold H. spontaneum 41-1 contributed tolerance to drought and cold to H. vulgare.

Trait

Table 3.2. Wild relatives contributing resistance/tolerance to abiotic stresses in barley, chickpea, rice, and wheat.

Gororo et al. 2002 Farooq et al. 1992, 1995; Wang et al. 2003 Noori 2005

Goodman et al. 1987 Valkoun 2001

Brar and Khush1997; Nguyen et al. 2003

Malhotra et al. 2003

Baum et al. 2003

Forster et al. 2004 Talame et al. 2004

Baum et al. 2003

Hadjichristodoulou 1993

Reference


195

C. Cytoplasmic Male Sterility Cytoplasmic male sterility (CMS) is a maternally inherited trait that prevents plants from producing normal pollen and is used as a tool to produce large-scale commercial F1 hybrid seeds in many crops. This system has been more frequently used for developing commercial hybrids in cross-pollinated crops (maize, sorghum, and pearl millet) than to inbreeders (with exception in rice and pigeonpea) to exploit hybrid vigor, largely because the flower structure of inbreeders does not permit an economical large-scale hybrid seed production. Although many CMS sources and fertility restorer genes are known in these crops, additional CMS sources and fertility restorer genes have been desirable since the Southern corn leaf blight (Bipolaris maydis) disease epidemic of the 1970s, which was precipitated by the wide-scale use of cms-T cytoplasm in the United States (Holley and Goodman 1989). Compared to the contribution of wild relatives in terms of abiotic/biotic resistance or tolerance genes, only a few examples exist that demonstrate the potential of wild relatives as sources of cytoplasmic male sterility and nuclear restorer genes (Table 3.3). ‘‘CMS lines having cytoplasm from the wild relative Oryza sativa f. spontanea have been used extensively for production of commercial hybrids in China (Yuan 1993).’’ New and stable CMS cytoplasm from ‘‘A’’ genome species has also been developed. In legumes, interspecific crosses between Glycine max and G. soja have given rise to a cytoplasmic-nuclear male sterile line and its maintainer; while crosses involving C. cajan with wild relatives gave to four sources of cytoplasmic nuclear male sterility in pigeonpea. Additionally, C. acutifolius contributed nuclear genes that interact with cytoplasm C. cajan to produce cytoplasmic nuclear male sterility system in pigeonpea.

D. Yield, Nutritional Quality, and Adaptation Traits Wild relatives are usually substantially inferior to modern cultivars with respect to agronomic and seed quality traits as well as yield; however, evidence suggests that wild accessions can contribute useful alleles for improvement of these traits if the appropriate breeding technique is used. It is worth noting that the favorable alleles found in wild relatives are often masked by deleterious genes that have been difficult to eliminate through conventional breeding techniques. Pioneering work in this regard was first reported in tomato, where Rick (1974) produced a high-soluble solid genotype from a green-fruited wild species, Lycopersicon pimpinellifolium. Other successful examples in tomato include the use of L. chmielewskii, L. hirsutum, L. peruvianum,

196

Soybean (Glycine max) A CMS line developed from wild soybean accession 035. A cytoplasmic nuclear male sterile line (cms A) and its maintainer (B line) developed from an interspecific cross involving G. max and G. soza. A male sterile cytoplasm discovered from wild soybean accession N23168.

Zhao and Gai 2006

Sun et al. 1994 Sun et al. 1997

Dalmacio et al. 1995 Dalmacio et al. 1996 Hoan et al. 1997

Lin and Yuan 1980

Ariyanayagam et al. 1995; Wanjari et al. 2001; Saxena and Kumar 2003; Saxena et al. 2005 Mallikarjuna and Saxena 2005

Pigeonpea (Cajanus cajan) Four CMS sources, A1 from C. sericeus, A2 from C. Scarabaeoides, A3 from C. volubilis, and A4 from C. cajanifolious, developed. Cytoplasmic nuclear male sterility system developed involving cytoplasm from cultivated pigeonpea and nuclear genes from wild relative, C. acutifolius.

Rice (Oryza sativa) Wild abortive (WA) type that has cytoplasm of O. sativa L. f. spontanea, and nuclear genome of O. sativa. IR66707A has cytoplasm of O. perennis and the nuclear genome of IR 64. IR69700A has cytoplasm of O. glumaepatula and nuclear genome of IR64. CMS lines containing cytoplasm of either O. rufipogon or O. nivara developed.

Reference

Cms Reported from Wild Species

Table 3.3. Wild relatives as a source of cytoplasmic male sterility (CMS) and/or fertility restorer gene in pigeonpea, rice, and soybean.


197

L. piminellifolium, and L. pennellii to improve fruit yield and processing quality traits, such as fruit size, color, firmness, soluble solids content, glucose and fructose concentration, and viscosity (de Vicente and Tanksley 1993; Fulton et al. 1997; Bernacchi et al. 1998a, b; Fridman et al. 2000; Yousef and Juvik 2001). Among cereals, there have been a number of studies reporting the successful introgression of genes from wild relatives for improved agronomic and seed quality traits (Tables 3.4 and 3.5). In oat, Avena sterilis and A. sativa crosses produced progenies with up to 30% increased grain yield over control due to increased growth rate from the wild accession. In sorghum, yield- and seed quality–enhancing traits were found in Sorghum virgatum and S.verticilliflorum and incorporated into cultivated genotypes. Similarly successful examples occur in pearl millet, rice, bread wheat, and barley. In contrast to cereals, wild species have been less utilized for agronomic and grain quality trait breeding in legumes. The few examples include Glycine soja used to increase grain yield and protein content in soybean; Cicer reticulatum and C. echinospernum for increased yield in chickpea; wild Phaseolus vulgaris to improve an Andean common bean cultivars; and Cajanus scarabaeoides for high protein content in pigeonpea. Halward et al. (1991) also reported yield increases in cultivated peanut from genes derived from Arachis cardenasii. Clearly, these gains demonstrate that wild species of legumes do contain gene(s) for agronomic and/or seed quality traits that can be exploited toward increased productivity through appropriate breeding strategy.

V. BIOTECHNOLOGICAL APPROACHES TO ENHANCE UTILIZATION OF WILD RELATIVES IN CROP IMPROVEMENT A. Chromosome-Mediated Alien Gene Transfer The wheat tribe Triticeae includes wheat, barley, rye (Secale cereale), triticale (Triticosecale), and their wild relatives. Bread wheat (Triticum aestivum) is an allohexaploid (2n ¼ 42) composed of A, B, and D genomes from Triticum urartu (Nishikawa 1983), Aegilops speltoides (Sarkar and Stebbins 1956; Dvora´k and Zhang 1990), and Aegilops squarrosa (Pathak 1940), respectively. Durum wheat is an allotetraploid (2n ¼ 28) with A and B genomes. The 21 chromosomes of wheat fall into seven groups of three chromosomes each, and the corresponding chromosomes of the A, B, and D genomes are homoeologues (Sears 1966). Ph1 gene on the long arm of chromosome 5B suppresses pairing

198

Wild Relatives Contributing Gene(s) for Enhanced Yield and/ or Seed Quality Traits

Pearlmillet (Pennisetum glaucum) Yield The introgressed progenies involving weedy and wild pearlmillet (P. americanum ssp. monodii) with P. typhoid produced segregants with increased growth rate and grain yield.

Oat (Avena sativa) Yield Introgressed lines from A. sativa A. sterilis recorded up to 30% grain yield advantage but with growth duration and harvest index similar to the A. sativa.

Chickpea (Cicer arietinum) Yield Few F6 lines [ILC 482 C. echinospernum] outyielded ILC 482 by 39% and free from undesirable traits found in wild relatives. Interspecific progenies involving C. reticulatum and C. echinospermum with chickpea produced greater biomass, more number of branches, and were earlier in maturity. Four desi and kabuli lines involving C. reticulatum with chickpea produced 6–17% increased seed yield over best control.

Trait

Bramel-Cox et al. 1986

Frey 1976; Takeda and Frey 1976

Singh et al. 2005

Malhotra et al. 2003

Singh and Ocampo 1997

Reference

Table 3.4. Wild relatives contributing genes for agronomic and/or grain quality traits transferred in chickpea, oat, pearlmillet, pigeonpea, sorghum, soybean, and wheat using conventional backcrossing and selection.

199

Wheat (Triticum aestivum) Agronomic traits Synthetic hexaploid from Huarani Aegilops tauschii produced backcrossed progenies with short plant stature, early maturity, high productive tillering, high spike productivity, and drought tolerance. Grain protein Gene(s) for higher grain weight and protein content incorporated from T. turgidum var. and/or grain dicoccoides to T. aestivum. weight A genomic region from T. turgidum var. dicoccoides associated with high grain protein content introgressed to T. aestivum. A gene for high protein content from T. turgidum var. dicoccoides transferred to T. aestivum.

Khan et al. 2000

Mesfin et al. 1999; 2000

Kushnir and Halloran 1984

Valkoun 2001

Diers et al. 1992

Cox et al. 1984

Sorghum (Sorghum bicolor) Yield Backcrossed derived lines involving virgatum and verticilliflorum with S. bicolor produced on average 13.5% greater grain yield than control cultivar.

Soybean (Glycine max) Protein content High grain protein content from G. soja transferred to G. max.

Reddy et al. 1979

Pigeonpea (Cajanus cajan) Protein HPL# 2, 7, 40, and 51 with high protein content were selected from a cross between A. scarabaeoides and C. cajan.

200

Wild Relatives Contributing Chromosomal Regions (Qtl) Containing Beneficial Alleles

Rice (Oryza sativa) Aluminum toxicity

Blair et al. 2006

Blair et al. 2003

von Korff et al. 2006

Pillen et al. 2004

Matus et al. 2003

Pillen et al. 2003

Baum et al. 2003

Reference

Relative root length (RRL) exerts significance influence on plants’ ability to tolerate Nguyen et al. 2003 aluminum toxicity in rice. O. rufipogon contributed favorable QTL alleles for RRL, a primary parameter for Al tolerance.

Common Bean (Phaseolus vulgaris) Yield and yield Wild P. vulgaris contributed 3 QTLs for increased yield to a Colombian variety of components cultivated common bean using an advanced backcross breeding strategy. A total of 13 QTLs for plant height, yield, and yield components along with a single QTL for seed size showed positive alleles from the wild parent, G24404, in BC2F3:5 introgression lines.

Barley (Hordeum vulgare) Drought and cold H. spontaneum 41-1 when crossed with Arta contributed positive alleles for days to heading, plant height, tiller number, and tolerance to drought and cold. In BC2F2 population [H. vulgare (Hv) x H. spontaneum (Hsp)] detected 29 favorable Yield and yield components; malting effects QTL from the Hsp. Lines homozygous for Hsp alleles were associated with 7.7% increased grain yield over Hv alleles. quality traits H. vulgare subsp. spontaneum, despite its overall inferior phenotypes, contributed favorable alleles for higher seeds per head, 1000-kernel weight, grain protein content, diastatic power, and b-glucan. In another BC2F2 population [H. vulgare (Hv) x ISR 101–23 (H. spontaneum (Hsp)], 52 QTL with favorable effects identified from Hsp. The Hsp QTL allele at EBmac0679[4H] was associated with 5.9% increase in yield across 6 environments. Wild barley accession ISR42–8 contributed several positive alleles associated with improved agronomic performance in H. vulgare: alleles on 4HL improved yield by 7.1% while allele on 2HS increased number of ears m2 by 16.4% and 1000-grain weight by 3.2%.

Trait

Table 3.5. Wild relatives contributing quantitative trait loci (QTL) associated with beneficial traits into progenies of the wild cultivated species crosses in barley, common bean, rice, sorghum, soybean, and wheat.

201

Stem elongation Yield and yield components

Grain quality

Drought

(continues)

Gene(s) for increased stem elongation ability transferred from O. rufipogon into rice. Brar and Khush 1997 O. rufipogon alleles at marker loci RM5 on chromosome 1 and RG256 on chromosome Xiao et al. 1996, 1998 2 were associated with enhanced grain yield in backcross progenies involving O. rufipogon, V20A (CMS), and V20B (maintainer), showing 1.1 to 1.2 t ha-1 superiority for the lines carrying O. rufipogon alleles at these loci. O. rufipogon contributed 13 QTLs for yield components, 4 for maturity, and 6 for Moncada et al. 2001 plant height in a backcross population involving Caiapo, an upland japonica cultivar from Brazil. O. glumaepatula contributed beneficial QTLs associated with increased tillering and Brondani et al. 2002 panicle number, known to substantially affect plant architecture. O. rufipogon contributed gene(s) for increased panicle length, panicles per plant, Septiningsih et al. 2003 percentage of seed set, grains per plant, grain weight and yield while reducing the duration into IR64 genetic background. O. rufipogon contributed favorable alleles for yield, grains per panicle, panicle length, Thomson et al. 2003 and grain weight in advanced backcross lines (O. rufipogon Jefferson). O. rufipogon contains 2 yield-enhancing QTLs, yld1.1 on chromosome 1 and yld2.1 Liang et al. 2004 on chromosome 2, capable of improving the yield of hybrid rice by 18 and 17%, respectively. BC3F1 (9311 O. rufipogon) produced lines that produced 24% to 43% greater yield over control. O. rufipogon contributed favorable alleles for days to heading, culm length, panicle Lee et al. 2004; 2005 exertion, panicle length, spikelets per panicle, and leaf discoloration associated with cold tolerance in RILs.

O. rufipogon contributed 2 QTLs, qSDT2-1 and qSDT2-2, associated with drought Zhang et al. 2006 tolerance in rice. Yoon et al. 2005 O. glandiglumis contributed alleles at the locus RM290 on chromosome 2 that increased grain weight, width, and thickness, and grain yield but decreased panicle length, while alleles at locus RM144 on chromosome 11 decreased the grain weight, grain width, and grain thickness but increased panicle numbers. Advanced lines with long, slender, and translucent grains were selected from BC2F2 www.ciat.cgiar.org/riceweb/ population (Bg90–2 x O. rufipogon), and O. rufipogon contributed alleles associated pdfs/poster_alleles.pdf with superior grain quality.

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O. rufipogon alleles had a beneficial effect on 74% of the 39 QTL associated with yield and yield related traits in interspecific BC2 progenies. O. grandiglumis contributed positive alleles for days to heading, spikelets per panicle, and grain shape traits into japonica cv. Hwaseongbyeo background. O. rufipogon contributed a QTL, gpa7, with pleiotropic effect on panicle structure in rice.

Wild Relatives Contributing Chromosomal Regions (Qtl) Containing Beneficial Alleles

Wheat (Triticum aestivum) Yield and yield A synthetic wheat W-7984 when crossed with Prinz contributed beneficial alleles components for grain yield and yield components in progenies. A synthetic wheat line XX86 when crossed with Flair contributed 24 QTL with positive effects on 1000-grain weight and grain weight per ear. Aegilops tauschii contributed positive alleles for earliness, short stature, ear length, grain weight, and spikelet number in T. aestivum.

Soybean (Glycine max) Seed protein content G. soja contributed 2 major QTL alleles that increased protein content in backcross progenies involving G. soja and G. max. Yield G. soja contributed yield-enhancing QTL alleles that demonstrated 9% yield advantage in backcross progenies.

Sorghum (Sorghum bicolor) Grain yield Chromosome regions associated with enhanced grain yield from S. arundinaceum identified and transferred to BC1F4 progenies involving S. bicolor.

Trait


Pestsova et al. 2006

Huang et al. 2004

Huang et al. 2003

Concibido et al. 2003

Sebolt et al. 2000

Jordan et al. 2004

Tian et al. 2006

Yoon et al. 2006

Marri et al. 2005

Reference


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(Sears 1976). This gene also suppresses pairing between wheat chromosomes and alien chromosomes of donor species, thereby restricting the incorporation of alien genes into wheat. However, there are methods that eliminate or suppress the activity of Ph1 to promote pairing between alien and wheat chromosomes, thus accelerating gene transfer from alien species to wheat. The methods to promote homoeologous chromosome pairing include use of (1) Ph1b/ph2b and ph1c mutants (Ceoloni et al. 1986), (2) 5-B deficient genetic stocks (Jauhar and Almouslem 1998), and (3) alien species that inactivate Ph1 (Sears 1976; Jauhar 1992). Using the above-mentioned approaches, transfer of several alien genes possessing beneficial traits has been effected into durum and bread wheat (Jauhar and Chibbar 1999). However, use of homoeologous chromosome pairing has also its own limitations as the recombination between homoelogous chromosomes of wheat and related species is either absent or drastically reduced in the proximal regions of chromosome arms, making it difficult to transfer a target gene from these regions (Lukaszewski 1995). In such cases, radiation followed by strong selection for the recovery of compensating translocations might prove an effective strategy (Sears 1993). B. Using Cloned Genes from Wild Relatives to Produce Transgenics As discussed, many wild species with desirable genes are either totally cross-incompatible with cultivated species or require complex bridge crossing and/or embryo rescue techniques for crosses to be successful. In practice this results in a long struggle to incorporate and retain desirable genes while eliminating linkage drag and deleterious genes from the wild accession. Traditional backcross breeding can be used for this process but is generally a tedious, time-consuming, and inefficient task even when marker-assisted selection is utilized (Tanksley et al. 1989). Molecular cloning and subsequent transfer through transformation has been suggested as the method of choice to overcome the problems associated with direct introgression through sexual crossing and selection although few examples of this approach exist to date. One notable transfer of a wild species gene through transgenesis to a set of cultivars is for bacterial blight, which is one of the most destructive diseases of rice. In this case, the broad-spectrum resistance gene Xa21 from Oryza longistaminata (Khush et al. 1990) was cloned through a map-based approach (Song et al. 1995) and transferred through Agrobacterium-mediated transformation to five rice cultivars (Zhai et al. 2000) and a widely used hybrid restorer line in China (Zhai et al. 2001). In both cases the transgenic lines and derived hybrids

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displayed high levels of broad-spectrum resistance to the disease and maintained elite agronomic traits of the parents. This approach has also been used to clone Hs1pro1 from wild sugarbeet (B. procumbens) and Rpi-blb1 from wild potato (S. bulbocastanum); and after being transferred to cultivated sugarbeet and potato, respectively, progenies showed high levels of resistance to nematode (Heterodera schachii) in sugarbeet and late blight (Phytophthora infestans) in potato (Cai et al. 1997; van der Vossen et al. 2003). Finally, for the legumes there is promise in the work of Bhattarai and Fettig (2005), who isolated a drought- and salinityinduced dehydrin gene, cpdhn1, from a cDNA from ripening seeds of Cicer pinnatifidum, a wild relative of chickpeas, which may be used for the transformation of commercial cultivars to create more stress-resistant chickpeas. C. reticulatum, another wild relative of chickpea, is also tolerant to drought (Singh et al. 1994) and could be used for studying the dehydrin genes (Thomashow 1993). Clearly, the transgenic approach can accelerate the introgression of the new alleles from wild relatives to crop species compared to conventional breeding, which in many cases can be a slower and less precise method due to linkage drag. The use of transgenic transfer of wild species genes could therefore increase the genetic diversity available for crop improvement. C. Developing Transgenics with Large-Scale Transfer of Exogenous DNA from Distant Relatives It has been difficult to produce hybrids between rice and some of its distant relatives from the tertiary gene pool even with marker-assisted selection and embryo rescue techniques; therefore, other methods of gene transfer are needed. One option is the use of microparticle bombardment with the unfractionated genomic DNA (Abedinia et al. 2000), which was used to introgress genes from Zizania palustris, a wild rice from North America that is adapted to extreme cold conditions (Oelke et al. 1997). In that study, transgenic plants were recovered with grain characteristics from wild rice, and AFLP analysis revealed that a significant amount of DNA from Zizania palustris was introduced. This method is not without drawbacks. Liu et al. (2004) found that alien DNA introgression into a plant genome could induce extensive alternations in DNA methylation and transcription of both cellular genes and transposable elements in a genotype-independent manner. Zizania latifolia possesses resistance to diseases and insects, cold and flood tolerance, and good grain quality. Using repeated pollination, Liu et al. (1999) produced a set of introgression lines with introgressed DNA segments from Z. latifolia that exhibited a high level of resistance to blast and


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bacterial blight. Wang et al. (2005) studied the impact of alien gene introgression on a recipient plant genome by examining 6,000 unbiased genomic loci of three stable recombinant inbred lines (RILs) derived from intergeneric hybridization between ‘Matsumae’ and the wild relative Zizania latifolia. Although the lines each contained less than 0.1% of the wild species genome, they had extensive and genome-wide de novo variations with up to 30% of the analyzed loci for the three lines studied. Further analysis using BLAST (Basic Local Alignment Search Tool) revealed that the genomic variations occurred in diverse sequences, including protein-coding genes, transposable elements, and sequences of unknown functions. Pair-wise sequence comparison of selected loci showed that the variation represented either base substitutions or small insertion/deletions. On the positive side, the same authors report the generation of new diversity with some of the introgression lines showing increased biomass, tolerance to low temperature and plant submergence, and immunity or high levels of resistances to several major rice diseases. It is evident that alien introgression can be highly mutagenic to a recipient plant genome. More recently, Chen et al. (2006) isolated eight resistance-gene analogs from Z. latifolia by using introgression lines and subsequently classified them into six distinct groups. Another method for creating interspecies transgenic hybrids was developed by Zhao et al. (1998), who used a ‘‘spike-stalk injection’’ method to transfer exogenous genomic DNA from wild rice, maize, sorghum, Echinochloa crusgalli, and Panicum maximum into the uppermost stem internode at the position just under the panicle base of recipient rice plants at flowering. Xing et al. (2004) developed many useful lines of the hybrid rice with this approach, including the restorer line RB207 with a 40% increase in the number of spikelets per panicle and 1000-grain weight and the maintainer line Yewei B with better tolerance to high temperature. These studies demonstrated that direct introduction of genomic DNA of distant relatives can be an effective approach for creating new rice germplasm and potentially for enhancing other crops with genes from wild relatives in species where highly efficient transformation methods are available. D. Marker-Aided Introgression Molecular marker-based genetic linkage maps have provided a method to monitor and facilitate interspecific gene transfer and to mitigate linkage drag, thus improving the prospects for successful introgression of desirable genes from wild species (Tanksley et al. 1989; Young and Tanksley 1989; Tanksley and McCouch 1997). An early combination of

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genetic mapping and wild species crosses was reported on by de Vicente and Tanksley (1993), who found that Lycopersicon pennellii could contribute quantitative trait loci (QTL) alleles for yield in cultivated tomato even though it did not display high yield itself. Using a RFLPbased genetic linkage map, they detected at least one QTL from L. pennellii that had an allelic effect opposite of what would be expected based on the phenotype of the wild plant. They concluded that by combining wild species crosses with molecular genetic linkage maps, it could be possible to identify and selectively introgress new alleles from wild species that normally were not considered to be sources of useful variation. Expanding on this, Tanksley and Nelson (1996) proposed advanced backcross QTL analysis as an efficient breeding methodology for the simultaneous discovery and transfer of valuable QTL from unadapted germplasm into elite breeding lines. In this method, compared to previous QTL analysis with the F2 or BC1 derived populations, QTL analysis is conducted in the BC2 or BC3 generation, and negative selection is practiced to reduce the frequency of deleterious donor alleles. Using this breeding scheme, unique wild gene introgressions for improved fruit yield, size, weight, shape, and firmness were transferred into cultivated tomato (Fulton et al. 1997, 2000; Bernacchi et al. 1998a, b). Among the legumes and cereals, advanced backcrossing and QTL analysis has been applied to successfully introgress higher yield from wild relatives into cultivated rice, wheat, barley, sorghum, common bean, and soybean (Table 3.5). The above-mentioned examples clearly demonstrate that wild relatives contain desirable alleles for agronomic traits even though their effect is phenotypically not evident in wild relatives. In future years there should be more successful exploitation of wild relatives as we progress toward saturating genetic linkage maps with user-friendly markers, such as SSRs and SNPs, and the technological cost of applying the marker technology is substantially reduced. E. Resynthesizing the Crop Progenitors to Capture Variability Lost During Crop Evolution and Domestication Polyploidization, a common event in the evolution of crop and noncrop plants alike, usually results in speciation and reproductive isolation and a genetic bottleneck between the new polyploid and its progenitor or related species. In spite of this, the existence of numerous wild polyploids and the frequent association of plant domestication with polyploidization testify that the polyploidy state must bring some significant selective advantages. Notable polyploids include such crops as


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wheat, rapeseed, peanut, and potato; furthermore, many crop species that were until recently considered typical diploids are, in fact, ancient polyploids that have undergone one or more rounds of chromosome doubling during their evolution but nevertheless behave cytologically as diploids (Leitch and Bennett 1997; Wolfe 2001). The duplication of genomes, either from the same species (autopolyploidy) or more frequently from divergent species (allopolyploidy or amphiploidy), is therefore a major force of evolution in plants, affecting genome size and gene copy numbers (Soltis and Soltis 2000; Wendel 2000). The narrow genetic base of many polyploid crop plants presents difficulties to the plant breeder trying to cope with evolving disease and insect pressures and variations in environmental stresses. This is compounded by the problems inherent in introducing novel alleles from nonpolyploid relatives to the polyploid crop as discussed earlier. This has led to the suggestion of ‘‘resynthesizing’’ polyploids to generate additional diversity and as a mechanism for gene introgression. The ‘‘resynthesis’’ pathway has, in some cases, proven a useful way to overcome the difficulties in breeding polyploids using genes from wild relatives. This pathway essentially attempts to artificially re-create evolutionary events similar to those that gave rise to the evolutionary speciation of the crop species. The most notable resynthesized crops have been the synthetic hexaploid wheat, produced by crossing durum wheat (2n ¼ 28, AB genomes) with Aegilops tauschii (2n ¼ 14, D genome). These hybrids have been used as an intermediary for transferring resistance/tolerance to many biotic and abiotic stresses as well as adding substantial variability for morphophysiological and reproductive traits to wheat (Fernandes et al. 2000; del Blanco et al. 2001 and references therein). A major program was developed at CIMMYT to create more than 1,000 synthetic wheats that could be used to broaden the genetic diversity of the modern wheat. Some of these or their derivatives have been recently released as cultivars (‘Chuanmai 42’ in China and ‘Carmona’ in Spain) (www.cimmyt.org/english/wps/news/wild_wht.htm). Another wheat strain developed by CIMMYT produces between 20 and 40% more grains under dry conditions than traditional wheat (www.nature.com/ news/2006). Synthetic polyploids have also been successful at generating new diversity in Brassicaceae. For example, resistance to Plasmodiophora brassicae (Diederichsen and Sacristan 1996), Leptosphaeria maculans (Crouch et al. 1994), and pod shattering (Summers et al. 2003), and variation in flowering time (Schranz and Osborn 2000; Pires et al. 2004) and oil quality (Lu et al. 2001) has been introduced into cultivated

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rapeseed (Brassica napus) germplasm. Using AFLP analysis, Seyis et al. (2003) showed that resynthesis generates an increase in genotypic variation caused by frequent intergenomic recombinatons between the diploid genome chromosomes in early generations of resynthesis. In legumes, polyploidization has been rarely associated with domestication. Two notable exceptions are the autopolyploid Medicago sativa and the amphiploid peanut, which originated via hybridization of two wild species with contrasting genomes that were possibly brought together through cultivation by humans. In the case of peanut, this hybrid would have been sterile, but would have regained fertility by a spontaneous duplication of the original number of chromosomes. As is the case with most amphiploids, the new amphiploid would have been reproductively isolated from its diploid relatives due to the differences in chromosome number between their genomes. As a result, the genetic variability of cultivated peanut is very low, and all cultivated germplasm is thought to be derived from only one or a few amphiploid plants, facts that hamper the efforts of plant breeders and limit the development of peanut genetics. In contrast, wild Arachis species are very diverse genetically (Halward et al. 1992; Galgaro et al. 1997; Moretzsohn et al. 2004) and have been selected during evolution in a range of environments ranging from marsh to semiarid, rocky terrain providing a rich source of variation for resistance to many biotic and abiotic stresses (Stalker and Moss 1987). It has been clear for some time that the introduction of wild genes into cultivated germplasm would present new possibilities for the crop. Various attempts to do this have been made, but until now, the most productive was through a cross that we now know approximates resynthesis (Simpson et al. 1993). In this cross a hybrid AA genome donor (A. cardenasii A. diogoi) was crossed with a BB genome species A. batizocoi, and the resultant sterile hybrid was treated with colchicine to double the chromosome number and regain fertility. Crosses and backcrosses of this synthetic amphiploid (named T AG-6) with peanut have resulted in the release of two peanut cultivars (Coan and NemaTAM) with wild genes for root-knot nematode (Meloidogyne arenaria (race 1) resistance (Simpson and Starr 2001; Simspon et al. 2003). In addition, it was the genetic variation of this amphiploid that allowed the construction of the only tetraploid peanut map available to date (Burow et al. 2001). Although hybrids of T AG-6 with peanut showed high fertility, meiosis was not totally normal, and there was substantial segregation distortion of about 25% in the backcrossed population used for making the tetraploid map. This is consistent with evidence, from both molecular and cytogenetic characterization, that none of the wild species used for the creation of


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T AG-6 is ancestral to A. hypogaea. In particular, the genome of A. batizocoi is very distinct cytogenetically, differing from the BB genome of A. hypogaea in the locations of one of its 18S–25S rDNA sites and of its centromeric heterochromatic bands in 9 of the 10 chromosome pairs. The most likely donor of the AA genome was probably A. duranensis and of the BB genome, A. ipaensis (Seijo et al. 2004). Recently an amphidiploid constructed from these two species has been shown to produce very fertile hybrids with A. hypogaea (Fa´vero et al. 2006). Preliminary mapping data indicates diploid genetics and a very low level of marker segregation distortion in an F2 population derived from a cross of this amphidiploid with A. hypogaea. This very strongly suggests that this amphidiploid is a genuine resynthesis of peanut. Clearly, resynthesis of A. hypogaea using ancestral and related species is an effective strategy for the introduction of new genes into the peanut crop while minimizing the problems of sterility and suppression of recombination, both major constraints in the utilization of wild species in breeding. Our understanding of the relationships of the wild genomes is improving in such a way that a routine resynthesis with a range of species now seems feasible. In parallel, advances in genetic mapping have been made that will allow plant breeders to work with complex hybrids more efficiently. Recently the first microsatellitebased genetic map of Arachis was published (Moretzsohn et al. 2005), which will allow the construction of a consensus map for Arachis in the future. New peanut cultivars incorporating wild Arachis genes promise improved resistance to biotic stresses and tolerance to abiotic stresses together with allelic combinations for enhanced yield potential and increased quality profiles that would never have been possible through conventional approaches. F. Developing Exotic Genetic Libraries Another approach for the enhanced utilization of wild relatives in crop improvement was suggested by Eshed and Zamir (1994) based on the development of exotic genetic libraries (also known as introgression lines) consisting of marker-defined genomic regions taken from wild species and introgressed onto the background of elite crop lines. In addition to providing a resource for the discovery and characterization of genes that underlie traits of agricultural value, Zamir (2001) emphasized the immediate utility of the introgression lines for the improvement of agricultural productivity of modern crop varieties and that the exotic libraries should consist of a set of lines, each of which carries a single, defined chromosome segment that originates from a donor

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species in an otherwise uniform elite genetic background and that provides systematic coverage of the entire genome. Once such permanent genetic resources have been generated for diverse donor accessions, they can be screened for multiple phenotypic characteristics to identify alleles of economic importance. Materials are readily available to other researchers interested in using the exotic genetic libraries in applied genomics and plant breeding. Development of introgression lines, more specifically called chromosome segment substitution lines (CSSLs), also known as contig lines or exotic genetic libraries, offers several advantages over conventional populations: (1) they provide useful stocks for highly efficient QTL or gene identification and fine mapping of these; (2) they can contribute to the detection of epistatic interactions between QTLs; and (3) they can be used to map new region-specific DNA markers (Eshed and Zamir 1995; Fridman et al. 2004). Crosses between the exotic genetic stocks can also enhance their trait value through pyramidization of beneficial loci and fixation of positive epistasis. For example, when tomato introgressed lines carrying three independent yield-promoting genomic regions were pyramided, lines produced more than 50% greater yields compared to controls under both wet and dry field conditions (Gur and Zamir 2004). Thus, surpassing yield barriers provides a rationale for implementing similar strategies in other crops that are important for global food security. In addition to the CSSLs developed in tomato (Eshed and Zamir 1995), there are CSSLs in rice (www.ciat.cgiar.org/riceweb/ pdfs/rice_2004_executive_summary.pdf; Sobrizal et al. 1999; Tan et al. 2005), wheat (Pestsova et al. 2001, 2002, 2003, 2006; Liu et al. 2006), and barley (Pickering 1992; Matus et al. 2003; von Korff et al. 2004). Fewer CSSL stocks have been developed for legumes, perhaps due to the greater difficulty of generating interspecific crosses compared to the cereals. One exception was the development of monosomic alien addition lines (MAAL) by Singh et al. (1998) from Glycine max by G. tomentella crosses in soybean. Glycine tomentolla belongs to tertiary gene pool of soybean and contains genes for resistance to rust (Phakopsora pachyrhizi Sydow) and cyst nematode (Heterodera glycines Ichinohe) as well as salt and drought tolerance. In that study, Singh et al. (1998) developed a total of 22 MAAL lines each with the addition of an extra chromosome of G. tomentella to the diploid complement of soybean. The MAAL lines presented new morphological traits and were favorable for broadening the narrow genetic base of cultivated soybean, demonstrating how exotic germplasm that had not been utilizable in the past could be incorporated into breeding programs.


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VI. OUTCOMES OF WILD RELATIVES USE IN GENETIC ENHANCEMENT OF CROPS Crop wild relatives have had important impacts on improving agricultural productivity. Their impact has been greater in cereals as compared to legumes as measured by germplasm and cultivar releases of genotypes with wild ancestry (Table 3.6). To date the largest number and greatest extent of production of wild-derived cultivars is found for rice with commercially grown rice cultivars possessing gene(s) for resistance to grassy stunt and tungro virus, brown plant hopper, and bacterial wilt and tolerance to acid soils in Asia. Indeed, the gene for grassy stunt virus is now routinely incorporated in all new cultivars of rice grown across more than 1 million square kilometers of rice production area. Chinese researchers have exploited yield-enhancing QTL from wild relatives to increase the yield potentials of the hybrids by approximately 1 t ha1, and these are now commercially grown in China (Xiao et al. 1998). In wheat meanwhile, four fusarium head blight or rustresistant wild-derived cultivars (‘Steele’, ‘Hope’, ‘H44–24’, and ‘Marquillo’), and one high-protein cultivar (‘Glupro’) have been released in the United States. ‘Sunnan’ wheat containing a rust-resistant gene from a wild relative has been released in Sweden. Similarly in barley, two cultivars with wild ancestory, ‘Athene’ and ‘Birgit’, were released in Germany. In peanut, four cultivars containing at least one wild Arachis species in their pedigrees have been released in the United States: ‘Spancross’, ‘Tamnut 74’, ‘Coan’, and ‘NemaTam’; the latter two resistant to nematode. ‘ICGV SM 86715’ with resistance to foliar disease was released in Mauritius. Nematode resistance has helped U.S. peanut growers save US $100 million annually (www.unep.org/documents/ default.asp?documentID=399&article). In common bean, a number of interspecific derived lines from crosses with Phaseolus acutifolius have been released in the United States (‘Tara’ and ‘Jules’ and derivates) and Canada (‘OAC Rex’), primarily for their high levels of common bacterial blight resistance. Genes from wild relatives have also been used to improve grain quality. For example, lines containing yield- and/or seed quality–enhancing alleles from wild relatives have been developed in rice, wheat, barley, soybean, and pigeonpea. The greatest amount of introgression has occurred in wheat with approximately 100 genes including traits for resistance to biotic and abiotic stresses and value-added products introgressed into this crop. The species contributing the largest numbers of these resistance genes are Thinopyrum intermedium, Lophopyrum elongatum, Aegilops squarossa, Ae. speltoides, Ae. umbellulata, Triticum monococcum, and T. timopheevii (Fedak 1999).

212 Reference

Munoz et al. 2004 www.manitoba pulse.ca/ Pulse%20Media/PulseBeatmag/ PB_winter-0405_web.pdfwww. ontariobeans.on.ca/OACRex.html

Yadav et al. 2004

ICGV 86699 and ICGV87165, possessing resistance to rust and late leaf spot, elite germplasm developed. A foliar disease–variety, ICGV SM 86715, released in Mauritius. Coan and NemaTAM with resistance to nematode released in USA.

Peanut (Arachis hypogaea) Spancross and Tamnut 74 with increased yield and good pod/seed characteristics released in USA.

Hammons 1970; Simpson and Smith 1975 Reddy et al. 1996; Moss et al. 1997 Moss et al. 1998 Simpson and Starr 2001; Simpson et al. 2003

Oat (Avena sativa) Rapida and Sierra oats, adapted to arid regions and involving A. fatua in their pedigrees, have been Suneson 1967a, b released for cultivation in USA.

Common Bean (Phaseolus vulgaris) Tara and Jules released in USA Resistance to common bacterial blight (CBB) transferred from tepary bean, a wild relative of common bean, into a line XAN159 and then into navy bean breeding lines HR67 and HR45.Another CBB resistance gene transferred from a different tepary bean into OAC Rex.

Chickpea (Cicer arietinum) BG 1100, BG 1101, and BG 1103, involving C. reticulatum and C. arietinum, produced 20% higher seed yield than the best-adapted cultivars and resistant to fusarium wilt (Fusarium oxysporum f. ciceri).

Barley (Hordeum vulgare) North Dakota (ND) 497 and ND 586, the former involving H. bulbosum and the later Schooler and Franckowiak 1981 H. brachyantherum and H. bogdanii, released as germplasm because of tolerance to barley yellow dwarf virus. 2 cultivars, Athene and Birgit, released in Germany had ssp. spontaneum as one of the parents in Arias et al. 1983 their pedigrees. 81882 derived from Vada H. bulbosum cross possess resistance to both powdery mildew and leaf rust. Pickering et al. 1998 72 agronomically useful recombinant inbred lines containing H. bulbosum chromosome segments Pickering and Johnston 2005 associated with resistance to several leaf diseases developed.

Elite Germplasm/Cultivars Containing Gene(s) from Wild Relatives

Table 3.6. Elite germplasm/cultivars developed from interspecific crosses in barley, chickpea, common bean, oat, peanut, rice, soybean, and wheat.

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Wheat (Triticum aestivum) A hexaploid wheat cultivar, Hope, was developed that contain Sr2 for resistance to stem rust from T. turgidum spp. dicoccum.

McFadden 1930 (continues)

www.irri.org/media/hotline/ hljun2003 www.irri.org/publications/ today/pdfs/3–1/feral.pdf

Zhang et al. 2006 Yoon et al. 2006

Song et al. 2005

Brar and Khush 1997 Brar and Khush 1997 Liu et al. 1999

Brar and Khush 1997

Soybean (Glycine max) SS201 and SS202, involving wild species G. soja, released as special purpose cultivars for the Fehr et al. 1990a, b production of soy sprouts and Japanese fermented product natto. Bean 13, selected for strong rooting capacity and tolerance to drought from an intergeric cross, released Li 1990 in Korea. Pearl soybean, involving unknown wild relative in its pedigree, released as a special-purpose cultivar Carter Jr et al. 1995 for the development of Japanese fermented product natto.

A tungro virus–resistant cultivar, Matatag 9, released in Philippines.

Rice (oryza sativa) IR28, IR29, IR30, IR32, IR34, and IR36, containing gene for grassy stunt virus, released for cultivation in many countries. IR2701-625-3, a sister selection of IR36, resistant to grassy stunt virus, released in Kerala, India. MTL98, MTL103, and MTL105, all containing gene(s) for brown plant hopper, released in Vietnam. Tong 31 and Tong 35, containing genes from Zizania latifolia, with improved grain quality and resistance to blast and sheeth blight, are grown in more than 600,000 ha in China. O. rufipogon contributed genes for cold tolerance and other abiotic stresses in well-known Chinese cultivar, Zhongshan No. 1. IL23 containing genes for drought tolerance from Oryza rufipogon developed. An introgression line, HG101, a backcross derivative involving japonica cv. Hwaseongbyeo and O. grandiglumis, outperformed Hwaseongbyeo for a few grain characterstics. IR 73678–6–9-B released as AS996 for cultivation under acid sulfate soils in Vietnam.

11 interspecific derivatives possessing resistance to early leaf spot, root-knot nematode, southern corn Stalker and Lynch 2002; Stalker rootworm, corn earworm, fall armyworm, and velbetbean caterpillar released as improved germplasm. et al. 2002a, b Advanced lines selected from interspecific crosses possessing resistance to root-knot nematode and Holbrook et al. 2003 tomato spotted wilt virus in A. hypogaea. 5 rust and late leafspot–resistant peanut germplasm developed Singh et al. 2003

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Chuanmai 42, a derivative from synthetic wheat and local wheat, released in China. A synthetic wheat derivative, Carmona, that has better grain quality, resistance to foliar diseases, and adapted to zero tillage, released in Spain.

Sharma and Gill 1983

A hard red winter wheat cultivar, Plainsman V released in USA, has high protein content probably transferred from Aegilops ovata. A wheat cultivar, Amigo, contains both wheat-rye and wheat-Aegilops elongatum translocations in its pedigree. 4 synthetic hexaploid wheat germplasm lines derived from T. tauschii and T. turgidum possess immunity to karnal bunt. A new salt-tolerant cereal, containing salt-tolerant gene from Thinopyrum bessarabicum, performed much better at 150 mol m3 concentration. An amphidiploid, OK 7211542, derived from hybrids of wheat with Th. intermedium or T. ponticum, is immune to BYDV, a serious disease that has no source of resistance in wheat primary gene pool. Swedish wheat cultivar, Sunnan, contain a translocation from Th. ponticum with leaf rust–resistant gene Lr19; Marquillo and H 44–24, both released in USA, contain T. dicoccoides in their pedigrees. An agronomically useful wheat-Ae. geniculata line, 2K-11-1, possesses resistance to leaf and stripe rust. Crossing the substitution line LDN (DIC-3A) to 4 T. tauschii accessions developed 4 synthetic hexaploid wheat germplasm lines resistant to fusarium head blight. Several wheat-alien species derivatives exhibited resistance to fusarium head blight (FHB) as that of Sumani 3. A hard red spring wheat cultivar, Steele-ND, resistant to fusarium head blight, released in USA. Kansas State University released 30 hard red winter wheat or durum wheat germplasm possessing resistance to hessian fly; green bug; soil-borne mosaic and spindle streak mosaic virus; leaf, stem, and stripe rust; powdery mildew; tan spot; and fusarium head blight, all derived from crosses involving wild relatives. Plainsman V has Aegilops ovata and Agent has Agropyron elongatum in their pedigrees, both released in USA. The 3n chromosome carrying aluminium-tolerant gene from Ae. uniaristata transferred into a model wheat cultivar ‘‘Chinese Spring’’ with no undesirable trait (spike neck break) from Ae. uniaristata.

www.oznet.ksu.edu/library/ crpsl2/srl126.pdf www.dfid-psp.org/ccstudio/ publications/annualreport/ 2004_aluminium.pdf www.cimmyt.org/english/ wps/news/wildWht.htm

Mergoum et al. 2005 www.k-state.edu/wgrc/ germplasm/grmplasm.html

Oliver et al. 2005

Aghaee-Sarbarzeh et al. 2002 Berzonsky et al. 2004

Bartos et al. 2002

Ayala et al. 2001

King et al. 1997a, b

Villareal et al. 1996

Cai 1994

Reference

Elite Germplasm/Cultivars Containing Gene(s) from Wild Relatives



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Wild relatives have contributed in development of cytoplasmic male sterility sources and/or fertility restoration systems that have been widely used in the production of hybrids in rice and pigeonpea. Similarly, genes for promiscuous nodulation and seed longevity, which led to the success of soybean cultivars in West Africa, were introgressed from wild sprawling soybean accessions mostly from Indonesia (Ortiz 2004).

VII. FUTURE OUTLOOK Wild relatives have been extensively used to transfer resistance to many pests and diseases in agriculturally important crops. Furthermore, exploitation of wild relatives in rice, wheat, barley, oat, sorghum, common bean, soybean, potato, and tomato have demonstrated that although wild ancestors may be phenotypically inferior to modernday cultivars, they harbor genes that can contribute to increased yield and/or seed quality traits. The demand for food will continue to increase as the world population approaches 7.5 billion people in 2020 (Pinstrup-Anderson et al. 1999). As gains from conventional breeding are gradually exhausted, further yield growth will be generated by the combination of conventional breeding with wide crossing, genomics, and transgenic technologies to tailor better-adapted crop cultivars to diverse growing conditions (Rosegrant et al. 1995). With the development of user-friendly markers and availability of saturated genetic linkage maps in crop species, it is likely that there will be greater exploitation of wild relatives to identify the alleles associated with yield-enhancing traits. When these genes are introgressed/pyramided into new genetic backgrounds, they could bring large increases in yield, as already demonstrated in pioneering studies with tomato. It is essential that CGIAR institutes concentrate on developing chromosome segment substitution lines targeting chromosomal introgressions from wild relatives that can be provided to plant breeders as a resource for the discovery and utilization of genes associated with improved agronomic traits. In conclusion, wild species germplasm holds great promise both for broadening the genetic base of many crops and for enhancing sustainable crop production.

VIII. ACKNOWLEDGMENTS The authors thank Vincent Vadez of ICRISAT for his critical review of the manuscript and the staff of ICRISAT library for their efforts in

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conducting literature searches and arranging for reprints. Funding support from Generation Challenge Program to Sangam Dwivedi is gratefully acknowledged.

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4 Allelopathic Crop Development: Molecular and Traditional Plant Breeding Approaches Cećile Bertin McGill University Department of Plant Science Raymond Building, 21111 Lakeshore Road Ste. Anne De Bellevue, Quebec H9X 3V9 Canada Leslie A. Weston Cornell University Department Of HorticultureIthaca NY 14853 USA Harlene Kaur Max Planck Institute for Chemical Ecology Molecular Biology Program, Jenna, Germany

I. INTRODUCTION II. COMMON BREEDING TECHNIQUES FOR SELECTION OF WEED-SUPPRESSIVE CROPS A. The Fate of Competitive Traits in Plants B. Use of Molecular Markers III. USE OF TRANSGENES TO PRODUCE ALLELOPATHIC CROPS A. Enhancement of Allelopathic Crops by Stimulating Allelochemical Production B. Identification of Key Enzymes Involved in Biosynthetic Pathways C. Forward Genetics: From Phenotype to Gene D. Introducing a New Gene in a Crop that Does Not Produce Alleochemicals IV. USE OF WHOLE-GENOME TECHNIQUES TO UNDERSTAND ALLELOPATHIC PHENOMENA A. Genomic Approaches B. Metabolomic Approaches C. Proteomic Approaches V. CONCLUSION VI. LITERATURE CITED


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I. INTRODUCTION Many plants release secondary metabolites into the environment that influence the growth and establishment of co-occurring species and alter competitiveness amongst the existing vegetation for limited light, water, and nutrient resources (Inderjit and Weston 2003; Weston and Duke 2003; Bais et al. 2004). Secondary metabolites can also serve as defensive tools against herbivores, pathogens, and parasites (Lovett 1990). The release of defensive secondary metabolites into the environment is referred to allelopathy, and the chemicals involved in these reactions are known as allelochemicals. Allelochemicals may influence growth patterns either by their direct release or after undergoing chemical/microbial degradation to other bioactive metabolites (Inderjit and Keating, 1999). Allelopathic interference may influence the evolution of plant communities by inhibiting the growth of more susceptible species via chemical interference and by favoring plants that exhibit resistance to phytotoxins (Schulz and Wieland 1999; Bais et al. 2004). In addition, allelopathic species have been proposed to have effects on agriculture as weed suppressants when used as cover crops or within intercropping systems (Weston and Duke 2003). A more precise understanding of allelopathy is essential for practical applications related to the improvement of crop competitive ability using allelopathy for weed suppression. If effective, this could lead to reduced dependency on herbicides in both agricultural and turf settings. Allelopathy is generally not recognized as effective tool for weed management in agronomic crops as weeds are usually more allelopathic than crop species (Duke et al. 2001). Weed management during the twenty-first century has relied essentially on the use of synthetic herbicides, primarily because of their low cost and their effective weed control. However, concerns about the impacts of pesticides on human health and the environment have resulted in limitations in usage of synthetic herbicides and renewed interest in the development of alternative weed management strategies (Duke et al. 2001; Bertin and Weston 2004). To meet the future needs of agricultural producers and landscape managers, it will be important to evaluate the underlying mechanisms that govern interaction between plants and determine the potential for biorational weed management as well as the degree to which it has been achieved in various settings. Skepticism about allelopathy in the past may have limited our ability to evaluate and optimize the processes that influence implementation of ecologically based pest management (Weston and Duke 2003). Extensive mechanistic understanding of the ecological principles mediating plant-plant interactions may enable us

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to develop biorational pest management practices that are effective. Toward this goal, a specific model plant system that would enable the study of allelopathy could assist in the development of biorational weed management through the integration of common conventional methods with recently developed molecular approaches. Therefore, the utilization of allelopathy through breeding and/or genetic manipulation in crop species may provide specific opportunities for successful implementation of alternative weed management systems. It seems that allelopathy can play an important role in the development of natural communities and dominance by non-native invasive species (Bais et al. 2003; Weston and Duke 2003; Weston et al. 2005). Despite the potential ecological and agronomic importance of allelopathy, relatively little is known about allelochemical mode(s) of action and the molecular target sites mediating their toxicity (Weston and Duke 2003). Relatively little emphasis has been associated with breeding and molecular approaches and their application in the development of crops with enhanced weed-suppressive ability (Duke et al. 2001). Recently genome-scale approaches to plant science and particularly to crop selection have expanded our knowledge of plant protection and some defense interactions. By specifically examining genome scale changes that occur in a plant under stress conditions—for example, pest-pathogen interactions, changes in edaphic factors, and abiotic changes—we can gain useful information regarding plant defensive responses and its mechanisms. The application of genomics and appropriate selection of model crop systems possessing allelopathic characteristics could also be an efficient tool with which to further address the study of plant-plant interactions, despite recent concerns about use of genetically modified organisms (GMOs) in crop production systems. This review provides a summary of the current efforts in traditional plant breeding and molecular approaches (collectively referred to as molecular plant breeding) utilized in the field of allelopathy, as well as an overview of the techniques that could be used to improve our knowledge in the area of plant interference(s). It is clear from our review of the literature that enhanced collaboration and interaction among weed scientists, ecologists, molecular biologists, and plant breeders will be necessary to advance the utilization of allelopathy in agriculture and natural settings for weed suppression. It is also clear that advances in gene isolation and gene insertion have resulted in increased understanding of secondary plant product sequestration in plant cells and offer opportunities for enhanced development of allelopathic or weedsuppressive crops. However, due to the social and political pressures associated with GMOs and their release into the environment at this

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time, traditional plant breeding approaches for selection of enhanced competitive ability or interference with weeds may offer the greatest opportunities for development of crops with enhanced weed-suppressive abilities. Additional research in the areas of plant-plant interactions and chemical ecology of higher plants is just now beginning to offer opportunities for commercial crop development.

II. COMMON BREEDING TECHNIQUES FOR SELECTION OF WEED-SUPPRESSIVE CROPS A. The Fate of Competitive Traits in Plants In contrast to breeding for plant resistance to insects and diseases, improving competitive or weed-suppressive ability in crops using traditional plant breeding has received little attention. This may be due to a number of factors, including the relative lack of understanding of the mechanisms of plant interference and the absence of a means of reliably selecting for enhanced weed-suppressive traits. The competitive ability of crops is assumed to be associated with exploitation of resources, differential resource allocation, or allelopathic interference (Aarsen 1983). Plant morphological traits that can affect resource allocation include interception of radiation and subsequent photosynthetic capacity (Eisele and Kopke 1997), early vigor (Acciaresi et al. 2001), plant height, tillering capacity, seed size, and initial shoot as well as root growth rates. These morphological traits are usually linked to exploitation of resources while interference competition is attributed to allelopathy and factors limiting access to resources (Bertholdsson 2005). Most large breeding corporations have continued to put major breeding emphasis on yield and disease resistance in edible crops. However, in nonedible crops such as turfgrass, selection for turf quality and ability to compete effectively with surrounding vegetation has been important for low-maintenance cultivars. Before beginning any plant breeding program to enhance weedsuppressive ability, it is important to utilize a practical and effective screening method for measurement of allelopathic potential. For example, various methods including the stair-step method for collection of hydroponically produced exudates (Bonner 1950; Liu and Lovett 1993), hydroponic culture assays involving whole plants (Einhelig 1985), relay-seeding techniques (Navarez and Olofsdotter 1996), and agar medium testing (Fujii 1992; Wu et al. 1999), cluster analysis using HPLC (Mattice et al. 2001), aqueous extraction of plant residues (Kim et al. 1999;


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Ebana et al. 2001) and small well plate bioassays (Rimando et al. 1998) have been widely utilized for evaluation of allelopathic potential of several plant species. Some methods have drawbacks, such as the stairstep collection of exudates, which can be costly and time-consuming to complete. However, the creation of a reliable and inexpensive method for screening allelopathic plants under controlled conditions is of utmost importance before further exploration of the plant’s secondary chemistry or genome is undertaken. Furthermore, consistent field verification of allelopathic traits would be critical in developing rationale for undertaking a long-term selection and breeding project. Unfortunately, plant traits associated with interference competition and morphological traits leading to enhanced competition are often negatively correlated with crop yield or plant productivity and therefore have not been greatly exploited for use in crop selection programs (Olofsdotter et al. 2002). For example, Bertholdsson (2005) has recently shown that barley (Hordeum spp.) land races and cultivars from northern Europe, which span the time frame of 100 years of production, generally show a decreasing tendency to exhibit allelopthic potential over time as they become modernized. One could hypothesize that recent selection pressure and breeding have resulted in the dilution of genes associated with the production of allelochemicals from cultivars, leading to declining weed-suppressive activity. Despite these obstacles, it is conceivable that one could develop breeding strategies where plant competitive traits are closely evaluated and included in trait selection. For example, a rice (Oryza spp.) plant type has been recently selected with favorable competitive traits from O. glaberrima and high-yield traits from O. sativa (Jones et al. 1996). Compared with traditional cultivars, this cultivar exhibited not only substantial yield increases (24%) but also showed increased competitive ability against weeds (Olofsdotter et al. 2002). The competitive ability of barley and wheat can also be improved by utilization of traditional breeding techniques (Christensen 1995; Acciaresi et al. 2001). In a recent study, Bertholdsson (2005) identified traits in spring barley (H. vulgare) and spring wheat (Triticum aestivum) that appeared to be closely related to the competitive ability of the crops. In both crops, the only parameters besides straw length, heading, and maturity that significantly contributed to competitiveness were early vigor and allelopathic potential (Bertholdsson 2005). Other researchers have also noted that, over time, the development of new cultivars from land races has apparently resulted in loss of potential to produce the allelochemicals DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) and DIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one) (Sicker et al. 2000). Glycosyl

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transferase enzyme activities have also been observed to decrease in recent cultivars, possibly as a result of gene loss (Frey et al. 2003). Moreover, it is hypothesized that the gene loss process has resulted in DIBOA as a climax benzoxazinoid metabolite in rye (Secale cereale) compared to DIMBOA in wheat and maize (Frey et al. 2003). Breeding programs directed toward increasing yields are not, however, solely responsible for the loss of competitive ability within a plant species. Environmental conditions and agricultural selection pressure may also play a significant role in impacting the evolution of allelopathic traits over time. Recent evidence suggests that invasive weedy plants may utilize their enhanced allelopathic traits in their nonnative environments to influence invasive success (Bais et al. 2003; Weston et al. 2005). Interestingly, certain secondary chemicals are absent in a given taxon irrespective of their presence in neighboring or ancestral taxa (Wink 2003). This observation reveals that differential expression of specified genes resulting from interaction with an ecological component is notable. The absence of trait expression in the phylogenetically derived groups compared to ancestral groups may be due to loss of enzyme activity responsible for expression of a particular trait. Wink (2003) has proposed that this inability to express an allelopathic trait may be associated with gene inactivation rather than total gene loss. Further phylogentic studies are required to confirm whether gene inactivation or total gene loss is associated with lack of allelopathic expression in genetically improved crops. Although traditional breeding techniques may have influenced the weed-suppressing ability of crop plants, Zhou et al. (2005) have shown conversely that there is no correlation between allelopathic traits and competitive agronomic growth characteristics in nine allelopathic rice cultivars produced in southern China. Therefore, multigenic traits involved in plant interference may prove difficult to select for using traditional breeding methods. B. Use of Molecular Markers Since the competitive ability of a crop can be positively modulated by increasing the fitness of a plant in a particular ecosystem, genetically encoded traits such as nutrient acquisition capacity and nutrient utilization efficiency in the plant can be optimized and could eventually lead to enhanced competitive ability (Kirk et al. 1999). Enhanced competitive ability could also be achieved by selecting plants for reduced susceptibility to biotic and abiotic stress resulting in healthier crops. Finally, plant defense mechanisms including plant microbial symbioses


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and allelopathy could potentially be incorporated in certain crops to improve the competitive ability of valuable cultivated plants. In addition, competitive ability of plants could likely be enhanced with additional study of complex plant-microbial interactions (Olofsdotter et al. 2002). Recently plant breeders have found that genetically based variation in the allelopathic activity among individuals in plant populations can successfully be evaluated using molecular tools such as RAPD (Random Amplified Polymorphic DNA) markers identifying the specific QTLs (quantitative trait loci) that are involved in gene expression (McRoberts et al. 1999). Genotypic population diversity among O. sativa accessions (250 recombinant inbreds between IAC 165, highly allelopathic japonica upland cultivar and CO 39, weakly allelopathic indica irrigated cultivar) was resolved using genetic markers. These findings suggest the involvement of only four QTLs related to three chromosomes (Jensen et al., 2001). However, Ebana et al. (2001) reported the involvement of seven QTLs (with phenotypic variability ranging between 9.4% and 16.1%) in rice allelopathy, located on six chromosomes as assessed through RFLP (Restriction Fragment Length Polymorphism) markers. Employment of RFLP, AFLP (Amplified Fragment Length Polymorphism) and SSR (simple sequence repeats or microsatellite) markers along with composite internal mapping distinguished only two QTLs positioned on chromosome 2B in wheat, which contributed to its allelopathic activities (Wu et al. 2003). QTL mapping with molecular markers (linkage maps) has also proven useful in determining chromosomal location and potential genetic variation related to ecological fitness traits at the ecosystem level (Mitchell-Olds and Pedersen 1998; Olofsdotter and Andersen 2004; Thomas and Klaper 2004). It has been shown that if a high number of QTLs with little effect on trait selection are implicated in weed suppression or successful competition, a more traditional breeding method can be a reasonable alternative for crop improvement from a weed-suppressive standpoint (Courtois and Olofsdotter 1998). The principle behind the use of traditional breeding for genetic studies of plants is relatively simple: Two parents chosen based on their contrasting traits are crossed, giving rise to recombinant inbred lines (RILs) using the single-seed descent (SSD) method. This method, which has the disadvantage of requiring considerable time and labor, consists of advancing the F2 without selection for two or three generations (sometime four to five generations) in such a way that each F4 or F5 seed traces back to a different F2 plant. In this case, only one seed is retained from each plant in each generation. This process is typically repeated for two or more generations so that traits

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are fixed and the plants having desirable characteristics can be further selected. Once a reasonable degree of fixation is obtained, the allelopathic potential of the RILs can be evaluated. Seed can then be increased and tested under field conditions for use as allelopathic cultivars, under varying degrees of weed infestation. The SSD method is frequently simpler than the pedigree method. The SSD method of plant selection has several advantages, including a quick increase of the additive variance among families the need for only a small breeding space and savings in terms of time and labor, and suitability for selection of traits with low heritability, such as allelopathy, where visual selection is not effective (Moreno-Gonza´lez and Cubero 1993). The three-line hybrid rice that is popularly cultivated in China is thought to be a successful competitor with weeds because of its rapid and profuse vegetative growth in comparison with many inbred lines. Additional suppression could potentially be provided if allelopathic traits could also be introduced into an elite restorer line to further develop three-line hybrid rice. Lin et al. (2000) attempted a simultaneous backcrossing and selfing breeding method to develop hybrid rice with allelopathic activity and its counterpart, isogenic hybrid rice with nonallelopathic effects on weeds. Three lines of rice, Kouketsumochi, Rexmont, and IR24, were used as the donors of allelopathy, nonallelopathy, and restoring genes, respectively. The backcrossing method was used to develop restorer lines with allelopathic activity and its isogenic line with nonallelopathic genes. Allelopathic potential and heterotic performance were analyzed in both the laboratory and greenhouse using the water-extract method and density-dependent method, respectively (Richardson and William 1988; Lin et al. 2000). Interestingly, results suggested that the heterotic effect on allelopathy in rice was positively significant, showing higher heterosis over the midparent. In addition, this specific hybrid rice had an additive suppressive effect on the target weed of barnyardgrass (Echinochloa crus-gala l.), exhibiting a large deviation from the resource competition curve generated with the progenitors (Lin et al. 2000). This approach seems to be a promising method that could potentially incorporate allelopathic traits into hybrid crops exhibiting high-yield capacities, but to date this technique has only been applied to rice selection. Another traditional but effective breeding method involves the production of isogenic lines by crossing two parents with contrasting traits through backcrossing to produce near-isogenic lines (NILs) carrying different genes. The allelopathic potential of the NILs can later be determined when a reasonable degree of fixation is obtained. Results in rice have shown that QTLs could be introgressed into elite backgrounds


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through marker-aided backcrosses and that NILs could be produced (Shen et al. 2001). In addition, NILs introgressed with QTLs for allelopathy could potentially be used to demonstrate the effects of allelopathy in field settings without being encumbered by confounding effects of competition. In addition, this method could also be utilized to evaluate allelopathic potential under various conditions as well as the effect of genotype-environment interactions (Olofsdotter et al. 2002). Further experimentation with other crop germplasm could potentially result in successful utilization of NILs in ongoing breeding programs. Despite the influence of biotechnology and genetic engineering on crop production and selection of new cultivars, traditional plant breeding research and breeding methodology will likely continue to play a significant role in future breeding programs. Further refinement of these methods and better knowledge of classical plant breeding are a prerequisite for the rational incorporation of new tools, such as molecular markers, in traditional breeding programs. Collaboration between geneticists and plant breeders is critical when conducting allelopathic research to identify the genes that encode for the weed-suppressive ability and to discuss and develop strategies for improving plant characteristics. It will be important to continually consult breeders throughout the research process in order to successfully identify trait indicators for the feature of interest that will be useful in a breeding program. Communication is crucial because breeders generally do not include plant features, such as allelopathy or successful resource competition that are not selected for in a typical breeding program. With continued collaboration with skilled breeders and molecular biologists, rapid advances in selection for weed-suppressive traits are likely.

III. USE OF TRANSGENES TO PRODUCE ALLELOPATHIC CROPS Since traditional breeding methods, as described earlier, have not been particularly successful in producing crops with allelopathic ability without affecting crop yield or have not been utilized to select for weed-suppressive abilities in many crops, genetic engineering may be viewed as a novel strategy to improve competitive ability of crops without subsequently affecting crop yields. Since biocontrol agents or efficacious organic products are not yet widely available for weed management in high-value crops, the successful development of allelopathic crops in some species may be dependent on the appropriate use of biotechnology.

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Genetic engineering has been proposed as one means to develop new strategies for weed management in traditional agricultural production as well as landscape settings (Weston and Duke 2003). Various novel strategies have been identified, including: 1. Creating allelopathic crops producing sterile seeds by transferring ‘‘terminator’’ or floral development inhibitor genes so that the weed-suppressive gene is unable to transfer to related weedy species. The technique involves the development of a gene that can be turned on or off in a developmentally regulated fashion and a procedure for controlling the expression of an engineered gene using a chemical inducer or other factor. 2. Decreasing weed competitiveness and fitness by inducing certain ‘‘unfitness factors’’ that could spread throughout the weedy population. This technique is generally linked to the utilization of a specific selection pressure introduced to the crop environment, such as a herbicide. 3. Improving crop competitiveness by introgression of favorable genes, which would increase plant vigor and accessibility to water, nutrients, and light. 4. Developing allelopathic crop cultivars with weed-suppressive potential through genetic manipulation. Allelochemicals are often not the ideal compounds to manipulate with enhanced selection in crop germplasm. Generally, allelochemicals are complex molecules and are dependent on a multigene system for biosynthesis. In addition, they generally have a short half-life in the rhizosphere because of complex interactions with biotic and abiotic components of soil (Inderjit and Weston 2000; Bertin et al. 2003). Therefore, the strategy of transforming a crop so that it possesses enhanced allelopathic characteristics might be more challenging than one would suspect. A multigene expression system may have to be introduced into a higher plant, and its regulation must be optimized so that the transformed crop can successfully produce the allelochemical(s) and then localize in the proper tissue or cellular compartment. Also, consideration must be given to the idea of continuous production of the allelochemicals of interest to overcome issues associated with short residual life. However, the continuous production of secondary metabolites may occur at a considerable metabolic cost to the plant, which may result in an overall decrease in crop yield. Although allelochemicals are natural plant products and some consider ‘‘natural products’’ to be safer to introduce into the environment than synthetic pesticides,


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allelochemicals can still exhibit potent mammalian toxicity or carcinogenicity. Therefore, it would be appropriate to advocate implementing some basic toxicity testing for widely utilized natural products in a manner similar to that utilized for herbicide screening (Duke et al. 2001). The public concern about GMOs and release of chemicals into the environment may also result in enhanced scrutiny or limitations in the development of allelopathic crops, even if these products are naturally produced and released by living plants. The ultimate choice of an ideal allelopathic crop to manipulate for enhanced secondary product production will be critical to ensure successful production and release of the phytotoxin(s). Therefore, an ideal allelopathic crop should release into the environment (1) highly potent phytotoxin(s) that do not require a high metabolic cost to the plant for their production, (2) phytotoxin(s) to which the crop is highly tolerant or resistant to avoid autotoxicity, (3) allelochemical(s) with more than one potential molecular target site to lower the probability of the development of resistance, (4) phytotoxin(s) that are produced and secreted directly into the soil rhizosphere by the living root system, and finally (5) phytotoxin(s) with high specific activity to other plants, some residual activity in the soil rhizosphere, and limited effects on other micro- or macrobiota (Duke et al. 2001). Obviously, it is difficult to find a specific plant/allelochemical production system that meets all of these requirements, but there is some progress being made in selection of weed-suppressive traits in certain crops at this time (Weston 2005). A. Enhancement of Allelopathic Crop by Stimulating Allelochemical Production If the biosynthetic pathway of the allelochemical of interest is known, its synthesis can potentially be modified by targeting the specific genes involved using a posttranscriptional gene silencing approach. Furthermore, research has shown that gene overexpression and antisense knockout techniques can be used to alter the quality and quantity of secondary metabolites. For example, overexpression of a cDNA for a key enzyme in a pathway that produces isopentenyl pyrophosphate used in monoterpene synthesis resulted into a 50% increase of monoterpene production while the oil composition remained unchanged. A 50% reduction in an undesired component of peppermint oil and as its precursor was observed as well as an increase of one of the end products of interest when antisense knockdown techniques were applied (Wagner et al. 2004). This approach could be utilized with allelopathic crop development such that the secondary pathways for production of

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allelochemicals or phytoalexins could be altered resulting in an increase in allelochemical production (Wagner et al. 2004). Increasing production of an endogenous phytotoxin through overexpression of a rate-limiting enzyme appears on the surface to be a relatively simple strategy. However, since the control points in the biosynthetic pathway of allelochemicals are generally not well understood, this strategy may not in reality be particularly easy or straightforward. The process is further complicated by the fact that allelochemicals are usually synthesized in very small quantities and their distribution within a plant may be restricted to a specific tissue. For example, the allelochemical sorgoleone, a potent inhibitor of several primary plant processes including electron transport in photosynthesis and respiration, is produced only in living root hairs of sorghum species (Czarnota et al. 2001; Czarnota et al. 2003). Achieving successful suppression or knockout of specific genes involved in the sorgoleone biosynthetic pathway has required manipulation of the environment surrounding root hairs (Yang et al 2003). The development of biorational approaches for the successful production and utilization of natural products, especially of medicinal drugs, has also unfortunately not been particularly successful (Borevitz et al. 2000). This failure is likely associated with stringent spatial and temporal transcriptional controls of the biosynthesis of toxic natural products during plant development (Fowler 1983; Robins 1994; Facchini and Deluca 1995). Transgenic manipulation to override these controls may be the key to enhance production of secondary metabolites. However, one must ensure that autotoxicity to the plant tissue itself is prevented. With recent advances in transgenic manipulation and incorporation of multigene traits in plant germplasm, future selection and utilization of crops producing potent allelochemicals may not be far off. A strategy based on Agrobacterium-mediated transformation and referred to as activation tagging is associated with the insertion of an enhancer from the cauliflower mosaic virus 35S transcript promoter that results in strong activation of the adjacent gene (Weigel et al. 2000). The T-DNA (transfer DNA) carrying the 35S at its right border is spliced into the plant genome at random sites. To survey the genome of the plant of interest as well as to isolate genes that affect allelochemical production, large collections of independent activation-tagged lines need to be screened. This requires investment in time and labor. However, the TDNA technology allows the identification of regulatory genes without the need to understand the individual enzymatic steps of the pathway, which are in many cases not elucidated. Used to characterize transcriptional regulators of natural product pathways, this approach has been


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successfully used to identify a jasmonate-responsive transcription factor involved in the rapid response to the stress hormone jasmonate (Van der Fits and Memelink 2000). Because the biosynthesis of many secondary metabolites is induced by jasmonate, the regulation of this gene, which influences many other genes involved in jasmonate-responsive metabolism, may lead to the opportunity to produce higher concentrations of secondary metabolites in plant systems (Borevitz et al. 2000; Van der Fits and Memelink 2000; Dixon 2001). The alteration of environmental conditions has been shown to further modify the production and release of allelochemicals and secondary products into the environment (Reigosa et al. 2002). For example, the level of UV radiation on rice leaves influences allelopathic activity since newly synthesized metabolites with allelopathic characteristics as well as increased production of known allelochemicals have been detected under enhanced radiation. To determine if a specific promoter confers responsiveness to environmental stressors such as UV irradiation, Shin et al. (2000) examined the expression of GUS activity of tobacco plants transformed with the constructs of various CASC (Capsicum annuum sequisterpene cyclase) promoters fused into the GUS gene so that they would easily and rapidly be able to investigate the effective motifs’ response to enhanced UV light. The levels of GUS activity for transgenic plants with pBl121-KF-1 and pBl121-KF-6 were significantly increased by UV irradiation, in comparison with nonirradiated plants (Shin et al. 2000). These results suggest that CASC promoters of KF-1 and KF-6 may contain cis-acting elements capable of conferring quantitative expression patterns that are associated with UV irradiation. This promoter, which may be specific to UV, can be used for overexpression of specific promoters constructed for allelochemical-producing genes (Shin et al. 2000). Also, Yang et al. (2004) showed that environmental conditions will affect development of root hairs in sorghum species. Since those root hairs are the site of release of sorgoleone, those modified growth conditions did affect sorgoleone production and release. Recently Dayan (2006) demonstrated that sorgoleone biosynthesis in sorghum can be induced by exposure to extracts of weedy plants. Consequently, the regulation of genes associated with allelopathy can be achieved by enhancing a specific response to plant-weed competition or environmental stresses. For example, since the cinnamic acid 4-hydroxylase (CA4H) gene likely plays a critical role in the elevation of the allelopathic potential of rice plants, CASC promoters were fused to the CA4H gene (Kim and Shin 2003). Introduced in a binary plant expression vector, the investigation of the regulatory effects on gene coding for this enzyme, which

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catalyzes the conversion of cinnamic acid to p-coumaric acid, may be feasible. This conversion is a key reaction in the biosynthesis of a large number of phenolic compounds in plants. Such a strategy could then be extended to the field of allelopathy and would allow the investigation of the regulatory effects on gene coding for allelochemical-producing enzymes. The environmental and hormonal regulation associated with the biosynthesis of inducible allelochemicals, as opposed to constitutively produced products, should potentially be considered when considering alteration of the allelochemical production within a crop. It should be noted that enhancement of allelochemical production is not always beneficial. For instance, increased glucosinolate production in Brassica species caused an increase in mortality of ladybird larvae when feeding on aphids produced on this tissue (Siemens et al. 2002). This example demonstrates the importance of plant allelochemicals on tertiary trophic levels. Potential interactions must be considered when manipulating plants, whether by gene modification or by conventional breeding. Furthermore, alteration of a biosynthetic pathway may have unintended consequences. For example, the overexpression of tryptophan carboxylase in periwinckle (Catharanthus roseus) with the goal of increasing the production of anticancer compounds actually resulted in a significant reduction of plant growth, potentially due to the disruption of overall plant metabolism by a marked reduction in tryptophan pools (Canel et al. 1998). B. Identification of Key Enzymes Involved in Biosynthetic Pathways The diversity and complexity of biosynthetic pathways in higher plants has been seen as a barrier to understanding the function of plant secondary metabolites, especially allelochemicals. Another approach that could be potentially considered is the isolation and identification of specific gene(s) encoding for allelochemical production through purification of the corresponding enzymes associated with these processes and later identification of responsible genes. Unfortunately, isolation and purification of minute quantities of bioactive enzymes from plant tissues is not a trivial matter. However, there have been some successful applications of genetic and genomic approaches to identify genes involved in plant secondary metabolite biosynthesis. These approaches have also been used to study phytotoxic bacteria, revealing some specific enzymes of secondary metabolism and their corresponding DNA sequences (Lydon 1996). However, the biosynthesis of a particular phytotoxin usually consists of a multistep pathway, and a clear understanding of the regulation of biosynthesis is a prerequisite for steering


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such a pathway toward a desired outcome (e.g., enhanced product synthesis, accumulation of the end product, deletion of side reactions, etc.). One key limiting factor in such an approach is the low abundance of the participating enzymes contained in plant cells. Generally plant enzymes can be initially detected in specialized cells or tissues, which can be difficult to fractionate. Plant enzymes involved in the biosynthesis usually operate as single proteins rather than protein complexes. Additionally, their genes are usually not clustered, in contrast to prokaryotic cells (i.e., for most antibiotics), making genetic characterization of pathways in plants generally more difficult than in bacteria or fungi. Purified proteins can be either sequenced to later obtain the gene sequence using PCR techniques or can be subjected to reverse genetics. In the case of reverse genetics, the purified protein will be used to screen a cDNA expression library for a clone expressing the protein of interest. Efficient and effective reverse genetic approaches, such as differential display or subtractive hybridization, have assisted in isolating, cloning, and characterizing novel induced genes with respect to allelochemical production (Yang et al. 2004). One example of this approach is the elucidation of the 10-step biosynthetic pathway of the antiarrhythmic alkaloid ajmaline at the enzyme level (Gao et al. 2002). The identification of two HIS cytochrome P450s that catalyze the entry-point reaction into isoflavanoid plytoalexin biosynthesis was also realized, thanks to the use of comparative EST database mining (Jung et al. 2000). In another example, the Mutator transposable element was used to map and clone the first enzyme specific for DIMBOA biosynthesis in maize (Frey et al. 1997). Another example of secondary product manipulation involves the lipid resorcinol pathway with the goal to enhance the allelopathic potential of rice cultivars. Lipid resorcinols are secondary metabolites not only found in rice but also probably responsible for the allelopathic potential of other monocot species. Yang et al. (2004) and Dayan et al. (2005) identified genes that are putatively involved in the ring formation of sorgoleone, an allelochemical produced by root hairs of sorghum spp.; these genes are likely involved in the biosynthetic pathway of sorgoleone and other lipid resorcinols. Dayan et al. (2005) also identified polyketide synthase (PKS) candidate orthologues from rice using the protein-protein blasting technique with a sorghum PKS that has been shown to be involved in the formation of 5-alkyl resorcinols. The full-length cDNA amplified from a rice cDNA library was cloned into a vector so that it could be expressed and purified. The identified gene that actually codes for the enzyme of interest was verified by a substrate specificity assay.

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All of the techniques described involve differential screening: mRNA isolated from tissue expressing the gene of interest is compared to mRNA from tissues that do not express the trait. Those methods include the employment of complementary DNA subtraction (cDNA) and differential display. In both methods, isolated clones should be tested to see whether they are truly differentially expressed. The major pitfall with differential screening is in the choice of tissue used, since differences can be detected in gene expression if the tissues compared are of different genetic make up or genotypes or originate from two different developmental stages. Consequently, the choice of tissues for comparison using differential screening is crucial (Yang et al. 2004, 2006). Allelochemicals of interest in agriculture are often produced by root systems. Roots, like other organs, will have common genes expressed for general cellular functions, but a large number of genes that will be differentially expressed may have no connection to allelochemical production. Ideally, in this case, one should use specific tissue from a plant producing the allelochemicals and root tissue from a plant not expressing production. Yang et al. (2004) for instance utilized sorghum root hair tissues that expressed sorgoleone production and sorghum root tissues that did not produce root hairs or expressed sorgoleone production. Differences may also be found in allelochemical production when plant species are subjected to different environmental conditions. Cloned or inbred lines should be then used to reduce genotypic differences unrelated to the production of allelochemicals for clearer evaluation of gene expression related to allelopathy. Once a gene has been isolated by either of the techniques just described, it is crucial to determine if the particular gene isolated is actually associated with a key step in the biochemical pathway of the allelochemical of interest. Three approaches can then be used: (1) express the clone, isolate the resulting protein, and confirm the enzymatic activity of this protein on precursor(s) of the allelochemicals; (2) use transposon tagging or mutagenesis to demonstrate that the cloned gene corresponds to a mutated gene; and (3) influence the level of synthesis of the phytotoxin by creating a construct that would either decrease the expression or enhance the production of the toxin (antisense and overexpression constructs, respectively) (Jorgensen et al. 1996). While these approaches have the advantage that the isolated gene clearly encodes a critical protein involved in allelopathy, the discovery, isolation, and purification of sufficient quantities of plant enzymes or related bioactive metabolites are often difficult and laborious tasks (Duke et al. 2001).


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C. Forward Genetics: From Phenotype to Gene Screening mutant plant populations for the identification of genes is a classical approach successfully used for years in many plant species. Forward genetic strategies include T-DNA insertional mutagenesis, such as activation tagging (upregulation of genes) (Hayashi et al. 1992) and use of gene knockout libraries (disruption of gene function) (Krysan et al. 1999). In Arabidopsis, T-DNA insertional mutagenesis has become routine using the in planta floral dip method (Clough and Bent 1998). Allelochemical-resistant Arabidopsis have been identified using a phytotoxin screen (survivors are resistant). Even though transformation methods could be helpful in assaying allelopathic genes, epistasis and redundancy could cause further confusion. Insertional mutagenesis disrupts gene function based on the insertion of a T-DNA tag into a genome (Weigel et al. 2000). The T-DNA not only disrupts gene expression but also serves as a marker to identify the mutation. The nature of the TDNA tag as well as its localization of insertion in the genome will have variable consequences. For instance, a T-DNA construct that carries a constitutive promoter such as cauliflower mosaic 35S promoter will be able to increase the expression of genes adjacent to the site of insertion and could also silence a gene if its insertion occurs in the coding region of the gene. Because allelochemicals have a target molecule within susceptible plants, activation insertion mutagenesis seeks to disrupt this phytotoxin-target interaction by altering the regulation of the target through transcription modulation or by altering metabolism that affects the target. Along similar lines, one could discover potential phytotoxin resistance genes by screening an existing library of T-DNA tagged or chemically induced (EMS) A. thaliana mutants with the allelochemical of interest to isolate phytotoxin resistance genes and genes critical for allelochemical mode of action. However, this technique requires screening for a mutation in thousands of individual plants, and will not be likely if there is no rapid screen for the trait of interest (Duke et al. 2001). Once mutants are identified and reconfirmed, resistance genes can then be isolated and identified using thermal asymmetric iPCR to identify the flanking region of the insertion site (Duke et al. 2001). Random primers and primers specific for the 35S insert can be used to isolate the flanking region. By comparison of the PCR product with the A. thaliana and other well-characterized species databases, the sequences around the insertion can be identified. The characterization of the location of insertion of the T-DNA tag in the transgenic plant is useful to determine

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the gene involved. Such analysis would also be useful to determine the mode of action of an allelochemical (Duke et al. 2001). D. Introducing a New Gene in a Crop that Does Not Produce Allelochemicals The insertion of genes to promote allelochemical production may eventually lead to the production of genetically engineered crops with novel weed-suppressive or allelopathic potential. Even though this approach is one of the promising molecular techniques under consideration for development of crops producing enhanced levels of secondary products, several objections have been raised related to the concept of creating allelopathic crops by engineering natural product pathways. The first objection is related to the fact that one might have to transfer and coordinately regulate a large number of genes to introduce an effective allelopathic activity since allelopathy is usually a multigenic trait, as already discussed. Even though several single-step conversions can produce phytotoxins from ubiquitous or common metabolic intermediates, this approach still represents a technological challenge at this time. A second consideration is the ability of the plant to overcome the effects of allelochemicals by rapidly evolving detoxification mechanisms, which often involve P450 enzymes. Resistance evolves for several reasons. In most cases, the more persistent the selection pressure is, the more rapidly resistance appears (Monaco et al. 2002). Consequently, the continuous selection pressure by an allelopathic compound may lead to the future development of resistance. Furthermore, some molecular target sites of synthetic herbicides are more susceptible to resistance development. Therefore, we believe that certain plants may develop resistance or tolerance more or less rapidly to phytotoxic compounds, depending on the allelochemical mode of action. However, very few studies have investigated the mechanisms of allelochemical resistance. Certain specific allelochemicals or related mixtures of natural products may have several modes of action, and resistance is potentially less likely to develop if multiple modes of action are implicated. For instance, sorgoleone, a quinone produced by Sorghum spp. root hairs, has three elucidated molecular target sites, hydroxyphenylpyruvate dioxygenase (Meazza et al. 2002) as well as photosynthesis (sorgoleone is a competitive binder to the D1 protein in photosystem II) and respiration (sorgoleone inhibits electron transport in both photosynthesis and respiration) (Czarnota et al. 2001). Although no commercial herbicides have been clearly identified as possessing more than one mode of action, specific pesticide mode of action is often


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not elucidated before pesticide registration occurs. Therefore, the potential low probability of natural and evolved resistance to phytotoxins with more than one target site may render the use of crops producing allelochemicals for weed management a more viable long-term strategy than the use of many synthetic herbicides (Weston and Duke 2003). A third problem that must be considered when engineering an allelopathic crop is the possibility of autotoxicity. However, autotoxicity may be more likely to develop when a novel biochemical pathway is inserted in a plant since the plant may not have an inherent means or mechanism to protect itself against the allelochemical produced. These mechanisms could include vacuolar sequestration, secretion, resistance at the target site, or metabolic inactivation (Duke et al. 2001). However, autotoxicity could also develop when the production of specific allelochemical is greatly enhanced since the appropriate mechanisms of avoidance might be overwhelmed in specific plant tissues. If so, enhancement of the native protective mechanism or introduction of a new mechanism of resistance would be an option to consider. Another important question that can be raised with the introduction of a new biochemical pathway in a plant is the consideration of the cost of partitioning a significant amount of metabolic resources into secondary metabolite production. It has been shown that the metabolic cost for a plant to synthesize secondary metabolites is often higher than that of primary metabolites (Gershenzon 1994). Therefore, one might suppose that allelochemical production must be, in some way, extremely beneficial to the plant. However, if this cost is associated with yield reduction, crops with inherent weed-suppressive abilities may not be beneficial for the producer as crop yield might be negatively influenced. Depending on the ecosystem, the balance between secondary metabolite production and crop yield production varies (Duke et al. 2001). IV. USE OF WHOLE-GENOME TECHNIQUES TO UNDERSTAND ALLELOPATHIC PHENOMENA A. Genomic Approaches DNA microarray technology has been used extensively in gene expression profiling and in the identification and genotyping of polymorphisms (Aharoni and Vorst 2001). Microarray analysis is now being explored and successfully applied in the field of allelopathy. Little is known about the effect of allelochemicals or plant defense responses on the entire plant genome and, in particular, on the expression of genes

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encoding the primary target. However, given the recent advances in molecular biology, enhanced understanding of such complex processes in allelopathy, including the biosynthesis, mode of action, and detoxification of allelochemicals, is imminent. Over the last few years, researchers have investigated the degree of power in microarray analysis, and microarray resources and affordability are ever increasing (Duke et al. 2005). This technique will allow the detection of global expression responses of plant genomes following a treatment with phytotoxins, and any alterations in the transcriptome should be detected after treatment. These changes should also be specific for various phytotoxins, even those with the same or similar molecular target sites (Eckes et al. 2004; Duke et al. 2005). In a recent study, microarray technology has been used to examine physiological response, detoxification pathways, and transcriptome responses in Arabidopsis when plants were exposed to the allelochemical benzoxazolin-2(3H)-one (BOA). Investigators determined that the major mechanism of detoxification in Arabidopsis seedlings when exposure to BOA was by O-glycosylation. By identifying genes involved in a chemical defense network, plant biologists are able to determine plant transcriptional responses to environmental toxins. Further biochemical and genetic experiments to enable researchers to draw a comprehensive picture of detoxification pathways and chemosensory mechanisms allowing plants to survive in the presence of environmental toxins such as allelochemicals are sorely needed (Baerson et al. 2005). One key limitation in the use of microarray technology is that heterologous probing from a wide range of taxa (from monocot to dicot arrays) is not possible due to sequence differences when using microarrays. At this point in time, the impact of specific toxins on global gene expression can best be investigated in species with published genome. The Arabidopsis and rice genomes are sequenced and characterized, and nearly full-genome oligonucleotide microarrays are commercially available, making potential utilization of recently characterized information is easily accessible (Sandermann 1999). A list of plant genome sequencing efforts, the progress of the efforts, and other information can be found at Plant Genomes Central (www.nvbi.nlm.nih.gov/genomes/ PLANTS/PlantList.html). Using oligonucleotide microarrays, several research groups have investigated the mode of action of agricultural fungicides and herbicides. It appears that certain phytotoxins affect specific gene clusters. Recent results may provide some insight as to their specific mode(s) of action but may not be applicable to the diversity of plant species under investigation (Agarwal et al. 2003; Gutteridge et al. 2005).


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In addition to limitations on the species that can be studied, there are several potential caveats associated with the use of the whole cDNA chips for microarray analysis. Since a particular toxin may affect a large set of genes at various doses and times after exposure, it is crucial to standardize or determine the proper treatment dose and time after treatment to critically observe alterations of gene transcription that might be directly related to the mode of action. Otherwise, the differences observed in gene expression might be easily misinterpreted. In addition, treatment with most phytotoxins can result in stimulation of stress and protective responses in sensitive plant tissues. Therefore, although microarray analysis can identify differences in gene regulation, motifs observed may actually be general stress response and not necessarily related to physiological processes linked to phytotoxin exposure (Duke et al. 2005). At certain times, genes related to stress and protection from xenobiotics may strongly be up-regulated, masking those that are directly related to the mode of action of a particular phytotoxin. Furthermore, many target sites are known to be associated with well-regulated and expressed sets of genes, which do not exhibit much variation. Therefore, one needs to be aware that microarray techniques may be challenging to employ when attempting to develop a complete understanding of the impacts of phytotoxins on plant growth, in particular when sifting through the huge amount of data generated using microarrays. However, by using this powerful strategy, one might be able to generate a much more complete transcriptional profile for various phytotoxins with differing modes of action. Once such a database is available, the use of microarrays could be particularly useful in providing clues on the specific mode of action of other phytotoxins under evaluation, including allelochemicals. B. Metabolomic Approaches Investigating plant-plant interference using metabolic fingerprinting, the unbiased identification and quantitation of all plant metabolites, is also emerging as a counterpart to proteomics (Weckwerth 2003; Goodacre et al. 2004). Metabolic fingerprinting involves the development of a detailed biochemical ‘‘fingerprint’’ of a plant cell or tissue sample. This laborious and exacting process is highly dependent on the skill and precision of the analyst. Depending on the precision and resolution needed, some sample prepararion of the tissues to be compared may be necessary. Ultimately, the identification and quantification of the individual metabolites is performed using analytical methods such as nuclear magnetic resonance (Lindon et al. 2000), Fourier

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transformed-infrared spectroscopy (FT-IR) (Goodacre et al. 1998; Johnson et al. 2000), or electrospray ionization mass spectrometry (Vaidyanathan et al. 2001; Goodacre et al. 2002). FT-IR fingerprinting coupled with chemometrics via cluster analysis has been suggested as a tool to correlate global metabolic changes resulting from abiotic or biotic stresses and/or interactions and could have excellent applications for further study of allelopathic interactions (Gilman et al. 2003). Transcription profiling techniques, coupled with metabolite analysis, have already been used to map novel structural and regulatory genes in the biochemical pathways of specific plant metabolites, including alkaloids, flavonoids, and isoprenoids (Ohlrogge and Benning 2000; Forkmann and Martens 2001). Linking changes in metabolite profiles to gene expression through DNA microarray analysis is one potential route to assign gene functions based on cellular dynamics (Dixon 2001). C. Proteomic Approaches In the field of allelopathy, a recent study has used a proteomic approach to better understand allelopathic phenomenon in rice (Lin et al. 2004). Researchers successfully employed the proteome analysis technique as a tool to investigate differential expression of proteins in allelopathic rice exposed to barnyardgrass stress. Results have provided useful information about the induced rice proteins, which are closely related to the pathways of isoprenoid and phenylpropanoid biosynthesis, a potential increase in metabolites of allelochemicals in the plant’s defense response. These results suggest also that proteomics could be an effective tool for physiological and genetic studies in allelopathy, especially in rice allelopathy. It is thought that allelopathy in rice is quantitatively inherited, and QTL techniques have been employed to intensively map gene-conferring allelopathic traits (Lin 2000; Lin 2003). However, mapping techniques such as these rarely lead to gene discovery since QTL mapping to an interval of 5 to 10 cm on a chromosome can be associated with hundreds of genes. The genes noted then become ‘‘positional candidate genes’’ for the QTL, and it is often difficult to identify actual functional genes. Proteomics can positively contribute to the identification of positional, functional, and expressional genes. Comparison of two-dimensional protein patterns obtained for key tissues in stressed and control plants generally results in identification of a set of stress-responsive proteins encoded by the respective candidate genes. Sequencing of these stressedresponsive proteins generally reveals that some of these proteins have functions clearly consistent with the stress-tolerant trait. The encoded


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genes will thus be both expressional and functional candidate genes. These encouraging results should allow future researchers to ascertain gene function more easily.

V. CONCLUSION Although molecular approaches will not likely replace traditional breeding methods for plant selection and the study of plant interactions in the near future, molecular and physiological approaches will enhance the type and quality of information that can be obtained from laboratory and field experiments on plant interactions with other plants, microbes, or the environment. Genomic approaches coupled to physiological studies on allelopathic species could provide new and unique insights into current models of plant interference. The study of allelopathy will benefit from collaboration with ecologists, plant breeders, and molecular biologists leading to the utilization of new tools for selection of crop plants with weed-suppressive traits. In turn, scientists studying allelopathic interference and mechanisms of plant competition can provide unique perspectives for crop selection and also provide significant contributions to the study of plant secondary products and functional genomics in higher plants. The continued elucidation of novel phytotoxins and biosynthetic pathways for secondary products in higher plants and other organisms will no doubt lead to the enhanced probability of incorporating these characteristics into targeted plants by traditional or molecular approaches. Our greatest challenge, however, will be the continued procurement of funding and support for research involving the study of novel secondary products and their role in complex and diverse ecosystems. Multidisciplinary approaches, although complicated to develop and administer, will certainly be required to further our advancement toward the development of biorational weed management strategies, including the selection and development of weed-suppressive crop species. For the foreseeable future, traditional approaches to breeding and selection offer the most likely means for development of crops with enhanced weed-suppressive ability. In the future, as our knowledge of secondary product metabolism increases, molecular approaches to the study of plant interference will become more common. In any case, the next 10 years will result in the rapid proliferation of our knowledge of the mechanisms surrounding plant interference. We will apply this knowledge to the development of more competitive crop plants and to understanding successful invasion of noxious weedy species.

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5 Molecular Genetics and Breeding for Fatty Acid Manipulation in Soybean Andrea J. Cardinal Department of Crop Science, Box 7620 North Carolina State University Raleigh, North Carolina 27695-7620 USA

I. INTRODUCTION II. GLYCEROLIPID BIOSYNTHESIS IN PLANTS III. BREEDING FOR IMPROVED OIL QUALITY, MAPPING, AND MOLECULAR BASIS FOR ALTERED FATTY ACID COMPOSITION A. Saturated Fatty Acids 1. Breeding for Reduced Palmitic Acid Content 2. Breeding for Increased Palmitic Acid Content 3. Breeding for Increased Stearic Acid Content B. Unsaturated Fatty Acids 1. Breeding for Decreased Linolenic Acid Content 2. Breeding for Increased Linolenic Acid Content 3. Breeding for Increased Oleic Acid Content IV. GENETIC RELATIONSHIPS AMONG LOCI, PLEIOTROPY, AND ENVIRONMENTAL EFFECTS ON FATTY ACID COMPOSITION A. Genetic Relationship among Different Fatty Acid Loci B. Phenotypic Correlations among Fatty Acid Components of Seed Oil C. Environmental Influence on Fatty Acid Composition D. Pleiotropy: Fatty Acid Content in Other Tissues V. RESEARCH NEEDS VI. ACKNOWLEDGMENTS VII. LITERATURE CITED

ABBREVIATIONS ACP CAPS DAGAT

Acyl carrier protein Cleavage amplified polymorphic sequence Diacylglycerol acyl transferase


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DGDG Digalactosyl diacylglycerol EMS Ethyl-methane-sulfonate ER Endoplasmic reticulum FATA Oleoyl-ACP thioesterase gene FATB Palmitoyl-ACP thioesterase gene G3P Glycerol-3-phosphate GPAT Glycerol-3-phosphate acyl transferase KAS I 3-ketoacyl-ACP synthetase I KAS II 3-ketoacyl-ACP synthase II KAS III 3-ketoacyl-ACP synthetase enzyme III LPA Lysophosphatidic acid LPAAT Lysophosphatidic acid acyltransferase MGDG Monogalactosyl diacylglycerol NMU N-nitroso-N-methyl urea 18:1-ACP Oleoyl-ACP 16:0-ACP Palmitoyl-ACP PA Phosphatidic acid PC Phosphatidylcholine PDAT Phosphatidylcholine:diacylglyceriol acyltransferase PE Phosphatidylethanolamine PG Phosphatidylglycerol PI Phosphatidyl inositol PS Phosphatidyl serine QTL Quantitative trait loci 18:0-ACP Stearoyl-ACP SL Sulphoquinivosyl diacylglycerol TAG Triacylglycerol

I. INTRODUCTION Soybean (Glycine max (L. Merr.) Fabaceae) provides about 30% of the world’s oil production (US Department of Agriculture statistics, 2006; www.nass.usda.gov/Publications/Ag_Statistics/agr06). Soybean oil is mostly composed of triacylglycerols. The relative composition of saturated and unsaturated fatty acids in the seed triaclyglycerols determines quality. Saturated fatty acids are not considered healthy, and polyunsaturated fatty acids are unstable due to their susceptibility to autoxidation and, consequently, the production of undesirable flavors and odors. In contrast, monosaturated fatty acids are desirable for both human health and oil stability. On average, soybean oil is composed of 110 g kg 1 palmitic acid (16:0), 40 g kg 1 stearic acid (18:0), 240 g kg 1 oleic

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acid (18:1), 540 g kg 1 linoleic acid (18:2), and 70 g kg 1 linolenic acid (18:3) (Cherrak et al. 2003). Soybean breeders have been genetically altering the fatty acid composition in soybean seeds to improve the nutritional and functional quality of the oil by conventional hybridization and selection methods for the last 30 years. Several lines with decreased linolenic acid, increased oleic acid, decreased palmitic acid, or increased stearic acid contents have been developed through recurrent selection, mutagenesis, or screening for natural mutations. In parallel with advances in breeding, the understanding of the biosynthesis of fatty acids in plants has improved tremendously over the last 10 years. Recently most of the genes involved in the fatty acid synthesis pathway in plants have been cloned and sequenced in several plant species, including soybeans. However, few studies relate a particular fatty acid mutant (phenotype) with a specific gene in the fatty acid biosynthetic pathway in soybeans. Sequence information of genes involved in the lipid biosynthetic pathway in soybeans provides a tool to develop allele-specific markers to investigate the relationship between quantitative trait loci (QTL) for seed fatty acid components and specific genes of the pathway and their interactions. The objective of this article is to review the genetics, breeding, QTL mapping, and molecular biology of genes involved in fatty acid manipulation in soybean.

II. GLYCEROLIPID BIOSYNTHESIS IN PLANTS Lipids, an essential constituent of plant cells, are major components of membranes, cuticular waxes, and storage tissues. The most abundant types of lipid in most cells are derived from the fatty acid and glycerolipid biosynthetic pathway. Membrane lipids (glycerolipids) are composed of glycerol with fatty acids esterified to the sn-1 and sn-2 positions and a polar head group attached to the sn-3 position. Storage lipids (triacylglycerols) have fatty acids attached to all three positions of glycerol. Depending of the type of polar head groups, glycerolipids can be divided into glycolipids (monogalactosyl diacylglycerol [MGDG], digalactosyl diacylglycerol [DGDG], sulphoquinivosyl diacylglycerol [SL]) and phospholipids (phosphatidylcholine [PC], phosphatidylethanolamine [PE], phosphatidylglycerol [PG], phosphatidyl inositol [PI], phosphatidyl serine [PS]). The five major fatty acids present in soybean triacylglycerols are palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), and linolenic acid (18:3) (Ohlrogge et al. 1991; Ohlrogge and Browse 1995; Kinney 1997).

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Biosynthesis of C16 and C18 fatty acids occurs in the plastids of both leaves and developing seeds. The first step in this pathway is the synthesis of malonyl-CoA from acetyl-CoA catalyzed by acetyl-CoA carboxylase. Malonyl-CoA is used exclusively in fatty acid synthesis, and the malonyl group is transferred from CoA to a protein cofactor, acyl carrier protein (ACP). The first condensation reaction catalyzed by a 3ketoacyl-ACP synthetase enzyme III (KAS III) requires as substrates malonyl-ACP and acetyl-CoA. Subsequent condensation steps between acyl-ACP and acetyl-CoA are catalyzed by 3-ketoacyl-ACP synthetase I (KAS I) until the production of palmitoyl-ACP (16:0-ACP). The last elongation step to produce stearoyl-ACP (18:0-ACP) is catalyzed by 3ketoacyl-ACP synthase II (KASII). The initial product of each condensation step is a 3-ketoacyl-ACP; this product is reduced by 3-ketoacyl-ACP reductase, dehydrated by 3-hydroxy-acyl-ACP dehydratase, and reduced again by enoyl-ACP-reductase. The final product of each round in the cycle is a saturated acyl-ACP lengthened by two carbons. The primary products of fatty acid synthesis in the plastids are 16:0-ACP and 18:1-ACP. 18:1-ACP is produced when 18:0-ACP is desaturated by stearoyl-ACP desaturase (Ohlrogge et al. 1991; Ohlrogge and Browse 1995; Kinney 1997). When an acyl group is removed from an acyl-ACP, elongation is terminated. Depending on which of two enzymes catalyze this reaction, the fatty acids are utilized in different pathways in different cellular compartments. First, if an acyl-ACP thioesterase catalyzes the reaction, free fatty acids are released, leave the plastid, and then acyl-CoA synthetases convert them to acyl-CoA thioesters to be incorporated into lipids in the endoplasmic reticulum (ER) or directly in triacylglycerols. However, if an acyltransferase catalyzes the removal of the acyl group, the fatty acid is transferred from ACP to glycerol-3-phosphate (G3P) or to monoacylglycerol-3-phosphate to be used for the synthesis of PA and plastid lipids. Thioesterases are specific for fatty acids of certain chain lengths. Soybean has two classes of thioesterases. One preferentially uses palmitoyl-ACP (16:0-ACP thioesterase) as a substrate and the second prefers C18 fatty acids (mostly oleoyl-ACP and, with lower efficiency, stearoyl-ACP). Plastid acyltransferases have distinct substrate specificities. PA made in the plastid has 16:0 in the sn-2 position and mostly 18:1D9 at the sn-1 position. PA is used for the synthesis of PG or is converted to DAG by a PA-phosphatase. This DAG pool is a precursor for the synthesis of plastid lipid membranes MGDG, DGDG, and SL. The ER acyltransferases that use acyl-CoA substrates in the ER to produce PA preferentially insert C18 fatty acids at the sn-2 positions, and 16:0 is


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confined to the sn-1 position. The PA in the ER can be used to produce PC, PE, and PI for incorporation into extrachloroplast membranes. The DAG from PC can return to the plastid envelope, where it can be used for the synthesis of plastid lipids. There is evidence that this lipid exchange between the ER and the chloroplast is reversible (Ohlrogge et al. 1991; Miquel and Browse 1992; Browse et al. 1993; Ohlrogge and Browse 1995; Somerville and Browse 1996; Kinney 1997). In all plant tissues, the major glycerolipids initially synthesized use 16:0 and 18:1 acyl groups. Then membrane-bound desaturases of the chloroplast (plastids) and the ER catalyze the subsequent desaturation reactions. A highly specific plastidial delta-3 desaturase inserts a trans double bond into the 16:0 at the sn-2 position of PG. Another highly specific plastidial delta-7 desaturase inserts a double bond into the 16:0 of MGDG. Two other plastidial desaturases (16:1/18:1 and 16:2/18:2) have no specificity for the length of the fatty acid chain, its position in the glycerol molecule, or the headgroup attached to the glycerol. The ER 18:1 and 18:2 desaturases act on the fatty acids esterified to PC at both sn-1 and sn-2 positions. Some of the steps of storage triacylglycerol synthesis in developing oilseeds (Fig. 5.1) are the same as for membrane lipid biosynthesis in leaves (Ohlrogge et al. 1991; Kinney 1997). The synthesis of saturated acyl-ACPs (16:0-ACP, 18:0-ACP) first occurs in the plastids, as described. Oleoyl-ACP (18:1-ACP) is synthesized from 18:0-ACP by an acyl-ACP delta-9 desaturase. Free fatty acids are released into the cytoplasm by the action of soluble acyl-ACP thioesterases. Released fatty acids are esterified to CoA and form an acyl-CoA pool in the cytoplasm. Acyl chains are attached to each carbon of the glycerol molecule by the action of three microsomal acyl transferases: (1) glycerol-3-phosphate acyl transferase (GPAT) catalyzes the formation of sn-1 acylglycerol-3-phosphate (or lysophosphatidic acid, LPA); (2) LPA is converted to phosphatidic acid (PA) by LPA acyltransferase (LPAAT); soybean LPAAT is selective for unsaturated acyl-CoAs. PA is dephosphorylated to DAG by PA phosphatase. DAG can be used to produce phospholipids or triacylglycerol. Catalyzed by choline phosphotransferase, DAG-containing oleate chains can react with CDP-Choline to form oleoyl-PC. Microsomal membrane-bound desaturases catalyze the synthesis of 18:2-PC and 18:3-PC. Finally, (3) diacylglycerol acyl transferase (DAGAT) attaches a third acyl chain in the glycerol molecule to form triacylglycerol (TAG). This last reaction is an exclusive step of TAG synthesis. Unsaturated fatty acids at position sn-2 of PC can be exchanged with 18:1D9 groups from the acyl-CoA pool, providing a means of increasing 18:2D9,12 and 18:3D9,12,15 in the acyl-CoA pool that

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Fig. 5.1. Triacylglycerol synthesis in soybean. (1) Choline phosphotransferase; (2) phosphatitylcholine:sn-1,2 diacylglycerol acyltransferase. Source: Adapted from Ohlrogge et al. 1991.


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can be used for TAG synthesis. This acyl exchange between acyl-PC and acyl-CoA pool is catalyzed by forward and a reverse reaction of acylCoA:PC acyltransferase. Additionally, PC can donate its entire DAG portion for TAG synthesis. The synthesis of PC from DAG and CDPcholine is rapidly reversible as catalyzed by choline phosphotransferase (Dewey et al. 1994). In addition, phosphatidylcholine:diacylglyceriol acyltransferase (PDAT) can transfer the fatty acids from position sn-2 of PC to position sn-3 of TAG (Dahlquist et al. 2000). In summary, newly synthesized 18:1 from plastids are incorporated into PC to be further desaturated or modified. Then the DAG moiety of PC and/or polyunsaturated FA from PC can be used for TAG synthesis. A small percentage of desaturated FA found in TAG may be derived from plastid desaturases (Kinney 1997). III. BREEDING FOR IMPROVED OIL QUALITY, MAPPING, AND MOLECULAR BASIS FOR ALTERED FATTY ACID COMPOSITION A. Saturated Fatty Acids 1. Breeding for Reduced Palmitic Acid Content. Palmitic acid (16:0) is the predominant saturated fatty acid in soybean oil. Oil from typical soybean cultivars contains 110 to 120 g kg 1 palmitic acid (Burton et al. 1994; USDA-ARS, National Genetics Resources Program). Cultivars with reduced palmitic acid content are desirable to reduce the health risks associated with cholesterogenic properties of saturated fatty acids (Hu et al. 1997). Several lines with reduced palmitic acid concentration have been developed by mutagenesis. C1726 (85.4 g kg 1 16:0) was developed by ethyl-methane-sulfonate (EMS) mutagenesis of ‘Century’. A22 (78.4 g kg 1 16:0) was developed by N-nitroso-N-methyl urea (NMU) mutagenesis of A1937; ELLP2 (71.4 g kg 1 16:0) was developed by EMS mutagenesis of ‘Elgin87’; and J3 (57.4 g kg 1 16:0) was developed from Xradiation of ‘Bay’ (Wilcox and Cavins 1990; Fehr et al. 1991a; Rahman et al. 1996a, Stojsˇin et al. 1998a). In addition, a natural mutation was discovered in line N79-2077-12 (64 g kg 1 16:0) from the fifth cycle of recurrent selection for high oleic concentration (Burton et al. 1983, 1994). The mutation in C1726 is at an independent locus from the mutations in A22, N79-2077-12, ELLP2, and J3 (Schnebly et al. 1994; Wilcox et al. 1994; Kinoshita et al. 1998b; Stojsˇin et al. 1998a). The mutation in A22 is at the same locus as N79-2077-12 but independent of the locus in ELLP2 (Primomo 2000; Primomo et al. 2002). The loci

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conferring low palmitic acid in these lines were given different names: fap1 (in C1726), fap3 (in A22), fapnc (in N79-2077), fap or fapx (in ELLP2), and sop1 (in J3) (Wilcox and Cavins 1990; Schnebly et al. 1994; Kinoshita et al. 1998b; Stojsˇin et al. 1998a; Primomo et al. 2002). However, the allelic relationships between fapnc/fap3 with sop1, and fap with sop1 are unknown. Molecular data suggest that fapnc and fap3 are the same locus (Dewey, pers. comm. 2006). The fap1 and fap3 alleles have additive gene action, and the Fap allele has partial dominance over fap (Wilcox and Cavins 1990; Fehr et al. 1991a; Stojsˇin et al. 1998a). No epistasis was detected between the Fap1 and Fap loci (Stojsˇin et al. 1998a). However, epistatic interactions were observed between fap3 and fap (Primomo 2000). In general, no maternal or cytoplasmic effects have been detected for low palmitic acid content; therefore, the genotype of the embryo determines the phenotype of the seed (Wilcox and Cavins 1990; Wilcox et al. 1994; Kinoshita et al. 1998b). Several lines have been developed to combine at least two low palmitic acid loci. C1943 and N94-2575 are homozygous for both the fap1 and fapnc alleles, and their 16:0 content is approximately 40 g kg 1 (Burton et al. 1998). A18 combines the fap1 and fap3 alleles and has a 16:0 content of 40 g kg 1 in the oil (Schnebly et al. 1994). LPKKC-3 combines the fap1 and sop1 alleles and also has a 16:0 content of 40 g kg 1 in the oil (Kinoshita et al. 1998b; Rahman et al. 2004). RG1 and RG3 combine the fap1 and fap alleles and have a 16:0 content of approxi1 mately 40 g kg in the oil (Primomo et al. 2002). In addition to the major genes already mentioned, minor/modifier genes influence the low palmitic acid content in soybean oil (Horejsi et al, 1994; Rebetzke et al. 1998b; Stojsˇin et al. 1998a, Rebetzke et al. 2001; Li et al. 2002). However, heritability estimates for 16:0 content on a plot basis ranged from 0.37 to 0.73 and on an entry-mean basis from 0.83 to 0.96 (Rebetzke et al. 1998b; Cherrak et al. 2003). Therefore, selection in few environments for both major and minor genes should be successful (Rebetzke et al. 1998b). Several studies have shown that the presence of major fap alleles that reduce the palmitic acid content in seed oil correlates with a reduction in yield. Agronomic changes have been observed in low palmitic acid lines that were homozygous for the fap1 and fap3 alleles (< 41.5 g kg 1 16:0) when compared to normal lines (> 100 g kg 1 16:0) derived from the same population (Ndzana et al. 1994). Low palmitic acid lines yielded significantly less, had less oil content, were taller and had more protein (in two populations out of three), had less 18:0 and more 18:1, 18:2 (in three populations), and 18:3 contents (in two out of three populations) (Ndzana et al. 1994). When lines homozygous for the


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fap1 and fap3 alleles were backcrossed to high-yielding donor parents, the yield drag observed in low palmitic lines was reduced but the oil content was also significantly reduced (Horejsi et al. 1994). However, lines with yield higher than the recurrent parent were obtained (Horejsi et al. 1994). When N87-2122-4, a low palmitic line homozygous for the fapnc allele, was crossed to two high-yielding normal palmitate lines, progenies that inherited the major fapnc allele had a 6 to 15% reduction in stearic acid (Rebetzke et al. 1998b; Rebetzke et al. 2001) and a 4 to 10% increase in oleic acid content (Rebetzke et al. 2001), an increase in linolenic acid content, a 10% decrease in yield in all crosses, and an increase in oil content in one cross only (Rebetzke et al. 1998a). No differences in linoleic acid and protein content were observed between progenies that inherited the major fapnc allele and those that inherited the normal allele (Rebetzke et al. 1998a). Palmitate content, due to the effect of modifier genes detected in these studies, was negatively correlated with 18:1 content and positively correlated with 18:3 content but was not correlated with yield (Rebetzke et al. 1998a). Weak positive correlations between palmitic acid content and seed oil or seed yield (0.13 and 0.12, respectively) were observed when N97-3708-13 was crossed to ‘Anand’ (Cherrak et al. 2003). N97-3708-13 inherited the low 18:3 trait from ‘Soyola’ and the low 16:0 trait from C1726 and N90-2013. Palmitate content in soybean oil was negatively correlated with linoleate content in a population that was segregating for the fap1 and fan loci. Linolenate was negatively correlated with oleate and linoleate content in the same population (Nickell et al. 1991). Mapping of Genes and QTLs for Reduced Palmitic Acid Content. QTL analysis of F2:3 families from the cross of N87-2122-4 to ‘Cook’ (a highyielding normal palmitate line) revealed the presence of a major QTL in linkage group A1 distal to Satt684 that accounted for 31% of the palmitic acid variation and a minor QTL in linkage group M near Satt175 and Satt306 that accounted for 9% of the palmitic acid (Li et al. 2002). The two QTLs had a significant epistatic interaction, and when lines were homozygous for alleles from N87-2122-4 at both QTLs, their palmitic acid was reduced to 60 g kg 1. A multiple regression model that included both QTLs accounted for 43% of the phenotypic variation for palmitic acid content observed among F2:3 families (Li et al. 2002). N87-2122-4 was derived from N79-2077-12 so it is assumed to carry the fapnc mutation. In addition, QTL analysis of a BC1F2 population from the cross of Cook and C1726 has revealed the presence of two QTLs on linkage groups B2 and H that show epistatic interactions (Pantalone et al. 2004).

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QTL analysis in a G. max (A81356022) by G. soja (PI468916) F2 population detected two genomic regions associated with palmitic acid content. One region was on linkage group B2 near RFLP loci A343_1 and A018_1, and the second genomic region was on linkage group J near RFLP locus K375_1. The reduced palmitic acid alleles were inherited from A81356022 at both loci (Diers and Shoemaker 1992; http://soybase.ncgr.org/cgi-bin/ace/generic/search/soybase). All the QTLs detected in this population result from segregation of alleles present in lines with normal palmitic acid profile in the seed oil. Biochemical and Molecular Basis for Reduced Palmitic Acid Content. Several steps in the biosynthetic pathway could affect the palmitic acid concentration in TAG. In the case of low palmitic acid mutants, changes in the activity and/or substrate specificities of 3-ketoacyl-ACP synthetase I (KAS I), 16:0-ACP thioesterase, lysophosphatidic acid acyltransferase, glycerol-3-phosphate acyltransferase, or diacylglycerol acyl transferase could produce this phenotype. A biochemical study comparing C1726 (fap1fap1), N79-2077-12 (fapncfapnc) and Dare found no evidence that glycerolipid acyltransferase activities or any other biosynthetic step after the formation of acyl-CoA was altered in these lines. The TAG composition of these lines appeared to be controlled by the amount of 16:0 produced in the plastids (Wilson et al. 2001a, 2001b). Northern analysis revealed that fapncfapnc genotypes have reduced accumulation of transcript corresponding to a 16:0-ACP thioesterase (FATB) gene but no differences in transcript accumulation for the 18:1ACP thioesterase (FATA) or 18:0-ACP desaturase genes were observed (Wilson et al. 2001b). Southern analysis using the FATB cDNA as a probe revealed that several copies of the 16:0-ACP thioesterase gene are present in the soybean genome but one copy is deleted in the fapncfapnc genotype (Wilson et al. 2001c). Studies determined that the soybean genome has four isoforms of the 16:0-ACP thioesterase, and allelespecific PCR markers have been developed for the isoform that is deleted in the fapncfapnc genotype (Cardinal et al. 2007). The allelespecific markers are ideal markers for marker-assisted selection because they are completely linked to the gene of interest. Sequence analysis of the fap3fap3 genotype has revealed a putative deleterious point mutation in the same FATB isoform that is deleted in the fapncfapnc genotype, indicating that these two mutations are allelic (R.E. Dewey pers. comm., 2006). In contrast, the molecular basis of the fap1 mutation has not been determined. No differences in transcript accumulation for fap1 fap1 genotypes were observed for 16:0-ACP thioesterase (FATB gene), 18:1-ACP


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thioesterase (FATA gene), or 18:0-ACP desaturase (Wilson et al. 2001b). cDNAs of three FATB isoforms have been sequenced from C1726 and other normal palmitate lines, but no sequence difference could be correlated with the fap1 mutation (Camacho-Roger 2006). Additional sequencing efforts are under way for the fourth FATB isoform. Transgenic experiments have helped to elucidate the role of specific genes in the oil biosynthetic pathway. Transgenic manipulations of the FatB (16:0ACP thioesterase) gene produced results as expected in soybean; a reduction of 16:0 content in soybean oil was observed when FatB was silenced, and the opposite phenotypic effect (30% 16:0) was obtained when the same gene was overexpressed (Kinney 1995, 1997). In contrast, it was expected that overexpression of KASII would reduce the amount of 16:0 content in seeds, but this was not observed in transgenic experiments (Kinney 1995, 1997). 2. Breeding for Increased Palmitic Acid Content. Cultivars with elevated palmitic and/or stearic acid contents would be useful for producing margarines and shortenings without unhealthy trans isomers of unsaturated fatty acids that are produced during hydrogenation of normal soybean oil to manufacture semisolid fat products (Kok et al. 1999). Several soybean lines that contain higher 16:0 content than normal cultivars have been developed through mutagenesis. For example, C1727 (172 g kg 1 16:0) was developed by EMS treatment of ‘Century’ (Wilcox et al. 1990); A21 (200 g kg 1 16:0) was developed by NMU treatment of A1937; A24 (180 g kg 1 16:0) was developed by EMS treatment of ‘Elgin’ (Fehr et al. 1991b; Schnebly et al. 1994); A25 (170 g kg 1 16:0) and A27 (160 g kg 1 16:0) were developed by EMS treatment of ‘Kenwood’ (Narvel et al. 2000; Stoltzfus et al. 2000a); A30 (148 g kg 1 16:0) was developed from NMU treatment of A89-144026 (Stoltzfus et al. 2000b); and J10 (156 g kg 1 16:0) and KK7 (135 g kg 1 16:0) were developed from X-ray irradiation of ‘Bay’ (Rahman et al. 1996a; Rahman et al. 1999). The high palmitic acid mutations in A21 and J10 are allelic to the mutation in C1727. The mutation in C1727 was named fap2 and the mutations in A21 and J10 were designated fap2-b and fap2(J10) (or sop2 in some studies), respectively (Wilcox et al. 1990; Fehr et al. 1991b; Schnebly et al. 1994; Rahman et al. 1999). The mutation in A24 is at an independent locus different from fap1, fap2, and fap3; it was denominated fap4. The mutation in A25 is at an independent locus different from fap1, fap2, fap3 and fap4; it was named fap6 (Narvel et al. 2000). The mutation in A27 is at an independent locus from fap1, fap3, fap4, and fap6 but closely linked to fap2-b; it was named fap5. The mutation in A30 is at a locus independent of fap1, fap2-b, fap3, fap4, and fap5 but closely linked to

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fap6; it was designated fap7 (Stoltzfus et al. 2000b). The mutation in KK7 is at an independent locus from fap2, fap2(J10) or sop2, and fap1; it was named fapx. The allelic relationships between fapx and fap4, fap5, fap6, and fap7 are unknown. No maternal effects were detected in crosses with most of these mutants (Schnebly et al. 1994; Rahman et al. 1999; Narvel et al. 2000; Stoltzfus et al. 2000a, b). No cytoplasmic effects were detected in two studies (Rahman et al. 1996a, 1999). No dominance was detected in most crosses (Narvel et al. 2000; Stoltzfus et al. 2000a, b). However, partial dominance for low palmitic acid content was evident in the crosses C1726 A21, Bay C1727, Bay KK7, C1726 J10, C1727 KK7, C1726 A27, and Kenwood A27 (Schnebly et al. 1994; Rahman et al. 1996a, 1999; Stoltzfus et al. 2000a). In addition, partial dominance for high palmitic acid content was evident in the crosses between A22 A30 and A27 A30 (Stoltzfus et al. 2000b); complete dominance for the C1727 (fap2) allele was evident in the cross C1727 J10 (Rahman et al. 1999); and overdominance for the high palmitic acid content was detected in the cross of A25 A27 (Stoltzfus et al. 2000a). Soybean lines have been developed that combine two high-palmitate loci. A19 with >250 gkg 1 16:0 (fap2-bfap2-b fap4fap4) was developed from the cross A21 A24 (Schnebly et al. 1994, 1996), and HPKKJ10 and HPKKC-7 with 220 gkg 1 16:0 (fap2(J10) fap2(J10) fapx fapx) were derived from the cross of J10 KK7 (Rahman et al. 1999, 2003, 2004). Agronomic changes were observed when lines with the fap2-b fap2-b fap4fap4 genotype (250 gkg 1 16:0) were crossed to high-yielding normal lines. Lines with increased palmitic acid content had lower yield, seed weight, and 18:0, 18:1, and 18:2 contents; reduced lodging; increased height, protein, oil, and 18:3 contents; and matured later (Hartmann et al. 1996). Ninety cultivars with different combinations of fap genes that increase the 16:0 content (fap2-b, fap4, fap5, fap6, and fap7) were studied for changes in agronomic performance. There was a significant positive phenotypic correlation between palmitic acid content and protein, stearate, and linolenate content (Stoltzfus et al. 2000c). There were significant negative phenotypic correlations between palmitate and oil, 18:1 and 18:2 contents (Schnebly et al. 1996; Stoltzfus et al. 2000c). The positive correlation between high palmitate and linolenate content impedes the development of cultivars with high 16:0 and low 18:3 contents (Bravo et al. 1999; Stoltzfus et al. 2000c). Mapping of Genes and QTLs for Increased Palmitic Acid Content. Very little information is available about the genomic location of genes increasing the palmitic acid content in soybean oil. Only the fap2 locus


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from C1727 has been mapped to LG D2 between RFLPs A_537 and A_611 of the original USDA/ISU map and the LG D1b+w of the integrated map (Nickell et al. 1994; Cregan et al. 1999). Biochemical and Molecular Basis for Increased Palmitic Acid Content. Alteration of several enzymes in the biosynthetic pathway of TAG could potentially affect the palmitic acid concentration. In the case of high palmitic acid mutants, reduced activities in 3-ketoacyl-ACP synthetase II (KAS II) and 18:1-ACP (18:0) thioesterase or increased activity of 16:0ACP thioesterase could be responsible for the elevated palmitic acid trait. A biochemical study comparing C1727 (fap2fap2) and Dare found no evidence that glycerolipid acyltransferase activity or the biosynthetic steps after the formation of acyl-CoA was altered in these lines. Like the low palmitic acid fapnc mutant, the TAG composition of these lines was due to the amount of 16:0 produced in the plastids (Wilson et al. 2001a, b). Kinetics studies suggest that fap2fap2 genotypes had reduced KASII activity (Wilson et al. 2001b). Northern analysis revealed that fap2fap2 genotypes had no difference in transcript accumulation corresponding to the 16:0-ACP thioesterase (FATB gene), 18:1-ACP thioesterase (FATA gene), or 18:0-ACP desaturase (Wilson et al. 2001b). The soybean genome has at least two isoforms of the KAS II gene. C1727 has a single-base mutation in the KASIIA isoform that creates a premature stop codon and a nonfunctional enzyme in the seed (Aghoram and Dewey 2006). A cleavage-amplified polymorphic sequence (CAPS) marker has been developed to detect lines inheriting the fap2(C1727) allele (Aghoram and Dewey 2006). The CAPS marker is completely linked to the target gene. 3. Breeding for Increased Stearic Acid Content. The confectionary industry requires an oil with high solid-fat index, which is directly related to the concentration of saturates in the oil. This is also needed to produce margarines and shortenings. The oil processing industry uses hydrogenation to increase the solid-fat index of soybean oil, but this process creates trans fatty acid that are undesirable for human health. Recently the industry has determined that to produce margarine without hydrogenation, it needs an oil with 30% saturates (Spencer et al. 2003). Of the two saturated fatty acids in soybeans, palmitic and stearic acid, only palmitic acid has been associated with increased cholesterol levels in humans (Kris-Etherton and Yu 1997). Therefore, to meet the requirements of this industry and to produce soybean oil for healthier human consumption, it is desirable to increase the levels of stearate in the oil and decrease palmitate.

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The 18:0 concentration in soybean oil is most commonly around 35 g kg 1 (USDA-ARS, National Genetics Resources Program;www.arsgrin.gov/cgi-bin/npgs/html/desclist.pl?51; verified November 2006). Several lines with increased seed stearic acid levels in the seed were developed through mutagenesis. A6 (281 g kg 1 18:0 and 20 g kg 1 20:0) was developed by treating FA8077 with sodium azide (Hammond and Fehr 1983). A81-606085 (187 g kg 1 18:0) and FA41545 (155 g kg 1 18:0) were developed by EMS treatment of FA9886 and ‘Coles’, respectively. KK-2 (66 g kg 1 18:0) and M25 (199 g kg 1 18:0) were developed by X-ray radiation of cv. ‘Bay’ (Rahman et al. 1997). In addition, a natural recessive mutation was discovered in FAM94-41 (90 g kg 1 18:0) (Pantalone et al. 2002). Crosses between A81-606085, FA41545, FAM94-41, and A6 indicated that their high stearic acid mutations were controlled by one locus (Fas). A81-606085, A6, FA41545, and FAM94-41 carry the fas, fasa, fasb, and the fasnc alleles, respectively (Graef et al. 1985; Bubeck et al. 1989; Pantalone et al. 2002). Several other lines developed through EMS mutagenesis carried mutant genes that were allelic to the Fas locus (Bubeck et al. 1989). However, one mutant line, ST2, has an allele for high stearic acid content at a different locus than Fasa (Bubeck et al. 1989). Lines A81-606085 and FA41545 were later named A9 and A10, respectively (Lundeen at al. 1987). Crosses between KK-2, M25, and ‘Bay’ determined that these mutants each carry a recessive allele at independent loci, designated st1 in KK-2 and st2 in M25 (Rahman et al. 1997). The allelic relationships between the Fas and St loci are unknown. The presence of minor genes for high stearic acid was observed in two studies (Lundeen et al. 1987; Bubeck et al. 1989). No maternal or cytoplasmic effects were detected in crosses with these lines (Graef et al. 1985; Bubeck et al. 1989; Rahman et al. 1997). Partial dominance for low stearic acid content was observed in most crosses of the mutant lines with their normal parent (Graef et al. 1985; Bubeck et al. 1989; Rahman et al. 1997). The fasa and the fasb alleles were completely dominant to the fas allele (Graef et al. 1985). The st1 allele is partially dominant over the st2 allele (Rahman et al. 1997). A line homozygous for the st1 and st2 alleles (>300 g kg 1 18:0) was developed; however, it produced irregular seeds that failed to grow into plants after germination (Rahman et al. 1997). Agronomic changes were observed in lines that carry the fasa (A6), fas (A9), or the fasb (A10) allele (Lundeen et al. 1987). BC1F2 high stearic and normal stearic acid sister lines for each of the fas alleles were grown in replicated trials. The high stearic acid lines had significantly lower content of 16:0, 18:1, and higher 18:2 for each of the three alleles. The 18:3 content of high stearic acid lines varied in each population. The


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mean stearic acid from the BC1F2 high stearic acid lines was significantly lower than their respective parents indicating the presence of minor genes. BC1F2 lines that inherited the fasa (A6) yielded less than the low stearic acid lines, but no yield drag was associated with the fas (A9) or the fasb (A10) alleles (Lundeen et al. 1987). Generally, the high stearic lines matured earlier, lodged less, had higher protein content, and were shorter than the low stearic lines (Lundeen et al. 1987). A subsequent study concluded that high stearic lines inheriting the fasa (A6) allele yielded and lodged less; matured earlier; were shorter; had lower oil, protein, palmitate, oleate, and linoleate content; and had higher linolenate content that the normal stearate lines (Hartmann et al. 1997). Mapping of Genes and QTLs for Increased Stearic Acid Content. Linkage analysis indicated that the Fas and the Fan loci are linked 21.7 cM apart on LG B2 (Rennie and Tanner 1989b) (http://soybase.ncgr.org/cgibin/ace/generic/search/soybase; verified 1/30/2006). An F2 population between Dare and FAM94–41 (fasncfasnc), a high stearate line (70 g kg 1 18:0), was used to perform linkage analysis between the stearate locus and SSR markers. The Fas locus was distally linked to Satt070 (12.3 cM), Satt474 (14.2 cM), and Satt556 (17.8 cM) on LG B2 (Spencer et al. 2003). Biochemical and Molecular Basis for the Increased Stearic Acid Content. No study has been published in soybean that demonstrates the molecular basis of the high stearic acid mutations. A likely candidate gene for the increased stearic acid content in some soybean mutants is the D9 stearoyl-ACP desaturase. Recently it has been shown that soybean has at least two isoforms of this gene, and both are highly expressed in seeds (Byfield et al. 2006). Studies are under way to determine if any mutation in these isoforms are responsible for the increased stearic acid content in several mutant lines (Byfield et al. 2006; R.E. Dewey, pers. comm., 2007). Preliminary results indicate that A6 has a deletion in one of the D9 stearoyl-ACP desaturase isoforms and that FAM94-41 has a deleterious point mutation in the same isoform (R.E. Dewey, pers. comm., 2007). Silencing of a delta-9-desaturase gene in transgenic rapeseed resulted in the expected substantial increase of 18:0 in the oil; and a reduction of seed germination was observed in transgenic plants with accumulation of 18:0 higher than 30% (Thompson and Li 1996). B. Unsaturated Fatty Acids 1. Breeding for Decreased Linolenic Acid Content. The linolenic acid content in soybean oil is undesirable because it is oxidatively unstable,

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and it is responsible for poor flavor and odor in cooking oil. Hydrogenation is used to improve the oil stability by reducing its linolenic acid content; however, during this process trans fatty acids are produced, and they have been associated with increased risk of coronary disease (BeareRogers 1995). Reducing the content of linolenic acid content in soybean oil would increase oxidative stability and reduce the need for hydrogenation. Most of the soybean accessions deposited in the National Plant Germplasm Collection have an oil linolenic acid content between 70 and 90 g kg 1 (USDA-ARS, National Genetics Resources Program). Several soybean lines with decreased linolenic acid content have been developed through recurrent selection, EMS mutagenesis, X-ray irradiation, and selection of naturally occurring phenotypes by breeders. For example, line N78-2245 was derived from the fifth cycle of a recurrent selection program for increasing high oleic acid content initiated by Dr. Charles Brim at the USDA-ARS in the 1970s (Wilson at el. 1981). This line possesses 100 g kg 1 16:0, 40 g kg 1 18:0, 347 g kg 1 18:1, 450 g kg 1 18:2, and 40 g kg 1 18:3 fatty acid composition in the oil (Wilson at el. 1981). Interestingly, not only the TAG fatty acid composition of this line was changed, but the seed phospholipid and diacylglycerol composition were altered as well, when compared to ‘Dare’ (a normal line). C1640, a line with 30 g kg 1 linolenic acid, was derived by EMS mutagenesis of cultivar ‘Century’. Genetic studies suggested that the recessive fan allele controlled the linolenic acid content in C1640 (Wilcox and Cavins 1985, 1987). Other low linolenic acid lines developed by EMS mutagenesis, selection of natural mutations, or X-ray irradiation (A5, PI361088B, PI123440, RG10, M-5, IL-8) each have a major gene that is allelic to the fan gene in C1640 (Rennie et al. 1988; Rennie and Tanner 1989a, 1991; Rahman et al. 1996c; Stojsˇin et al. 1998b). Independent loci, fan2 and fan3, for reduced linolenic acid content were observed in A23 (60 gkg 118:3) and in a line developed from EMS treatment of A89-144003 (Fehr et al. 1992; Fehr and Hammond 1998). In addition, soybean lines KL-8 and M-24 each have an allele for low linolenic acid content that is independent of fan but allelic to each other. They were designated fanx and fanxa, respectively (Rahman and Takagi 1997; Rahman et al. 1998). The allelic relationships between fanx and fan2 or fan3 are unknown. Generally these major genes did not have maternal effects (except for A5), did not have cytoplasmic effects, and had additive effects (except for KL-8) (Wilcox and Cavins 1985; Stojsˇin et al. 1998b; Fehr et al. 1992; Rahman et al. 1994, 1996c, 1998; Rahman and Takagi 1997; Primomo et al. 2002). However, strong maternal effects were observed in crosses


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and in grafting experiments with N78-2245 (Wilson and Burton 1986; Carver et al. 1987). A16 and A17 (22 to 24 g kg 1 18:3) were developed by combining the fan(A5) allele and the fan2(A23) allele (Fehr et al. 1992). A line (A29) with 13 g kg 1 linolenic acid content was developed by combining the fan(A5) fan2(A23) and fan3 alleles (Ross et al. 2000). In addition, N852176, a line with 440 g kg 1 18:1 and 30 g kg 1 18:3 fatty acid composition in the oil, was derived from the cross of N78-2245 and PI123440. Inheritance studies in this population suggested that at least two recessive genes with major effects were involved in conferring the low linolenic acid phenotype of this line; one gene was inherited from each parent (Burton et al. 1989). Backcrossing of these low linolenic genes into a high-yield genetic background resulted in the cultivar ‘Soyola’ (Burton et al. 2004). In summary, there are at least three major loci responsible for low linolenic acid content in soybean seed oil (fan and fan2 and fan3). However, there could be up to five loci involved in conferring the low linolenic acid seed content if fanx and the gene from N78-2245 are distinct loci. In addition to the major genes, modifier genes for low linolenic acid content have been observed in several studies (Wilcox and Caviness 1985; Fehr et al. 1992; Ross et al. 2000). In order to obtain lines with 10 g kg 1 linolenate content, it is important to select both on the three major loci and on additional minor genes (Ross et al. 2000). The chance of obtaining 10 g kg 1 linolenate lines from a cross of a 10 g kg 1 linolenate line by a high-yielding line is very small. However, the odds of obtaining 10 g kg 1 linolenate lines are greatly increased if selection is performed in the progeny from a cross of 10 g kg 1 linolenate line by a 20 g kg 1 linolenate line. No correlation was observed between 18:3 content and yield or 18:3 content and oil concentration in a cross between N97-3708-13 ‘Anand’ (Cherrak et al. 2003). The estimate of heritability based on regression of F2:4 derived lines on their F2 parents was 0.73, indicating that F2 phenotypes are good predictors of their progeny line phenotypes (Cherrak et al. 2003). No significant differences in seed yield but significant differences in maturity, lodging, and height were observed between lines with 10 g kg 1 and 20 g kg 1 18:3 content in two out of three populations (Ross et al. 2000). Significant differences in oil and protein content were observed between lines with 10 g kg 1 and 20 g kg 1 18:3 content in all three populations (Ross et al. 2000). Lines with 20 g kg 1 18:3 content on average matured earlier, lodged more, were higher, and had reduced protein and increased oil content than lines with 10 g kg 1 content (Ross et al. 2000). However, in populations in which linolenate content varied from 25 g kg 1 to 85 g kg 1,

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it was significantly negatively correlated with oil content but not correlated with seed yield, maturity, lodging, height, seed weight, and protein content (Walker et al. 1998). Associations between the fan(C1640) allele and agronomic performance depended on the high-yielding parent used to develop a particular population (Wilcox et al. 1993). The differences in seed yield, maturity, and height between low linolenic lines and normal lines were opposite in effects in two populations (Wilcox et al. 1993). Mapping of Genes and QTLs for Decreased Linolenic Acid Content. The Fan locus has been mapped near the microsatellite marker loci Satt534 and Satt063 and RFLP markers pB194-1 and pB124 on linkage group B2 of the general soybean consensus map by several authors (Brummer et al. 1995; Cregan et al. 1999; Reinprecht 2003). It accounted for 85% of the variation in linolenic acid in one population (Brummer et al. 1995). Molecular Basis for Decreased Linolenic Acid Content. A membraneassociated desaturase inserts a double bond in the omega-3 position of linoleoyl-PC to produce linolenoyl-PC. The FAD3 gene encodes this omega-3 desaturase. The reduced linolenic acid content in A5 was caused by a partial or full deletion of this gene. Cosegregation studies between the absence of a FAD3 RFLP fragment and linolenic acid content in a segregating population explained 67% of the variation observed for that trait (Byrum et al., 1997). Four microsomal FAD3 isoforms, FAD3-1a, FAD3-1b, FAD3-2a, and FAD3-2b, have been isolated in soybeans (Bilyeu et al. 2003; Anai et al. 2005). Following Anai et al.’s (2005) nomenclature, FAD3-1a and FAD3-1b have 93% similarity at the cDNA level and they each have about 77% similarity at the cDNA level with FAD3-2a and FAD3-2b. Also, FAD3-2a and FAD3-2b are extremely similar to each other (Anai et al. 2005). The fan allele (Fan locus) corresponds to the FAD3-1b isoform and different lines (J18, M5, A5, C1640) carrying different alleles at that locus have mutations in different parts of the gene (Bilyeu et al. 2003; Anai et al. 2005; Chappell and Bilyeu 2006). In addition, the Fanx (M24) locus was demonstrated to correspond to the FAD3-1a isoform (Anai et al. 2005). The isoforms have different expression patterns. The FAD3-1b is expressed in immature seed coat, roots, germinating shoots seedlings, seedlings without cotyledon, leaves, shoots, and in all stages of seed development. The FAD3-1a isoform is expressed in the hypocotyls, immature cotyledons, immature pods, and developing seeds during all stages of development. The FAD3-2a isoform is expressed in the


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seed coat, roots, vegetative buds, immature flower, and seed during all the stages of seed development (Bilyeu et al. 2003; Anai et al. 2005). The FAD3-2b isoform is expressed in the early stages of seed development (Anai et al. 2005). The mutation in the FAD3-1b isoform caused a reduction of 50% in the linolenic acid content, and a mutation in the FAD3-1a isoform caused a reduction of 25% in the linolenic acid content. Therefore, mutations that reduce expression/activity of the FAD3– 1b isoform are more effective in reducing the 18:3 level in soybean seeds (Anai et al. 2005; Bilyue et al. 2005). Following Anai et al.’s, (2005) nomenclature, CX1512-44 carries two mutations, one in the FAD3-1b and one in the FAD3-2a isoforms (Bilyeu et al. 2005). The mutation in the FAD3-1b isoform causes an overall 50% reduction in the linolenic acid content, and the mutation in the FAD3-2a isoform causes an overall 17% reduction in the linolenic acid content (Bilyeu et al. 2005). No pedigree imformation was provided for CX1512-44; therefore, it is not possible to associate the FAD3-2a isoform with a specific mutant locus. Two isoforms, FAD3-1a and FAD3-1b, were isolated, from both cultivar ‘Soyola’ (a low linolenic acid line) and a normal line (R.E. Dewey, pers. comm., 2006). No sequence differences between the two lines were observed that would alter the amino acid sequences of the encoded enzymes. Gene-specific probes for the two isoforms were developed, and their expression was compared among several soybean lines. The FAD3-1b isoform is expressed at much higher levels than the FAD3-1a isoform during seed development in all soybean lines evaluated. The accumulation of FAD3-1b transcript in the low linolenic lines ‘Soyola’, ‘Satelite’, and N97-3363-4 was half of the amount observed in normal soybean lines. This provides the likely molecular explanation of the low linolenic phenotype of these lines (R.E. Dewey, pers. comm., 2006). The reduced accumulation of FAD3-1b transcript could be caused by mutations in the promoter or intragenic regulatory regions with no changes in the amino acid protein product sequence or by mutations in a separate gene that regulates the FAD3-1b expression. Evidence for the former was provided by the development of allele-specific PCR primer pairs for the FAD3-1b gene inherited from ‘Soyola’. In two segregating populations, more than 77% of the linolenic acid variation was explained by the segregation of FAD3-1b determined with these markers (Camacho-Roger 2006). Transgenic soybean plants in which the FAD3 gene has been completely suppressed had 14 g kg 1 18:3 in the seed oil, suggesting that a small amount of polyunsaturated FA present in the seed could be synthesized by the plastidial desaturases FAD7 and/or 8 (Hitz et al. 1995).

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2. Breeding for Increased Linolenic Acid Content. The development of highly polyunsaturated soybean oil has an application in the manufacturing of lubricants and drying oils. Few studies have evaluated the inheritance of high linolenic acid in soybeans (Diers and Shoemaker 1992; Pantalone et al. 1997a, b). All the studies evaluated crosses between the cultivated soybean (Glycine max) and its closest wild relative (Glycine soja) because high levels of 18:3 are not observed in the U.S. soybean germplasm collection. Four populations were derived from the cross of one of two low linolenic acid breeding lines to one of two high linolenic acid Glycine soja PIs (Pantalone et al. 1997a). The fatty acid analysis of the F3 progeny of these populations showed that the high linolenate trait from the wild soybean was determined by a set of desaturase alleles different from the Glycine max parents used in the study. In addition, a strong negative phenotypic correlation was observed between linolenic acid content and seed mass in the same populations (Pantalone et al. 1997b). Mapping of Genes and QTLs for Increased Linolenic Acid Content. Several QTLs for high linolenic acid content have been mapped in an F2:3 population derived from the cross of a normal 18:3 Glycine max line (A81–356022) and high a 18:3 Glycine soja line (PI468916) (Diers and Shoemaker 1992). One QTL mapped to linkage group E near RFLPs SAC7_1 and A242_2, a putative second QTL mapped to linkage group E near RFLPs K229_1 A454_1 and A203_1, a third QTL was mapped to linkage group K near RFLP A065_3, and a fourth QTL mapped to linkage group L near A023_1. The QTL on linkage group E near RFLP locus A424_2 could also be a QTL for oleate and linoleate since QTLs for those traits were mapped to the same genomic location (http://soybase. ncgr.org/cgi-bin/ace/generic/search/soybase). Given the biosynthetic pathway, it is very unlikely that the genes involved in conferring the reduced linolenic acid trait are the same genes or have the same molecular basis as those conferring the increased linolenic acid trait. Biochemistry and Molecular Basis of Increased Linolenic Acid Content. No study explaining the molecular basis or biochemistry of the increased linolenic acid content observed in Glycine soja lines has been published. However, an increased expression of FAD3 genes or increased FAD3 desaturase enzyme activity could explain the increased linolenic acid content observed in Glycine soja lines. Alternatively, these lines could have a higher copy number of the FAD3 gene. 3. Breeding for Increased Oleic Acid Content. A soybean oil with increased oleate content (>600 g kg 1) has greater stability during


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high-temperature heating and frying than an oil with normal oleate content (Warner and Knowlton 1997). Most of the soybean germplasms contain between 210 and 230 g kg 1 of oleic acid in the oil (USDA-ARS, National Genetics Resources Program). N78-2245, a mid-oleic acid content line, was the first line derived from Dr. Brim’s recurrent mass selection program to increase oleic acid (Wilson at el. 1981). Since then, several lines (N85-2176, N87-2122-4, N97-3363-4, N98-4445A) with mid-oleic and low–linolenic acid concentration (Fig. 5.2) have been developed by Dr. J.W. Burton (Wilson et al. 2002). The latest release, N98-4445A, has an oleic acid content of 540 g kg 1. It is a sister line of N97-3363-4 and was selected as a different F3 plant from the same cross from which N97-3363-4 was derived. The oleic acid content inheritance in this material seems to be a quantitative trait, and it is highly influenced by environmental conditions (Burton et al. 1983; Hawkins et al. 1983; Carver et al. 1987; Oliva et al. 2006). Independently, two mid-oleic acid mutants have been developed by X-ray irradiation of the cultivar ‘Bay’. The mid-oleic acid content in M23 mutant (527 g kg 1) was controlled by a single partially recessive gene, ol (Takagi and Rahman 1996). The mid-oleic acid content in M11 (387 g kg 1) was controlled by a single recessive gene, ola. ola is completely dominant to the ol allele and both alleles are at the same locus (Rahman et al. 1996b). The genetic relationships between genes governing the high oleic content in the lines developed at USDA-ASR-NCSU and ol locus are unknown. However, transgressive segregants with more than 700 g kg 1 of 18:1 have been obtained from the cross of N98-4445A and M23, indicating that these two lines each contain at least one high oleic allele at different loci (Alt et al. 2005b). The presence of modifiers genes was detected in the cross of M23 and Archer, indicating that the inheritance of this trait is complex and that selection for both major and minor genes is necessary to breed high oleate soybean lines (Alt et al. 2005a). Narrow sense heritability estimates for oleate from a cross of M23 and ‘Archer’ were 0.33 on an F3 seed basis and 0.44 on an F2 plant basis (Alt et al. 2005a). Broad sense heritability estimates were 0.37 to 0.46 on a plot basis and 0.82 on an entry-mean basis (Alt et al. 2005a). Mapping of Genes and QTLs for High Oleic Acid Content. QTLs for fatty acid composition were detected in replicated F2:3 families of a cross between a normal Glycine max line and a normal Glycine soja plant introduction. Most QTLs for linoleate coincided in their genomic location with QTLs for oleate on linkage groups A1 near RFLP A170_1 and E near RFLP A242_2, and they had opposite effects (Diers and

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Fig. 5.2. Pedigree of high–oleic and low–linolenic acid line N97-3363-4.


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Shoemaker 1992; Cregan et al 1999). Whichever allele increased the oleic concentration at a particular QTL also reduced the linoleate concentration, as expected from the knowledge of the biosynthetic pathway. One QTL for oleate was detected on linkage group B2 near RFLP A_619, and one QTL for linoleate mapped to linkage A1 near RFLP A118_1. All the QTLs detected in this population result from segregation of alleles present in lines with normal to low oleate fatty acid profile in the seed oil (Diers and Shoemaker 1992). Six QTLs were detected in a cross between a high-yielding normal oleate line and N00-3350, a high oleate line descendant from N984445A (H.R. Boerma, pers. comm., 2006). These results confirm the complex inheritance of this trait. Biochemical and Molecular Basis for the High Oleic Acid Content. Omega-6 desaturase encoded by the FAD2 gene catalyzes the desaturation of 18:1-PC to 18:2-PC. Soybean has at least two FAD2 genes. FAD2-1 is seed-specific and is maximally expressed during oil biosynthesis. FAD2-2 is expressed at relatively constant levels in all tissues and is thought to be responsible for the synthesis of linoleic acid for membrane lipids (Heppard et al. 1996). Two isoforms of FAD2-1 are present in soybeans, and they are 93.8% identical at the sequence and amino acid levels (Tang et al. 2005; Schlueter et al., 2007). Both isoforms are approximately 67% identical to the FAD2-2 DNA sequence. FAD2-1B is nearly twice as active as the FAD2-1A enzyme when expressed and assayed in yeast (Tang et al. 2005). In addition, the FAD2-1A enzyme is highly unstable at 308C, and two regions play a role in the destabilization of the protein at that temperature (Tang et al. 2005). In silico analysis of soybean expressed sequence tags (ESTs) corresponding to FAD2-1 and FAD2-2 isoforms verified their seed-specific and constitutive expression, respectively (Tang et al., 2005). However, expression studies of the two isoforms of the FAD2-1 gene provided evidence of transcript accumulation in both vegetative tissues and developing seeds contrary to previous suggestions for the seed-specific nature of the FAD2-1 gene. Furthermore, both FAD2-1A and FAD2-1B showed evidence of alternatively spliced transcripts that do not change the amino acid residues of the putative v–6 desaturase enzymes. FAD2-1A and FAD2-1B map on linkage groups O and I, respectively, in the soybean genome (Schlueter et al., 2007). For the FAD2-2 gene, three isoforms were reported, which were designated as FAD2-2A, FAD2-2B and FAD2-2C (Schlueter et al., 2007). FAD2-2A has a 100 bp deletion in the coding region which would render the enzyme inactive. FAD2-2A and FAD2-2B are localized on

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the same bacterial artificial chromosome and were mapped to linkage group L (Schlueter et al., 2007). Southern analysis using the FAD2-1 as the probe showed that line M23, which carries the high oleic acid allele ol, lacked one of the three bands observed in cultivar ‘Bay’ and M11 (Kinoshita et al. 1998a). No differences in Southern blot pattern were observed when the FAD2-2 was used as probe. The absence of the third band was completely linked to the high oleic acid content inherited from M23 in a population derived from the cross of this line with ‘Bay’. Similar results were observed in a F2 population derived from M23 and ‘Archer’ (Alt et al. 2005a). Results from three populations derived from mid-oleate lines N984445A and N97-3363-3 conclude that only minor oleate QTLs cosegregate with the FAD2-1 and FAD2-2 loci (Cardinal, unpublished results). Therefore, mutations in these candidate gene loci do not cause the mid-oleate phenotype in N98-4445A and N97-3363-3 lines. However, seed-specific gene silencing of FAD2-1 in soybeans resulted in oil with more than 85% oleic acid content and less than 1% linoleic acid as expected from the knowledge of fatty acid synthesis (Kinney 1996). IV. GENETIC REALTIONSHIPS AMONG LOCI, PLEIOTROPY, AND ENVIRONMENTAL EFFECTS ON FATTY ACID COMPOSITION A. Genetic Relationship among Different Fatty Acid Loci In order to develop soybean cultivars that have multiple altered fatty acid traits, it is necessary to first determine if different major mutations that affect different fatty acids are independent from each other. The high-palmitate fap2 allele is independent from the low-palmitate fap1, sop1, fap3, and fapx alleles (Erickson et al. 1988; Schnebly et al. 1994; Kinoshita et al. 1998b; Rahman et al. 1999). The high-palmitate fap4 allele is independent from the low-palmitate fap1 and fap3 alleles (Schnebly et al. 1994). The high-palmitate sop2 allele is at an independent locus from the low-palmitate sop1, fap1, and fapx alleles (Rahman et al. 1996a, 1999). The fapx and fap2 loci that control high palmitic acid are inherited independently from the high stereate locus st2 so it was possible to develop a High Total Saturate line (HTS) (> 380 g kg 1 saturates; 214 g kg 1 16:0 and 171 g kg 1 18:0, but 108 g kg 1 18:1), which combines the fap2, fapx and st2 loci from the cross of line HPKKJ10 to M25 (Rahman et al. 2003).


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The low linolenic acid fan allele is inherited independently from the low palmitic acid fap1 and fapx alleles and high palmitic acid fap2 loci, and no interaction was observed among loci (Nickell et al. 1991; Primomo et al. 2002). The low linolenic fan and fanxa loci are inherited independently from the high oleic ol allele, and germplasm with both characteristics is easily obtained (Rahman et al. 2001). The low-palmitate fap1 and sop1 are inherited independently from the locus controlling elevated oleate (ol) and the loci fan and fanxa controlling reduced linolenate, permitting the development of germplasm with 40 g kg 1 16:0, 510 g kg 1 18:1 and 29 g kg 1 18:3 (Rahman et al. 2004). The high-palmitate fap2 and fapx loci, the low linolenic acid fan and fanxa, and the high oleate ol locus are inherited independently (Rahman et al. 2004). However, the presence of high palmitate loci reduce the oleic acid content, and it was only possible to develop a germplasm with 170 g kg 1 16:0, 418 g kg 1 18:1, and 29 g kg 1 18:3 (Rahman et al. 2004). B. Phenotypic Correlations among Fatty Acid Components of Seed Oil Several studies have reported the phenotypic correlations, Pearson’s correlations, correlated responses to selection, or correlations between fatty acids of two groups that had been selected to differ in their 18:3 content. However, no study has been published that reported genotypic correlations among fatty acids (Burton at al. 1983; Walker et al. 1998; Ross et al. 2000; Cherrak et al. 2003). A recurrent mass selection program for increased oleic acid content in soybean oil increased the mean oleic acid content from 25% to 33% and resulted in correlated decreases in linolenic acid content from 8% to 6% and in linoleic acid from 53% to 47%. No changes were observed in palmitic and stearic acid content in this study (Burton at al. 1983). Linolenate content was negatively correlated with oleate content in three populations containing between 1% to 2.7% linolenic acid in the oil. No significant correlation was detected between linolenate and palmitate or stearate (Ross et al. 2000). However, in populations in which linolenate content varied from 2.5% to 8.5%, it was significantly negatively correlated with total oil, stearate, oleate, and linoleate content in three populations and positively correlated with palmitate in two populations (Walker et al. 1998). No significant correlation was detected between linolenate and palmitate in a population from a cross between a low palmitic and low linolenic acid line (N97-3708-13) and a high-yielding normal line (Anand) (Cherrak et al. 2003).

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In several populations derived from crosses of lines RG1 (fap1fap1fapx fapx fanfan) and RG3 (fap1fap1fapx fapx FanFan) to several normal or low palmitate lines, no phenotypic correlation between palmitic and linolenic acid content was observed (Primomo et al. 2002). Palmitic acid content was not significantly phenotypically correlated with oleic content but was positively correlated with stearic acid content and negatively correlated with 18:2 content. Linolenic acid content was negatively correlated with either 18:1 or 18:2 contents depending on the population and positively correlated with stearic acid in three out of six populations (Primomo et al. 2002). The correlations among fatty acid components will be better understood when studied in light of the biosynthetic pathway and the candidate genes that are segregating in a population. C. Environmental Influence on Fatty Acid Composition Environmental stability studies for unsaturated fatty acids that compared the performance of selected and unselected lines determined that maturity effects make interpretation of genotype-by-environment interactions difficult; therefore, analyses should be performed within maturity groups (Carver et al. 1986). Warmer environments were associated with higher oleic acid and lower linoleic and linolenic acid content. Lines with higher oleic and linoleic acid content were more sensitive to environmental changes compared to unselected lines. Lines with lower linolenic acid content tended to be less sensitive to environmental changes (Carver et al. 1986). A similar study evaluated the stability of palmitate and stearate content in the oil of 30 lines derived from the base, third, and sixth cycles of recurrent selection for higher oleic acid content. Five lines of each cycle were early-maturing and five lines were late-maturing with a mean maturity difference between the groups of 16 days (Rebetzke et al. 1996). This study concluded that mean temperature during the podfilling period primarily influences the palmitic acid content in soybean seed oil and had a smaller effect on stearic acid. Lines that were grown in locations with mean temperature of 268C during pod filling had higher palmitic acid content than when they were grown at 208C (Rebetzke et al. 1996). However, there were significant genotype environment interactions due to a change in the variance and ranking for both traits; and latematuring lines were more sensitive to changes in temperature (Rebetzke et al. 1996). In addition, the mean daily minimum temperature during the growing season influences the palmitic content in lines inheriting the fapnc mutation (Rebetzke et al. 2001). In another experiment, soybean lines with elevated palmitic acid, elevated stearic acid, reduced linolenic


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acid, or normal fatty acid content were evaluated in different years and planting dates (Schnebly and Fehr 1993). Each unique genotype had a different response to the environment. There was a positive association of average daily temperature with palmitic, stearic, and oleic acid content in two normal soybean lines. A negative association between average daily temperature with linoleic and linolenic acid content was detected in the same normal soybean lines. No relationship between temperature and oleic or linoleic acid content was observed in most genotypes with low linolenic acid content (Schnebly and Fehr 1993). This study concluded that genotypes with unique fatty acid contents were not necessarily more or less sensitive to different environments. Similar results were obtained when several genotypes with normal fatty acid composition, reduced linolenate, reduced palmitate, increased palmitate, increased oleate and reduced linolenate, reduced palmitate and linolenate were planted in two different dates at five very different locations from Mississipi to Missouri (Oliva et al. 2006). Their stability was calculated for each fatty acid from the regression of the fatty acid level on average temperature over the final 30 days of seed fill level across 10 environments. Genotypes differed in stability of fatty acid profile across environments. Mid-oleate lines N98-4445A and N97–3363 were the most unstable among the genotypes. However, the mid-oleate line M23 was relatively stable in oleate over the 10 growing environments. IA 3017 with lowest linolenate content (1%) varied least and was the most stable in linolenate content across environments while higher linolenate genotypes were less stable. In summary, environmental stability will have to be determined for each line that carries novel alleles for any fatty acid since all the previous studies have shown that genotypes react uniquely to the environment. Significant environment effects and year environment interaction were observed when A6 and several soybean lines within a normal range for stearic acid content were grown in multiple years or locations (Schnebly et al. 1993; Rebetzke et al. 1996). Mean temperature during the growing season had a small effect on stearic acid content; therefore, the genotype environment interaction must be accounted by other environmental factors (Rebetzke et al. 1996). In addition, studies have been done to determine the effect of temperature on the fatty acid composition of a normal soybean. For example, Fiskeby V was grown in a phytotron at five different temperatures from 188 to 338C during the day and 138 to 288C during the night. Linolenic and linoleic acids decreased while the 18:1 increased as temperature increased. However, palmitic and stearic acids remained the same (Wolf et al. 1982).

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D. Pleiotropy: Fatty Acid Content in Other Tissues Pleiotropic effects in root tissues have been shown to exist for the fan mutation in A5, C1640, and PI123440 and a low linolenic acid line of unknown origin because the low linolenic acid phenotype was also expressed in roots (Martin and Rinne 1985; Wang et al. 1989; Liu and Brown 1996; Camacho-Roger 2006). In addition, pleiotropic effects in roots, unifoliate and trifoliate tissues were observed for a low palmitic acid line (unknown pedigree) since those tissues also had a reduced palmitate content when compared to a normal line. No error estimates, however, were provided in the study to determine if the differences observed were significant (Liu and Brown 1996). The fatty acid composition of roots, leaves, and stems was compared between a normal palmitate cultivar ‘Elgin87’, a low-palmitate line A18 (fap1fap1fap3fap3), and a high-palmitate line A19 (fap2bfap2b fap4fap4) (Schnebly et al. 1996). A18 had the lowest palmitate content in those tissues from V2 to R6 stage of development. The decrease in palmitate was accompanied by an increase in oleate. A19 had the highest palmitate content in those tissues from the V2 to R6 stage of development. The increase in palmitate in A19 was accompanied by decrease oleate and linoleate. These results demonstrate that at least one of the two palmitate genes carried by each of these lines is constitutively expressed. Since the differences in palmitate content among the lines in roots, leaves, and stems were smaller than those observed in the seed oil, it is posible to speculate that only one of the two palmitate genes each line carries is expressed in those tissues. A study was undertaken to determine if the low-palmitate fap1 and fapnc mutations have pleiotropic effects on other tissues of the plant (Camacho-Roger 2006). The results of this study demonstrated that the fapnc and fap1 mutations have pleiotropic effects in leaf tissue and that the fan (PI123440) mutation has pleiotropic effects in both leaf and root tissues. In conclusion, breeding for enhanced seed oil quality could indirectly affect the fatty acid membrane composition in other tissues and adversely affect in very small and unpredictable ways the development of the soybean plant or their response to different environments.

V. RESEARCH NEEDS From our ongoing sequencing efforts, it is now clear that several isoforms of each candidate gene generally exist in soybeans. These isoforms may be located in duplicated regions of the soybean genome. The


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existence of these isoforms gives rise to a tremendous complexity in the fatty acid biosynthetic pathway. These isoforms have very similar sequences but may be expressed in different tissues, at different developmental stages, or respond to different environmental stressors. Further complex networks of epistatic interactions may arise from interactions among multiple isoforms; each isoform could interact epistatically with different isoforms of other genes. Transgenic experiments cannot help us elucidate this complexity. Isoforms are generally very similar in their sequences, and antisense transgenes would likely silence all the isoforms of a particular gene. Similarly, in overexpression experiments, plant researchers tend to select plants that show an extreme phenotype to study the effect of a particular gene, which makes it very difficult to detect their subtle interactions with other genes of the pathway. To understand the complexity of the fatty acid pathway, the effects of particular gene isoforms and their interactions with different genes and the environment need to be studied. We can accomplish these goals only by evaluating populations that are segregating for several natural genes (isoforms) in multiple environments. Results of QTL studies are helpful but alone do not indicate which gene is responsible for the phenotypic variation observed in a trait. However, we now have the tools (sequence information and biochemical information for most of the genes, several mutant and naturally occurring alleles for each fatty acid component) to implement cosegregation studies of particular candidate genes with phenotypic variation for all the fatty acid components in soybeans, an agronomically important crop. Studies of the stability of particular fatty acids in segregating populations are needed. This can be accomplished by testing if the effects of QTLs and candidate genes change with the environment (i.e., if QTLs by environment interactions are significant). Resolution of overall genotype-by-environment interactions into specific gene-by-environment interactions will demonstrate which specific genes or QTLs interact with the environment. This information will be critical for the development of environmentally-stable high oleic cultivars. In conclusion, tools needed to manipulate very effectively the fatty acid composition in the soybean oil by allele-specific selection will soon be available. Although genotypic selection will undoubtedly be effective for improving fatty acid content in soybean, the relative efficiency of genotypic selection compared to phenotypic selection remains unproven. Phenotypic selection on oil fatty acid composition measured by gas-liquid chromatography of the methyl esters is already very effective. In order for the allele-specific genotypic selection scheme

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to be more efficient than phenotypic selection, genotypic selection needs to permit selection of all the fatty acid traits simultaneously and needs to be cheaper or faster than gas-liquid chromatography. In order to achieve this goal, the candidate gene alleles that cause the most desirable and stable phenotypic effects across environments need to be determined for each fatty acid. This will require a detailed understanding of the relationships between mutations with observable phenotypes and specific candidate gene isoforms. Allele-specific markers will be required for each of these mutations in order to conduct full-scale genotypic selection. Optimally, multiple sources of alleles for each candidate gene isoform should be used in breeding to minimize fixation of alleles linked to target genes, which would ultimately reduce response to selection for other important agronomic traits.

VI. ACKNOWLEDGMENTS Funding for part of the research described in this review was received from: USDA, CSREES, National Research Initiative Competitive Grants Program. Plant Genome- 52.1 to A. J. Cardinal and the United Soybean Board. The author is grateful to Drs. J. W. Burton, R. E. Dewey, and J. B. Holland for their critical review of the manuscript.

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6 Improving Breeding Efficiency for Early Maturity in Peanut S. N. Nigam and R. Aruna International Crops Research Institute for The Semi-Arid Tropics (Icrisat) Patancheru 502 324 Andra Pradesh, India

I. INTRODUCTION A. General B. Cropping Practices and Crop Duration II. BOTANY A. Reproductive Biology B. Physiology 1. Reproductive Yield 2. Thermal Time and Phenological Development C. Methods of Maturity Estimation III. GENETICS AND BREEDING A. Inheritance, Heritability and Combining Ability of Early Maturity and Its Components B. General Breeding Procedures for Early Maturity C. Breeding Strategies for Increased Efficiency 1. Selection for Low Tb and CTT for Various Phenological Stages 2. Selection for Tolerance to High Temperature 3. Selection for Photoperiod-Insensitive Genotypes 4. Selection for High Crop Growth Rate and Partitioning 5. Selection for High Water-Use Efficiency 6. Diversification of Sources of Earliness in Maturity 7. Evaluation in Target Environments/Cropping Systems 8. Opportunities for Biotechnological Interventions D. Other Issues in Breeding for Early Maturity 1. Yield Potential 2. Seed Size 3. Resistance to Foliar Fungal Diseases IV. CULTURAL MANIPULATIONS TO SHORTEN CROP DURATION V. SUMMARY VI. LITERATURE CITED


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I. INTRODUCTION A. General Peanut, also referred to as groundnut (Arachis hypogaea L., Fabaceae), an annual legume, is an important source of edible oil and vegetable protein. Its seeds contain about 48 to 50% oil and about 26 to 28% protein. They also are a rich source of minerals, vitamins, and dietary fiber. Peanut seeds can be eaten raw, roasted, or boiled, or made into a paste, popularly known as peanut butter and chutneys, or can be used in the preparation of different confections. The haulms are used as fodder, and the cake, after extraction of oil, is used in the livestock feed industry. Peanut shells are used as fuel, as filler in the feed industry, and in cardboard making. Being a leguminous crop, peanut enriches the soil with nitrogen and is therefore valuable in cropping systems. It is simultaneously a food crop and a cash crop, providing smallholder families with dietary protein, high-grade fat, and cash income from sale on local markets. Peanut is cultivated in over a 100 countries in tropical, subtropical, and warm temperate regions of the world. The approximate limits of the present commercial production are between latitudes 40o N and 40o S. It is grown on 26.4 million ha with a total annual production of 36.1 million t and an average productivity of 1.4 t ha-1 (FAOSTAT 2004). Asia, with about 25 countries and 54.2 % of the area, produces 67.2% of the world’s production. It is followed by Africa (about 46 countries) with 41.5% area and 24.7% production. North America in 2.3% of the area and South America in 1.4% of the area produce 5.4% and 2.0% of the world’s production, respectively. The production of peanut in Europe is negligible. Important peanut-producing countries are China, India, and Indonesia in Asia; Nigeria, Senegal, and Sudan in Africa; the United States in North America; and Argentina and Brazil in South America.

B. Cropping Practices and Crop Duration Peanut is grown in high-input commercial, semicommercial, and subsistence farming systems. In the high-input system, cultivation is completely mechanized; in the subsistence system, it is mainly manual with minimum on-farm inputs. In the commercial system, the peanut is grown as the sole crop, whereas in semicommercial and subsistence farming, it may be grown as a sole crop, intercropped, or as a mixed crop. The semiarid tropical region, characterized by unpredictable

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rainfall, contributes more than 90% of the world’s peanut production. Generally, only one major crop of peanut is grown each year in the rainy season. However, in many Asian countries with climatic conditions (mainly temperature) favorable to peanut, two or three crops are harvested per a year, either with irrigation or on residual soil moisture. In a monocropping situation, duration of the crop depends on the number of days of favorable temperatures for crop growth and plant extractable soil moisture availability either through irrigation or rains. In some agroecologies in the United States and China, the crop duration may reach 160 to 180 days. In many agroecological situations—where there are short growing seasons, end-of-season droughts, early frosts, cultivation in residual soil moisture, or multiple cropping—however, short-duration cultivars are needed to escape adverse climatic conditions and late-season diseases and insect pests. In specific intercropping systems, early-maturing cultivars offer less competition to the latermaturing crops. Multiple cropping is more frequent in the rice-based cropping systems in East and Southeast Asia and intercropping in South Asia. Short duration of a genotype is a relative term. In terms of calendar days, it may be different for different situations. For example, a 140-day cultivar in the United States or a 120-day cultivar in China may be classified as a short-duration cultivar in these countries. However, in South and Southeast Asia, a short-duration cultivar should not exceed 100 days to maturity. Similarly, along the northern fringes of the SudanSahelian region in West Africa, which may have a very short rainy season, the duration of the cultivar should not exceed 90 days (Virmani and Singh 1986).

II. BOTANY Cultivated peanut is a member of Fabaceae, tribe Aeschynomeneae, subtribe stylosanthineae. The genus Arachis is divided into nine sections. Section Arachis contains Arachis hypogaea, which is divided into two subspecies, fastigiata Waldron and hypogaea Krap et Rig. Subspecies hypogaea contains two botanical varieties, hypogaea (Gregory et al.) and hirsuta Kohler, while subspecies fastigiata consists of four botanical varieties: fastigiata, peruviana Krapov. & W.C. Gregory, aequatoriana Krapov. & W.C. Gregory, and vulgaris C. Harz (Krapovikas and Gregory 1994). In subspecies hypogaea, the central axis is without flowers and reproductive and vegetative nodes alternate regularly (alternate ramification)

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on lateral branches. Genotypes belonging to subspecies hypogaea are generally late in maturity and have postharvest seed dormancy. In subspecies fastigiata, the central axis is with flowers, and reproductive and vegetative nodes show no order (sequential ramification) on lateral branches. Genotypes belonging to subspecies fastigiata generally mature early and lack fresh seed or postharvest seed dormancy. The other differences between the two subspecies are summarized in Holbrook and Stalker (2003). A. Reproductive Biology Inflorescences are borne in the axil of leaves of a peanut plant. They are either simple or compound monopodia, and each has up to five flowers. Generally, only one flower per inflorescence is open at any given time. Onset of flowering is independent of day length. However, short-day conditions or increasing mean temperatures up to 308C enhance number of flowers (Bagnall and King 1991a). Flowering in peanut can appear as early as 17 to 18 days after emergence and can last 85 to 100 days depending on the cultivar, climatic conditions and the cultural practices followed. Both bimodal and continuous flowering patterns are reported in the literature. Flowering with abrupt alternation of high and low frequencies during the major portion of the flower production period is observed in all botanical types. This cyclic behavior of flowering is not closely related with climatic conditions (Smith 1954). The number of flowers produced per plant may range from 40 to 250 in subspecies hypogaea (Seshadri 1962) and from 95 to 148 in subspecies fastigiata (Krishna Sastry et al. 1985). However, only 8 to 20% flowers develop into mature pods (Cahaner and Ashri 1974; Krishna Sastry et al. 1985). A large number of early-formed flowers develop into pods (Gregory et al. 1951; Cahaner and Ashri 1974). Pods initiated earlier have faster growth rate and greater number of seeds than pods initiated later in the season (Williams 1979). Flowers that appear 70 days after sowing do not form mature pods. Suppression of the late-formed flowers leads to better development of early-set pods (Har-Tzook 1970; Ono and Ozaki 1971). Most flowers are self-pollinated before or as they open, and crosspollination is rare. The pollen tube takes 10 to 18 hours (hr) after pollination to reach the ovary and effect fertilization (Smith 1956). After fertilization, the flowers wither rapidly and the intercalary meristematic cells that comprise the basal tissue of the ovary produce a geotropic stalklike structure called a peg (or a gynophore). Initially associated with embryo development, the peg grows at first slowly and then rapidly. The tip of the peg usually contains two (sometimes three to five, depending on the


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botanical variety) fertilized ovules. At the time of peg growth, the embryo is at the 8- to 12-cell stage and becomes quiescent (Pattee and Mohapatra 1987). Peg growth continues until penetration into the soil (after 8–14 days of fertilization), and when it receives mechanical stimulus the peg transforms into a pod (Zamski and Ziv 1976). Ovules and embryos then start growing, mature to form seeds within the pod, which later become dry and brittle to form the shell. In Spanish genotypes (var. vulgaris), pods are ready for harvesting after about 7 weeks; Virginia genotypes (largeseeded types of var. hypogaea) mature after about 11 weeks of underground development (Schenk 1961). B. Physiology Ong (1986) concluded that temperature is the dominant environmental factor that influences peanut development. The life cycle of a peanut plant has been divided into different vegetative (VE, V0, and V1 to VN) and reproductive stages (R1 to R9) based on discrete, objective, and visually identifiable events (Boote 1982). VE refers to emergence, V0 to flat and open cotyledons at or below the soil surface, and V1 to VN to development of corresponding nodes on the main axis. R1 refers to the beginning of the bloom, R2 to beginning peg, R3 to beginning pod, R4 to full pod, R5 to beginning seed, R6 to full seed, R7 to beginning maturity, R8 to harvest maturity, and R9 to overmature pod. An R stage remains unchanged until the date when 50% of the plants in the sample demonstrate the desired trait of the next R stage. The V and R stages in peanut overlap. Rate of node development is dependent on air and soil temperature, availability of soil water, and plant maturity. Stress, whether water, heat, or nutrient, can decrease the rate of plant development (Craufurd et al. 1993) including the rate of pod formation in peanut (Williams and Boote 1995). In addition, reproductive development is influenced by photoperiod. These effects have important consequences for the duration of developmental and growth stages. 1. Reproductive Yield. Duncan et al. (1978) described the reproductive yield (Yr) in this way: Yr ¼ C Dr p where C is the mean crop growth rate Dr is the duration of reproductive growth p is the mean fraction of crop growth rate partitioned toward the reproductive organs (assessed by harvest index, HI)

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Variations in mean growth rate (C) are dominated by environment (E) and genotype (G) E interactions because the photosynthetic variation in a species is small, while the scope for radiation interception is very large. Once light interception by a peanut crop is complete, the major sources of yield variation between cultivars lie in their partitioning and duration (Duncan et al. 1978). Various environmental challenges also influence C, p, and Dr. For example, drought will influence C and p, calcium deficiency will influence p, and foliar diseases will mainly influence C (Williams 1992). Temperature and photoperiod also have profound influences on vegetative and reproductive growth and development in peanut. Photoperiod has a marginal influence on time to flower initiation. Its main effects are observed on reproductive efficiency. Long days stimulate vegetative growth and reduce pod yield by affecting p (Wynne et al. 1973; Wynne and Emery 1974; Emery et al. 1981; Nigam et al. 1994). Flowers, pegs, pod number, and pod and seed weight are enhanced under short-day conditions by greater p and/or increased duration of effective pod-filling phase (Witzenberger et al. 1988; Bagnall and King 1991b; Nigam et al. 1994). However, the effect of photoperiod occurs only above a certain critical temperature lying between 22/188C and 26/228C regimes (Nigam et al. 1994). High soil temperature reduces dry matter accumulation, flower production, the proportion of pegs forming pods, and individual seed mass (Golombek and Johansen 1997; Prasad et al. 2000). Genotypic variation exists for photoperiod temperature interaction (Nigam et al. 1998). Temperature and irradiance play a major role in determining crop duration and p. Photo-thermal quotient (PTQ MJ m-2 degree-day-1, derived from total short-wave solar radiation incidence during the growing season and CTT) could largely explain the variation in HI across locations, provided the data were not confounded by the effects of photoperiod (Bell and Wright 1998). 2. Thermal Time and Phenological Development. In both photoperiodinsensitive and sensitive genotypes of annual crops, maintained in a given constant photoperiod, the rate of progress toward flowering is a positive linear function of temperature from a base temperature, Tb, at which the rate is zero, up to an optimum temperature, T0, at which it is maximal. Between these limits, the relation may be described as 1=f ¼ a þ bT where f is the time from sowing to first open flower T is the mean temperature


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The values of a and b are specific to the genotype but, in photoperiodsensitive genotypes held in any constant photoperiod, the value of a is, in addition, a function of photoperiod. Above T0, the values of constants will differ from those for the suboptimal range and the relation (and so the sign of the parameter b) will be negative (Summerfield et al. 1991). There is variation in Tb for different phenological stages and among genotypes. In their study, Bell et al. (1991) did not find differences in Tb values for seedling emergence, but for days to first flower, the Tb values for Spanish genotypes were higher than those for the Virginia and Valencia genotypes. However, thermal times for flowering were lower for the Spanish than for the Virginia and Valencia types. Variation in Tb values for seedling emergence has been observed among genotypes (ICRISAT, unpublished data). For peanut, Tb ranges between 98C and 138C and T0 between 278C and 328C (Williams and Boote 1995). The cumulative thermal time (CTT) is measured in day-degrees (8Cd) above the base temperature and is calculated on successive days by subtracting the base temperature from the mean daily temperature and adding each value to the subtotal accumulated since the seed was sown. In photoperiod-insensitive genotypes, the CTT for maturity does not differ across environments barring the influence of environmental factors other than photoperiod. For photoperiod-sensitive genotypes, the CTT will vary with photoperiod over the photoperiod-sensitive range. All associations, which are described earlier, function within the range of Tb and T0. At higher temperatures, the start of pod and seed filling can be delayed substantially in some genotypes (Craufurd et al. 2002), which will affect the crop duration. Genotypic differences for heat tolerance have been reported in peanut (Craufurd et al. 2003). Mills (1964), Emery et al. (1969), Williams et al. (1975), Ono (1979), Leong and Ong (1983), Mohamed (1984), Ong (1986), and Ketring and Wheless (1989) determined the CTT requirement of different phenological phases in peanut. Solar radiation, particularly during the first 75 days of growth, also affects the peanut maturity (Holaday et al. 1979). C. Methods of Maturity Estimation Determination of optimum maturity in peanut is critical as it affects marketable yield, quality, and flavor of the produce. The indeterminate and subterranean fruiting in peanut confounds the estimation of optimum maturity. Indeterminate fruiting characteristic of peanut results in seed of varying maturities on the plant as harvesttime approaches.

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Sanders et al. (1982) reviewed several methods to determine crop maturity in peanut. These methods are variations of four approaches: indirect methods (days after planting, heat units), relative color evaluations (internal pericarp color, oil color, methanol extract, pod maturity profile), weight and weight relationships (kernel density, seed to hull ratio maturity index), and quantification of a specific component (arginine maturity index and protein markers). Some methods are more complex than others to evaluate, and each method has deficiencies. General use of some of the methods, such as days after planting and oil color, is precluded as they are highly influenced by environmental conditions. Pod maturity profile and seed to hull ratio maturity index are some of the better methods for estimation of maturity. However, the most commonly used method by small farmholders in developing countries is internal pericarp color because of its simplicity. When 75 to 80% pods in cultivars belonging to subspecies fastigaita and 70 to 75% pods in cultivars belonging to subspecies hypogaea show internal pericarp darkening, the crop is ready for harvest.

III. GENETICS AND BREEDING A. Inheritance, Heritability, and Combining Ability of Early Maturity and Its Components The genetics of maturity in peanut is not well defined, largely because of the difficulty of defining maturity for an indeterminate, nonsenescent plant (Kvien and Ozias-Akins 1991). Although several authors have reported genetics of plant maturity, pod/seed maturity, and components of maturity, their inferences are inconclusive. Badami (1923 and 1928) reported earliness recessive to late maturity, whereas Patel et al. (1936) and Hassan (1964) found late maturity incompletely dominant over earliness. In both cases, a single gene was involved. On the other hand, Samooro (1975) reported four or fewer genes for seed maturity. When Holbrook et al. (1989) used the hull-scrape method to estimate pod maturity, they reported four or five genes with complete dominance of lateness over earliness and absence of reciprocal differences. Tai and Young (1977) studied inheritance of free arginine level in peanut seed and reported partial dominance of low arginine level (an indicator of seed maturity) with two major genes, several minor genes, and their interactions. Upadhyaya and Nigam (1994) reported a single gene with an additive effect for the control of days to first flower, three genes with two types of epistatis (dominant-recessive, 13 late: 3 early


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and duplicate-dominant, 1 late: 15 early) for days to accumulation of the first 25 flowers, and the absence of reciprocal differences for these traits. They also reported that the genes for early accumulation of flowers in ‘Chico’ and ‘Gangapuri’, the two early-maturing cultivars, were located at different loci. For days from seedling emergence to first flower, Vindhiyavarman and Raveendran (1996) reported two recessive genes acting in an additive manner. The inheritance of maturity and its components has also been studied using diallel, line tester, and generation mean analyses. Parents with good general combining ability for early maturity have been reported (Basu et al. 1986). Parker et al. (1970) found significantly higher variance for general combining ability (gca) than specific combining ability (sca) for time of emergence (measured in hours), time first leaf opening cotyledonary branch (in hr), time first leaf opening main stem (in hr), days to first flower, and number of flowers per plant (at 32 days) under a controlled environment. Similarly, Wynne et al. (1970) and Nigam et al. (1988) reported a higher magnitude of gca than sca variance for days to first flower under field conditions. Gibori et al. (1978) reported significant additive genetic variance with bidirectional dominance for days to first flower. Mohammed et al. (1978) observed highly significant additive genetic variance for pod and seed maturity indices. However, Ali et al. (1999) reported both additive and dominance genetic effects for maturity index (ratio of number of mature to total number of pods). They did not detect epistatis. Khalfaoui (1990b) studied heredity of extreme precocity (ability of a genotype to form highest proportion of ripe pods rapidly) in a cross between two Spanish cultivars, ‘73–30’ and ‘Chico’, using days from sowing to emergence (S-E) and from emergence to first flowering (E-F), number of flowers produced during the first four days of flowering (4F), and percentage of ripe pods 80 days after sowing (% RP, using internal pericarp color as pod maturity indicator) as parameters. He reported extremely limited variation for S-E between the two parents, making it difficult to study genetic effects. For E-F, highly significant additive and significant dominance, additive additive and dominance dominance effects, with duplicate digenic interaction were observed. For 4F, allelic interactions involving more than two genes or linkage effects between the genes were reported. For %RP, highly significant additive and significant dominance effects were observed. The additive effect for %RP was markedly greater than the dominance effect and two to three genetic factors were responsible for the difference between the two parents. Transgressive segregation was observed in favor of flowering precocity, intense flowering, and pod ripeness precocity. The genetic correlation between S-E and E-F

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(0.50 0.12) and S-E and 4F (0.41 0.13) was moderate, but there was no genetic correlation between S-E and %RP and the correlation between 4F and % RP (0.29 0.14) was slight. Thus, S-E, E-F, and 4F, being genetically independent or only loosely linked with %RP, cannot be used effectively to select for precocity of % RP. However, in eight cultivars previously studied (Khalfaoui 1990b), a close linkage between % ripe pods at 90 days and the flowering rapidity and intensity indicated that flowering characters favored genotype precocity. Several workers reported estimates of broad sense and narrow sense heritability of maturity and its components in peanut (Table 6.1). The studies just described indicate that maturity is not simply inherited. However, components affecting early maturity are highly heritable, suggesting that selection for earliness can be successful in early segregating populations. Table 6.1. Heritability estimates of maturity and its components in peanut.

Trait Days from sowing to seedling emergence Days from seedling emergence to first flowering

Broad-Sense Heritability Estimates 48–83% (51%) 49–81% (23–39%) 11–55% 96.9%

Days to 50% flowering

Days from emergence to accumulation of 10 flowers Days from emergence to accumulation of 25 flowers Number of flowers produced during the first four days of flowering Days to maturity

Fruit maturity based on oil pigmentation Low arginine level in seed (maturity) Maturity based on hull-scrape method Percentage of ripe pods 80 days after sowing Maturity index

96.3% 66.5–72.5% 22–65%

Reference Khalfaoui 1990b Khalfaoui 1990b N’Doye and Smith 1993 Mazumdar et al. 1969 Islam and Rasul 1998 Singh and Singh 1999 N’Doye and Smith 1993

17–61%

N’Doye and Smith 1993

16–44% (9–38%)

Khalfaoui 1990b

98.6% 91.7% 80.5%–96.7% 69–95% 69–93% 71% 13–41% (24%) Fairly high

Mazumdar et al. 1969 Islam and Rasul 1998 Singh and Singh 1999 Gupton and Emery 1970 Tai and Young 1977 Holbrook et al. 1989 Khalfaoui 1990b Ali and Wynne 1994

Values in parentheses are estimates of narrow-sense heritability.


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B. General Breeding Procedures for Early Maturity In most breeding programs, a predetermined crop duration (in calendar days) is defined depending on the local/regional/cropping system requirements. Generally, days to flowering, profuse early flowering, and days to maturity are taken into account while selecting parents as sources of early maturity. In segregating populations, a cutoff date based on predetermined crop duration is set and the populations are uprooted for visual observation of pod maturity based on internal pericarp color. Plants with a higher percentage of mature pods and high pod yield are selected for further laboratory evaluations for seed appearance, uniformity, and maturity. The selected material is advanced to the next generation. Advanced breeding lines are evaluated for two to three years in on-station replicated trials with staggered harvesting starting from a predetermined date. In addition to pod yield and pod and seed maturity, extent of further gains in pod yield, seed weight, and shelling turnover in the following staggered harvesting are taken into account while selecting advanced breeding lines. Those with high pod yield and pod and seed maturity and minimum gains in pod yield, seed weight, and shelling turnover over the preceding harvest are selected for further multilocation evaluation and selection. As temperature is the predominant environmental factor that influences the growth and development of peanut plant (Bell et al. 1994), estimation of crop duration in calendar days at a given season/location seldom relates with that of at other seasons/locations. Significant genotype environment/location year (Fincher et al. 1980; Roy et al. 1980; Pattee et al. 1980; Court et al. 1984; Knauft et al. 1986), harvest date sowing date and harvest cultivation (Mozingo et al. 1991) interactions on optimum harvest date are reported. CTT has been used in many crops to overcome the problems associated with the use of calendar days to describe the duration or optimum harvest time of a cultivar. However, doubts have been expressed on the ability of simple cumulative thermal time models to accurately predict phenological development in peanut (Mills 1964; Ketring and Wheless 1989; Hammer et al. 1992). The delays in phenological development after the first flowering could occur because of flower or embryo abortion (Bell et al. 1994), water deficit, or extreme heat (Ketring and Wheless 1989). Solar radiation, particularly during the first 75 days of growth, also affects the peanut maturity (Holaday et al. 1979). Since 1991, CTT (8Cd) is now used at ICRISAT Center (Patancheru, 188N, 788E) to determine the date of harvesting when breeding for early-maturing cultivars (Rao et al. 1991; Upadhyaya et al. 1992). Based on 23 years (1974–1996)

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meteorological observations, assuming 15 June as the sowing date, a peanut crop accumulates (on average) about 1239 8Cd in 75 days, 1474 8Cd in 90 days, 1717 8Cd in 105 days, and 1954 8Cd in 120 days in the rainy season (June–October). In the postrainy season (November–April), the number of calendar days required to accumulate these CTTs is about 30 days more than during the rainy season due to lower temperatures. For early-maturing cultivars, 14708Cd representing 90 days in the rainy season was selected, as it is the preferred duration in South and Southeast Asia. A computer program is used to calculate accumulated 8Cds each day from the date of sowing using 108C as base temperature (Tb) to estimate the date of harvesting. Early-maturing cultivars developed at ICRISAT have been released for cultivation in many countries (ICGV 86015 in Nepal, Pakistan, Sri Lanka, and Vietnam; ICGV 86143 in India, Vietnam, and Zambia; ICGV 86061 in Congo and Philippines; ICGV 86105 and ICGV 88023 in Guinea Conakry; ICGV 86065 in Mali; ICGV 86072 in Bangladesh; ICGV 86082 in Burkina Faso; ICGV 92195 in India; and ICGV 93382 in Myanmar). Use of CTT in breeding for earlymaturing cultivars is able to account for about 70 to 80% variation in crop duration across locations/seasons (ICRISAT unpublished data). In addition to temperature, other climatic factors, particularly solar radiation, need to be integrated in the model to improve prediction of maturity and stability of crop duration across years and locations. C. Breeding Strategies for Increased Efficiency In breeding for early maturity, it is helpful to partition crop duration into different segments/stages and examine the possibility of shortening their duration individually and collectively with an overall aim to reduce crop duration. These segments/stages include days to germination and emergence, days to first flower after emergence, days from opening of first flower to opening of a given number of flowers per plant, and days from opening a flower to maturation of seeds that develops from that flower. It is also important to take into account the time from seed maturation to major deterioration of the strength of the peg (Bailey and Bear 1973) to avoid seed losses during harvest. Cultivar differences exist in days to emergence and tolerance to cold temperatures during germination. Cultivars with large seeds generally take longer to emerge, and those with tolerance to cold temperature are able to emerge early under low temperature conditions. Early emergence hastens early flowering, which in turn hastens maturity. Due to the indeterminate growth habit, peanut produces more flowers on a plant than it can support to produce pods and seeds. In cultivars of


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differing maturity periods from very early to late, Bear and Bailey (1973) observed a high proportion of the first 25 flowers developing into mature pods. Days to flower (Yadava et al. 1981; Yadava et al. 1984; Khalfaoui 1990b; Islam and Rasul 1998) and days to production of first 50 flowers from planting (Khalfaoui 1990b) are related with maturity. A wide variation was observed among Spanish (var. vulgaris) genotypes in number of days required to produce 40 flowers; those that accumulated these flowers early produced higher plant yield (Krishna Sastry et al. 1985). In addition to temperature, the rate of peg growth and distance of its axil from the soil surface may have influence on this period. In case of erect growth habit (subspecies fastigiata), short plant stature and smaller internodal length may help to reduce this period by a few days. The latter will also help in case of runner and semirunner growth habit (subspecies hypogaea) types and will result in more uniform maturity. When the duration requirement is 120 to 125 days or above, it is not difficult to breed new cultivars with desired crop duration and high yield potential. However, it may entail some penalty in seed size in large-seeded types. Breeding for short duration becomes a challenging task when the duration requirement is less than 100 days with high yield potential. It generally limits the choice of botanical types to subspecies fastigiata var. vulgaris in a breeding program. Due to the alternate flowering habit in subspecies hypogaea, it is difficult to reduce crop duration below 115 days in this botanical group. Based on the botanical characteristics and physiological behavior of the crop, the following characteristics could be visualized for attaining short duration of the crop: short plant stature (plant height in case of subspecies fastigiata and plant spread in case of subspecies hypogaea) with smaller internodal length, faster germination and emergence, fewer days to first flowering, and accumulation of a maximum number of early flowers, more flowers per node, absence of late flowers, fewer days after fertilization for a peg to enter soil, faster pod and seed growth, high seed partitioning, and high shelling turnover. To capitalize on the full potential of the genotypes with the aforementioned traits, it would be essential to modify crop husbandry to accommodate larger numbers of plants per unit area to provide quick ground cover and to provide plants with required nutrients and other inputs. The following considerations in breeding strategy will help to achieve the objective of early maturity. 1. Selection for Low Tb and CTT for Various Phenological Stages. Although some genetic variation in Tb and CTT for different phenological stages among genotypes has been reported (Bell et al. 1991; Williams and Boote 1995), more studies are needed to discern these traits and identify

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genotypes with low Tb and CTT. Within the available genetic variation, selecting the genotypes with low Tb and CTT for different phenological stages will help reduce crop duration considerably. It may not be possible to select for these traits in segregating populations. But these criteria can be applied in selecting parents for hybridization and in evaluation of advanced breeding lines under controlled environmental conditions. 2. Selection for Tolerance to High Temperature. In situations/places where temperatures can go high during the cropping of peanut, tolerance to high temperatures will be essential to ensure high yields and required crop duration. Drought often results in high air and soil temperatures. High temperatures delay the start of seed filling; reduces total plant biomass, root/total biomass ratio, and seed yield (Wheeler et al. 1997; Craufurd et al. 2002); and delays crop maturity (Bell and Wright 1998). While Wheeler et al. (1997) did not observe adverse effects of high temperature on seed harvest index, Craufurd et al. (2002) found 0 to 65% reduction in this trait under high temperature. Prasad et al. (2000) observed significant reductions in total dry matter production, partitioning of dry matter to pods, and pod yield under high air and/or soil temperature. High air temperature did not affect flower production but significantly reduced the proportion of flowers setting pods, and pod numbers. However, high soil temperature significantly reduced flower production, pod set, and seed weight. The effects of high air and soil temperatures were mostly additive and without interaction. Various researchers have reported genotypic differences in tolerance to high temperature (Wheeler et al. 1997; Prasad et al. 2000; Craufurd et al. 2002). The genotypic differences in the response of peanut yield to episodes of high temperature stress are due to differences in the timing of seed filling (Wheeler et al. 1997; Craufurd et al. 2002). 3. Selection for Photoperiod-Insensitive Genotypes. For locationspecific genotypes, photoperiod insensitivity may not matter much, but for wide adaptation, it would be essential to ensure stability in yield potential and crop duration. Nigam et al. (1997) reported additive gene action in some crosses and partial dominance to dominance in other crosses for response to photoperiod in peanut. They further suggested that the selection for this trait be delayed to later generations because of practical difficulties in evaluating it in segregating populations. Genotypic variation exists for photoperiod temperature interaction (Nigam et al. 1998). 4. Selection for High Crop Growth Rate and Partitioning. As yield is a function of C, Dr, and p (Duncan et al. 1978), reducing the total crop


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duration while maintaining Dr will have little effect on pod yield of a genotype provided the required plant density is maintained in the field. However, the reduction in crop duration will have to come through reduction in duration of vegetative phase (i.e., early onset of flowering). For substantial reduction in crop duration, some sacrifice in Dr may also be required. The adverse effect of reduction in Dr can be neutralized if it is accompanied with increased C and p. Genotypic variation in HI and total dry matter is reported in peanut (Velu and Gopalkrishna 1985; Sharma and Varshney 1995; Dwivedi et al. 1998). Sharma and Varshney (1995) observed high broad sense heritability for HI and its component traits. Ntare and Williams (1998) reported low heritabilities for C, p, Dr, and yield, but higher heritability for p than yield, C was largely responsible for low heritability of yield, and indirect selection for yield via p was 22% more effective over direct selection. Their results also indicated that selection for yield and physiological components in segregating populations may be difficult. While biomass is controlled by both general (gca) and specific combining ability (sca) effects, HI is predominantly controlled by gca effects (Dwivedi et al. 1998). Both combining ability effects interact with environments. The sca effects for biomass and HI are insensitive to photoperiod, while the gca effects for HI are sensitive. Lal et al. (2006) observed highly significant gca variance for HI. Nigam et al. (2001) also reported significant additive, dominance, and additive additive genetic effects for HI. However, additive genetic effects were more important than the dominance effects. Tolerance to high temperatures will also help in maintaining C and p in high-temperature environments. While selecting parents for hybridization, it is desirable to select those that have high C and p across photoperiod regimes besides other traits associated with early maturity. Selection for biomass and HI can be practiced in early-segregating generations. 5. Selection for High Water-Use Efficiency. CTT works satisfactorily when other factors affecting growth are not limiting. Under rainfed conditions where long and frequent dry spells occur, CTT and other parameters may lose their significance and the soil moisture availability becomes the main determinant of crop duration. Although mild water stress promotes flower, peg, and pod production, the crop growth slows down under severe moisture stress and increases again as the soil moisture becomes available. Field experiences have shown that the crop duration may get prolonged depending on the severity, frequency, and duration of dry spells in rainfed cultivation. Under moisture limiting conditions, high water-use efficiency, ability to recover quickly from drought, and high partitioning will be paramount considerations along

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with CTT that will influence the crop duration. The two traits that are associated with high water-use efficiency, low specific leaf area (SLA) and high soil plant analytical development (SPAD) chlorophyll meter readings (SCMR), show high (66.0%) and moderate (40.7%) heritability, respectively (Vasanthi et al. 2004). SCMR is also strongly correlated with pod yield and other economic traits, such as 100-seed weight (Upadhyaya 2005). SLA is reported under the control of additive, dominance, and additive additive genetic effects. However, additive genetic effects are more important (Nigam et al. 2001). Lal et al. (2006) observed highly significant variance of general combining ability (gca) for SLA and SCMR. The specific combining ability (sca) variance was also highly significant for SCMR, but its magnitude was very low. They also found significant reciprocal effect for SLA. Thus, selection for these traits can be effective in the early generations. For SLA, the choice of female parent in the improvement of the trait would be crucial. 6. Diversification of Sources of Earliness in Maturity. ‘Chico’ has been used extensively as a source of early maturity in peanut breeding programs around the world. It belongs to subspecies fastigiata var. vulgaris and was released in 1975 in the United States (Bailey and Hammons 1975). ‘Chico’ is a selection from PI 268661 introduced into the United States in 1960 from Rhodesia (present Zimbabwe), where it was originally introduced from Krasnodar, Russia. It takes 80 to 85 days to mature in the rainy season at ICRISAT Center. The plants are small with very small twoseeded pods and low yield. Other sources of early maturity selected from introduced materials include ‘Shepharadi No. 9’, ‘Congo’, ‘Avir’ (Gibori et al. 1978) and ‘JL 24’ (Patil et al. 1980). These genotypes are reported to mature in 90 to 100 days. Mutation has also been induced to create sources of early maturity/early maturing cultivars. Examples for varieties include T AG 1, a gamma ray mutant of ‘Spantex’, in the United States (Simpson and Smith 1986) and ‘Luhua 6’, a gamma ray mutant of ‘Baisha 1016’, in China (Qui et al. 1990). Mutants have also been used in hybridization to develop sources of earliness such as TAG 2 (R 25 (a mutant) TPL 2066-1) in the USA (Simpson and Smith 1986) and TG 1E (Tall Mutant x TG 9) and TG 2E (Dwarf Mutant TG 3) in India (AICORPO 1983 and 1984). Simpson (1990) reported an extremely early-maturing wild species Arachis praecox Krapov., W.C. Gregory and Valls (Collection No. Valls Simpson Gripp 6416, A genome), which matures in 45 days. This has been crossed with B genome species, A. batizocoi Karp. & W.C. Gregory (GKP 9484), the chromosome number doubled, and then crossed to A. hypogaea at the Texas Agricultural Experiment Station, Texas, to introgress genes for early maturity into cultivated background. However, the A


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genome species took much longer to mature when grown at ICRISAT Center (ICRISAT, unpublished data). The sources of early maturity used in breeding program at ICRISAT include ‘Chico’, 91176, and 91776 (sister breeding lines from TG 3 8068 cross from Tindivanam, Tamil Nadu, India), TG 1E, TG 2E, and TG 3E (‘TG 17’ ‘Chico’) (all breeding lines from Bhabha Atomic Research Centre, Trombay, India), ‘Gangapuri’ (a landrace from Khargone, Madhya Pradesh, India), and ‘JL 24’ (a selection from EC 94943, released as Phule Pragati in India). These take 90 to 100 days to mature at ICRISAT Center. ‘JL 24’, a released cultivar in India, is popular among the farmers because of its early maturity, medium seed size, and high shelling turnover. Because of these desirable traits, it was also released in Myanmar, Congo, Philippines, Zambia, Mali, and Malawi. Among the several short-duration breeding lines developed at ICRISAT, ICGV # 92196, 92206, 92234, 92243, and 92267 have been registered as improved germplasm (Upadhyaya et al. 1998, 2002). Their maturity period at ICRISAT Center is similar to that of ‘Chico’, but they have higher pod yield and larger seed size than ‘Chico’. Some of the early-maturing cultivars (< 100 days) released/registered include ‘Pronto’ (Kirby and Banks 1980) and ‘Spanco’ (Kirby et al. 1989) from ‘Chico’ ‘Comet’ cross in the United States; ‘Dh 40’ (Dh 3–20 TG 2E), ‘TNAU 97’ [G 961 (‘CO 1’ PPG 3)] (Sridharan et al. 1991), ‘ALG (E) 57’ (‘CO 2’ ICGV 86687) (Vindhiyavarman and Raveendran 1991), and ‘GG 3’ (‘GAUG 1’ ‘JL 24’), ‘GG 5’ (27-5-1 ‘JL 24’), ‘GG 12’ (‘Shulamit’ ‘GAUG 10’), ‘TG 26’ (BARCG 1 TG 23), ‘R 9251’ (‘JL 24’ TG 23), ‘JL 220’ (JL 80 VG 77), ‘M 522’ (PG 1 F 334-AB-14), ‘RS 138’ (a selection from introduction from Brazil), ‘VRI 3’ (J 11 Robut 33-1) (extra early), and ‘Co 4’ (‘TMV 10’ ICGS 82) (Basu et al. 2002) in India; and ‘55-437’ (a selection from a population of South American origin, drought resistant), ‘73-30’ (61-24 59-127), ‘47-10’ (a selection from a Madagascar population), ‘Te’3’ (a selection from a local population from southern Burkina Faso), ‘TS 32-1’ (‘Spantex’ ‘Te 3’, moderately drought resistant), ‘KH 149 A’ (GH 119-7.I.II-III 91 Saria, rosette resistant), ‘KH 241 D’ (GH 1185.2 II 91 Saria), and ‘A-124 B’ (a selection from local ‘Loudima Red’ population) in West Africa (Bockelee-Morvan 1983). Except for ‘A-124 B’, which is a Valencia type, all other short-duration varieties in West Africa are of Spanish types. Recently the Groundnut Germplasm Project for West and Central Africa (Mayeux et al. 2003) recommended the following cultivars, which mature in 90 days or less: ‘ICG (FDRS) 4’ (‘Argentine’ PI 259747, rust resistant), ‘GC 8-35’ (‘55-437’ ‘Chico’, drought resistant), ‘55-21’ (‘55437’ ‘Chico’, drought resistant), ‘55-33’ (‘55-437’ ‘Chico’, drought

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resistant), ‘SRV 1-3’ (a selection from recurrent selection program, drought resistant), ‘SR 1-96’ (a selection from recurrent selection program, cultivar known as 11908-13, drought resistant), ‘Fleur 11’ (an introduction from China, drought resistant), and ‘78-936’ (an introduction from China, drought resistant). ‘GC 8-35’, ‘55-21’, and ‘55-33’ are reported to mature in 80 days and ‘78-936’ in 75 to 90 days in West and Central Africa. The genetic base for sources of early maturity remains narrow. Further, sources have not been studied in detail for various traits associated with early maturity. Khalfaoui (1990b) suggested the use of days from planting to first flower, from planting to production of more than three flowers per day, and days from planting to accumulation of 50 flowers per plant as potential selection criteria for early maturity among the germplasm lines. Among more than 15,000 germplasm accessions maintained in the RS Paroda Gene Bank at ICRISAT, 21 landraces (16 Spanish and five Valencia types) have been identified as sources of early maturity (ICRISAT 2003). Many of these with better agronomic traits (ICG # 3540, 3631, 4729, 9427, and 9930) are comparable to ‘Chico’ in maturity. Some of these outyield JL 24 in harvests at 1240 8Cd (ICG # 11914, 13585, 14728, and 14814) and at 1470 8Cd (ICG # 3631, 4558, 4890, 5560, 13585, and 14788). Limited information is available on genetic diversity for early maturity and its components. Phenology and physiology of the stable and agronomical superior sources of early maturity need to be studied in detail to identify genotypes differing in various components of earliness in maturity. Similarly, their genetic constitution and allelism should be determined. Those differing in genetic constitution for early maturity and its components should be crossed with each other to pyramid diverse genes in a single background. A review of the pedigrees of the released cultivars indicates that the genes for early maturity may also be available outside the known sources of early maturity. As segregation for lateness was observed in ‘Chico’ ‘Gangapuri’ (both sources of early maturity) and its reciprocal cross (Upadhyaya and Nigam 1994), there is a possibility of finding early-maturity segregants in crosses between two normal-maturing varieties. These genes for early maturity are likely to be different from those accessed from regular sources of early maturity. 7. Evaluation in Target Environments/Cropping Systems. In most breeding programs, varieties are evaluated for crop duration and yield under a sole crop situation. However, their evaluation in the target environment/cropping system would be desirable to identify the most suitable genotypes for maximum returns.


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Under rainfed conditions, incorporation of fresh seed dormancy in short-duration cultivars will be required to inhibit sprouting of mature seeds if rains occur at the time of harvest. Both monogenic with dominance or partial dominance (Stokes and Hull 1930; Ramachandran et al. 1967; Lin and Lin 1971; Bhapkar et al. 1986; Upadhyaya and Nigam 1999) and polygenic (Hull 1937; John et al. 1948) control for fresh seed or postharvest seed dormancy are reported. Khalfaoui (1991) observed additive, dominance, and digenic epistatic effects involved in the control of fresh seed dormancy. Several sources of fresh seed/postharvest seed dormancy in a Spanish background are now available (BockeleeMorvan 1983; Upadhyaya et al. 1997) and can be used in breeding programs. 8. Opportunities for Biotechnological Interventions. Compared to many other crops, the progress in genomics research in peanut has been slow. The search for polymorphic markers and those linked with different traits needs to be intensified to saturate the linkage maps and extend the use of marker-assisted selection in peanut. The identification of quantitative trait loci (QTLs) associated with difficult-to-measure physiological traits associated with early maturity and their consequent use in marker-assisted breeding will help to increase efficiency in breeding efforts. Comparative mapping studies of QTLs for such traits already identified in other legume crops should be useful. D. Other Issues in Breeding for Early Maturity Some other desirable traits may have a bearing on crop duration. These need to be given due consideration while breeding for short duration varieties. 1. Yield Potential. Compared to the medium- and long-duration cultivars, the yield potential of short-duration cultivars is lower (Khalfaoui 1990b). To improve yield potential, high growth rates and partitioning, early podding, modification of pod weight, and modification in planting pattern will be essential (Duncan 1975; Duncan et al. 1978). A modification in planting pattern to rapidly establish a full canopy to intercept incident photosynthetically active radiation (PAR) will be required for achieving good yield potential (Bell et al. 1994). A yield potential of 3.0 to 3.5 t ha-1 should be an achievable target in 90- to 100-day cultivars. 2. Seed Size. Breeding for large seed size while keeping the crop duration short is unlikely to succeed. Not only do large seeds take more time

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to develop and mature, they also take more time to germinate and emerge when sown. Other contributing factors being equal, the small and medium-size seeds will require less time to develop and mature and will have no prolonging effect on crop duration. 3. Resistance to Foliar Fungal Diseases. Early leaf spot (Cercospora arachidicola Hori), late leaf spot (Phaeoisariopsis personata [Berk. & M.A.Curtis] van Arx), and peanut rust (Puccinia arachidis Spegazzini) occur wherever peanut is grown. Genetic resistance to these diseases has usually been associated with low yields and late maturity. Aiming at higher levels of resistance in early-maturing background will be difficult to achieve. A moderate level of resistance will have only limited influence on crop duration and would also stabilize productivity in a cropping system.

IV. CULTURAL MANIPULATIONS TO SHORTEN CROP DURATION Cultural practices can hasten crop maturity by advancing phenological development through temperature in microenvironment. In North Vietnam, peanut seeds are soaked in lukewarm water for 24 h to sprout them. The sprouted seeds with protruding radicles are sown directly in fields with fine tilth. The sowing is followed by light manual irrigation on the ridges. This enables early emergence of the crop in spite of low temperatures prevailing during February in the country. Recently seed priming is receiving attention in India and Vietnam, where seeds are soaked for 8 h in water and then dried back to their original water content. A primed seed will germinate only if it takes up additional moisture from the soil after sowing. Apart from swelling slightly and weighing more, primed seed can be treated in the same way as nonprimed seed. Peanut cultivation under polyethylene mulch, commonly practiced in Central China and North Vietnam, among other advantages, also helps to reduce the crop duration by raising the soil temperature, which leads to early emergence of the crop, early flowering, and early maturity of the pods. Elevated soil temperature helps to compensate for low air temperature and results in higher radiation use efficiency (Awal and Ikeda 2003). Sowing depth is another factor that influences the days to seedling emergence. Deeper sowing delays emergence and weakens the seedlings, which in turn influences crop yield and duration. The normal recommended sowing depth is 5 centimeters (cm). The seeds can be sown at a shallower depth to hasten seedling emergence,


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provided the field is irrigated frequently until the seedlings are well established. In China, growth hormones such as Fosamine and 2,3,6– trichlorobenzoic acid (TCBA) are used to stop late flowers, thereby enhancing the filling of earlier-set pods and uniform maturity. For arresting excessive vegetative growth, Paclobutrazol (P 333) in China and Vietnam and Kylar in the United States are used. V. SUMMARY Following conventional approach based on calendar days to flower and maturity, location-specific early-maturing cultivars have been released. But it has been difficult to reduce the crop duration to less than100 days without sacrificing yield potential. Further, these cultivars did not always maintain their early-maturity trait across locations/seasons. The approach was improved by making use of cumulative thermal time (CTT) instead of calendar days while selecting for early maturity in segregating populations and staggered harvesting based on CTTs in trials of advanced breeding lines. This modification helped in creating stability in the early-maturity trait of breeding lines across locations/ seasons. To develop cultivars with less than100 days duration, which are required in South and Southeast Asia, further improvements in the approach will be necessary. Based on the botanical characteristics and physiological behavior of the crop, these characteristics could be utilized for attaining short duration: short plant stature (plant height in case of subspecies fastigiata and plant spread in case of subspecies hypogaea) with smaller internodal length, faster germination and emergence, fewer days to first flowering, accumulation of maximum numbers of early flowers, more flowers per node, absence of late flowers, fewer days after fertilization for a peg to enter soil, faster pod and seed growth, high seed partitioning, and high shelling turnover. Selection for low base temperature (Tb) and CTT for different phenological stages (germination and emergence, first flower after emergence, opening of first flower to opening of a given number of flowers per plant, and opening a flower to maturation of seeds that develops from that flower) can individually and collectively lead to a considerable reduction in crop duration. However, it should be ensured that the reproductive duration is not adversely affected. To compensate for any reduction in reproductive duration and to enhance yield potential of short-duration cultivars, it would be essential to select for high crop growth rate and partitioning, tolerance to high soil and air temperatures, high wateruse efficiency, and photoperiod insensitivity. Some of these physiolo-

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gical traits are not easy to measure in segregating populations. Until marker-assisted selection becomes possible for these traits in peanut, the emphasis should be on evaluating the parents chosen for hybridization and breeding lines in advanced generations. Early maturity and its components (pod/seed maturity, days to flower, days to 50% flowering, and days to accumulate first 10 or 25 flowers) are highly heritable traits, and selection in early generations should be successful. Similarly, physiological traits such as biomass, harvest index, and water-use efficiency (as measured by specific leaf area and SPAD chlorophyll meter readings) show enough additive and additive additive genetic variance for exploitation either in early or late generations. However, some of these traits may not be easy to measure in segregating generations; thus they could be evaluated in later generations. Selection for photoperiod insensitivity should also be done in later generations. There is a need to diversify sources of early maturity in peanut. Germplasm lines should be studied in detail for component traits of early maturity and other physiological traits so that parents with differing traits could be crossed to accumulate diverse desirable genes. As large seed size and resistance to foliar diseases will prolong the crop duration, only medium seed size and moderate levels of resistance should be incorporated in early-maturing varieties. To capitalize on the full potential of the genotypes with aforementioned traits, it is essential to modify crop husbandry to accommodate larger number of plants per unit area to provide quick ground cover and to provide required nutrients and other inputs. Some cultural practices can also hasten germination, emergence, flowering, and maturity by modifying microclimate. Along with cumulative thermal time and other desirable morphological traits, SCMR has been integrated in the breeding scheme for early maturity at ICRISAT. As a noninvasive surrogate of water-use efficiency, SCMR is easy to operate, reliable, fairly stable, and low cost. It is also strongly correlated with pod yield and 100-seed weight. It can be used to screen large numbers of breeding populations in the field. Meanwhile search for markers linked to specific traits, such as water-use efficiency, harvest index, and foliar disease resistance, is in progress.

VI. LITERATURE CITED AICORPO (All India Coordinated Research Project on Oilseeds). 1983. Annual Progress Report, Rabi/Summer Groundnut 1982–83. pp. 1–107. Directorate of Oilseeds Research, Rajendranagar, Hyderabad 500 030, Andhra Pradesh, India.


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AICORPO (All India Coordinated Research Project on Oilseeds). 1984. Annual Progress Report, Rabi/Summer Groundnut 1983–84. pp. 1–149. Directorate of Oilseeds Research, Rajendranagar, Hyderabad 500 030, Andhra Pradesh, India. Ali, N., and J.C. Wynne. 1994. Heritability estimates and correlation studies of early maturity and other agronomic traits in two crosses of peanut (Arachis hypogaea L.). Pakistan J. Bot. 26:75–82. (CAB Abstr.) Ali, N., J.C. Wynne, and J.P. Murphy. 1999. Estimation of genetic effects and heritability for early maturity and agronomic traits in peanut (Arachis hypogaea L.). Pakistan J. Bot. 31:323–335. (CAB Abstr.) Awal, M.A., and T. Ikeda. 2003. Effect of elevated soil temperature on radiation-use efficiency in peanut stands. Agri. Forest Meteorol. 118:63–74. Badami, V.K. 1923. Hybridization work on groundnut. pp. 29–30. In: Agricultural Department Report for 1922–23. Mysore, India. Badami, V.K. 1928. Arachis hypogaea (the groundnut). pp. 297–374. In: Inheritance studies. PhD Thesis, Cambridge Univ., UK. Bagnall, D.J., and R.W. King. 1991a. Response of peanut (Arachis hypogaea) to temperature, photoperiod and irradiance 1. Effect on flowering. Field Crops Res. 26:263–277. Bagnall, D.J., and R.W. King. 1991b. Response of peanut (Arachis hypogaea) to temperature, photoperiod and irradiance 2. Effect on peg and pod development. Field Crops Res. 26:279–293. Bailey, W.K., and J.E. Bear. 1973. Components of earliness of maturity in peanuts, Arachis hypogaea L. Am. Peanut Res. Educ. Soc. 5:32–39. Bailey, W.K., and R.O. Hammons. 1975. Registration of Chico peanut germplasm. Crop Sci. 15:105. Basu, M.S., A.L. Rathnakumar, and C. Lal. 2002. Improved groundnut varieties of India. All India Coordinated Research Project on Groundnut. p. 28. National Research Centre for Groundnut (ICAR). Junagadh 362 001, Gujarat, India. Basu, M.S., M.A. Vaddoria, N.P.Singh, and P.S. Reddy. 1986. Identification of superior donor parents for earliness through combining ability analysis in groundnut Arachis hypogaea L. Ann. Agr. Res. 7:295–301. Bear, J.E., and W.K. Bailey. 1973. Earliness of flower opening and potential for pod development in peanuts, Arachis hypogaea L. Am. Peanut Res. Educ. Soc. 5:26–31. Bell, M.J., R.C. Roy, M. Tollenaar, and T.E Michaels. 1994. Importance of variation in chilling tolerance for peanut genotypic adaptation to cool, short-season environments. Crop Sci. 34:1030–1039. Bell, M.J., R. Shorter, and R. Mayer. 1991. Cultivar and environmental effects on growth and development of peanuts (Arachis hypogaea L.). I. Emergence and flowering. Field Crops Res. 27:17–33. Bell, M.J., and G.C. Wright. 1998. Groundnut growth and development in contrasting environments 2. Heat unit accumulation and photo-thermal effects on harvest index. Expt. Agr. 34:113–124. Bhapkar, D.G., P.S. Patil, and V.A. Patil. 1986. Dormancy in groundnut-A review. J. Maharashtra Agr. Univ. 11:68–71. Bockelee-Morvan, A. 1983. The different varieties of groundnut, geographical and climatic distribution, availability, technical sheets for varieties extended. Oleagineux 38:73–116. Boote, K.J. 1982. Growth stages of peanut (Arachis hypogaea L.). Peanut Sci. 9:35–40. Cahaner, A., and A. Ashri. 1974. Vegetative and reproductive development of Virginia type peanut varieties in different stand densities. Crop Sci. 14:412–416. Court, W.A., R.C. Roy, and J.G. Hendel. 1984. Effect of harvest date on agronomic and chemical characteristics of Ontario peanuts. Can. J. Plant Sci. 64:521–528.

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7 Ploidy Manipulation for Breeding Seedless Triploid Citrus Patrick Ollitrault and Dominique Dambier CIRAD, UPR Amelioration Des Plantes a Multiplication Vegetative TA50/PS4 Boulevard De La Lironde, 34398 Montpellier, France Francois Luro INRA, GECA San Giuliano 20230 San Nicolao, France Yann Froelicher CIRAD, UPR Amelioration Des Plantes a Multiplication Vegetative San Giuliano, 20230 San Nicolao, France

I. INTRODUCTION II. NATURAL POLYPLOIDY IN CITRUS A. Mechanism of Spontaneous Formation of Polyploids 1. Triploid Formation 2. Tetraploid Formation B. Vegetative and Fruit Characteristics of Polyploids C. Meiosis and Fertility of Polyploids 1. Meiosis of Tetraploids 2. Meiosis of Triploids III. PLOIDY MANIPULATION FOR TRIPLOID SEEDLESS CULTIVAR BREEDING A. Selection of Spontaneous Triploids in 2x 2x Crosses 1. Biological Limitations and Practical Aspects 2. Segregation and Recombination B. Sexual Crosses between Diploids and Autotetraploids (Doubled Diploids) 1. Biological Limitations and Practical Aspects 2. Segregation and Recombination C. Sexual Hybridization between Diploid Females and Tetraploid Somatic Hybrids 1. Biological Limitations and Practical Aspects 2. Segregation and Recombination D. Endosperm Culture of 2x 2x Crosses 1. Biological Limitations and Practical Aspects 2. Segregation and Recombination Plant Breeding Reviews, Volume 30. Edited by Jules Janick Copyright © 2008 John Wiley & Sons, Inc.

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E. Diploid þ Haploid Protoplast Fusion 1. Biological Limitations and Practical Aspects 2. Segregation and Recombination IV. CONCLUSION V. LITERATURE CITED

I. INTRODUCTION Citrus ranks first in world fruit production with 108 million tonnes (t) produced in 2004 (FAOSTAT 2005). More than 3 million hectares are cropped with citrus fruit from warm temperate to tropical zones, between latitude 408 N and S. Major producing countries include Brazil (20.6 t), the United States (14.9 t), China (14.7 t), Mexico (6.5 t), and Spain (6.2 t). In 2004, sweet orange represented a very large part of this production (58%); followed by the small citrus group (tangerines, mandarins, Clementines: 21%); lemon and lime (11%); and pummelo and grapefruits (4%); and 5% not elsewhere specified (FAOSTAT 2005). About 30% of production is processed. In the Mediterranean Basin, 82% of production is oriented to the fresh fruit market as compared to 24% in the United States, 32% Brazil, but around 97% in China. The very large area of production; the diversity of consumers, demands, and preferences; and the adaptation to increasing abiotic and biotic stresses have prompted producing countries to adopt policies for the development of new cultivars. For the fresh fruit market, fruit quality is a central preoccupation worldwide. Among the different criteria of citrus quality, seedlessness is a major characteristic for the fresh fruit market. Considerable breeding work has been done to develop new seedless cultivars. The seedlessness of citrus varies among accessions and sometimes environmental conditions. ‘Navel’ orange and ‘Satsuma’ are usually seedless, but occasionally they produce seeds when pollinated. With cross-pollination fruit, some pummelos produce more than 100 seeds while they are seedless in self-pollination conditions. Seeds of numerous cultivars display polyembryony (see Plate 7.1). In such seed, one embryo results from sexual fertilization of the ovules while the others arise from somatic embryogenesis from nucellar cells, though most frequently the zygotic embryo is less vigorous than the nucellar ones and rarely survives (Kobayashi et al. 1982a). Pollination and fertilization are necessary for the development of nucellar embryos and seed production (Soost 1987). Only two Citrus species, C. maxima and C. medica, are strictly monoembryonic. Polyembryony is a constraint for

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sexual breeding. Moreover monoembryonic and polyembryonic cultivars display differential behavior regarding ploidy manipulation. Sterility can be divided into three types (Yamamoto et al. 1995): female sterility, male sterility, and self-incompatibility. Some degree of male and female sterility is found in ‘Satsuma’ mandarins, ‘Marsh’ grapefruit, ‘Washington Navel’, and ‘Hamlin’ and ‘Valencia’ sweet oranges. The degree of pollen sterility is variable in cultivated citrus, and usually pollen-sterile accessions produce seedless fruits or fruits with few seeds when cultivated in solid blocks. Male sterile or selfincompatible accessions have the ability to produce seedless fruits when cross pollination is prevented. However, those accessions may produce seedy fruits in mixed planting particularly if pollinating insects are present. For areas such as the Mediterranean Basin where the main seedless ‘‘easy-peeler’’ cultivar is the self-incompatible ‘Clementine’, selection is required for both male and female sterility. Indeed, cultivation of new seedless but pollen-fertile varieties close to ‘Clementine’ fields would results in the production of seedy ‘Clementine’ fruits; conversely ‘Clementine’ pollen should pollinated male-sterile or self-incompatible new cultivars. Self-incompatibility is very frequent in pummelo (Iwamasa and Oba 1980) and is found in several mandarin and mandarin hybrid cultivars. Here self-incompatibility is gametophytic (Soost 1969). Male and female sterility may be due to different genetic factors such as triploidy; sterility genes; and chromosomal abnormalities such as reciprocal translocation, the main cause of the reduced fertility of ‘Valencia’ sweet orange; and inversion, the cause partial pollen sterility of ‘Mexican’ lime (Iwamasa 1966). Some male-sterile and self-incompatible accessions cannot produce seedless fruits because of the lack of parthenocarpy. Thus, parthenocarpy is an indispensable trait for seedless fruit production, and this character seems to be widely present in citrus germplasm. Some seedy accessions can also produce parthenocarpic fruit (Sykes and Possingham 1992). Autonomic parthenocarpy where seedless fruit is produced without any external stimulation (pollination), is the main type of parthenocarpy in citrus (‘Navel’ orange or ‘Satsuma’ mandarin), but stimulative parthenocarpy has also been reported (Vardi et al. 1988). Three major breeding strategies have been developed for seedlessness: 1. The creation of new self-incompatible cultivars. ‘Clementine’, ‘Fortune’ mandarin, and ‘Afourer’ tangor are good examples of such self-incompatible cultivars. The main default of these cultivars is their ability to be cross-pollinated and to produce seedy fruits when they are cultivated in multicultivar orchards.

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2. Induced mutagenesis of seedy cultivars. Such programs have been developed in several countries and gave good results for seedless line selection (Hearn 1984; Spiegel-Roy et al. 1985; Chen et al. 1991; Froneman et al. 1996). ‘Mor’ tangor is a recent example of cultivar developed in Israel from ‘Murcott’ tangor. However, the seedless mutants should produce fruits significantly smaller than the parental seedy cultivar. 3. Triploid selection. The selection of triploid lines has been, and is already, a very interesting way to develop seedless cultivars. Indeed triploidy is generally associated with both male and female sterility. Thus, most of the trees under field evaluation present these characteristics, and an efficient selection can be carried out in other traits. Moreover larger fruit size associated with triploidy should correct the reduction of fruit size generally observed in seedless mutants of seedy cultivars. This review focuses on ploidy manipulation for triploid seedless breeding. This strategy has been developed by several groups worldwide (Khan et al. 1996; Grosser et al. 1998, 2000; Ollitrault et al. 1998a; Starrantino 1999; Guo et al. 2000; Chandler et al. 2001; Navarro et al. 2003 and 2004; Russo et al. 2004; Handaji et al. 2005; Wakana et al. 2005; Wu et al. 2005), and new avenues have been opened by biotechnology. Mechanism of natural polyploid citrus formation and characteristic of natural polyploids are reviewed as well as biotechnological methods for ploidy manipulation. The different strategies for triploid creation are discussed with emphasis on the genetic structures of the triploid population generated. II. NATURAL POLYPLOIDY IN CITRUS Diploidy is the general rule in Citrus and its related genera with a basic chromosome number x ¼ 9 (Krug 1943). However, some polyploid genotypes have been detected early in citrus germplasm. Longley (1925) was the first to formally identify a tetraploid wild form: the ‘Hong Kong’ kumquat (Fortunella hindsii Swing.). Triploid ‘Tahiti’ lime (Jackson and Sherman 1975) (see Plate 7.2), tetraploid strains of Poncirus trifoliata (Iwasaki 1943), allotetraploid Clausena excavata Burm. F. (Froelicher et al. 2000), tetraploid Clausena harmandiana and hexaploid Glycosmis pentaphylla are other examples of natural polyploidy found in the germplasm of Aurantioideae subfamily of Rutaceae (Froelicher 1999).


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A. Mechanism of Spontaneous Formation of Polyploids 1. Triploid Formation. The occurrence of spontaneous triploid seedlings has been reported early. Lapin (1937) mentioned about 4% triploidy in hybrid seedlings of Citrus limon crossed with eight other diploid species and cultivars. Frost and Soost (1968) reported that more than 5% of 1,200 hybrids from diploid parents growing at Riverside were probably triploids. Later studies have shown that most of the spontaneous triploids arising from diploid parents are found in small and abnormal seeds of monoembryonic female parents (Esen and Soost 1971; Esen and Soost 1973). The rates of spontaneous triploidy vary among cultivars; 1% for ‘Clementine’ and 6 to 7% ‘King’ mandarin have been observed both in California and Sicily (Geraci et al. 1975). ‘Wilking’ mandarin also presented a high rate of 14.6% triploid seedlings (Soost 1987). Triploid embryos have also been observed in immature seeds of polyembryonic cultivars with frequencies between 0 and 8% in Citrus deliciosa clones (‘Tardiva de Ciaculli’ and ‘Avana’) and between 0 and 11.5% in different Citrus limon cultivars (Geraci et al. 1977; Geraci 1978). The frequency of triploid embryos is very high in several sweet orange cultivars. Oiyama et al. (1980) found between 26 and 30% small seeds and between 8 and 33% triploid seedlings in sweet orange cultivars and hybrids of sweet orange. High percentages of triploid embryos were also found in presumed interspecific hybrids of sweet orange such as ‘Ortanique’ tangor (25%) (Wakana et al. 1981), ‘Temple’ tangor (6.8%), and ‘Sugeka’ orangelo (23%) (Esen and Soost 1971). So it appears that there is a genetic control of triploid embryo formation as well as environmental factors because the same cultivar may produce different rates of triploid hybrids in different years (Geraci et al. 1977). Cytogenetic studies (Esen and Soost 1977; Esen et al. 1979; Wakana et al. 1981) have shown that triploid embryos are associated with pentaploid endosperm, which is a strong indication that triploid hybrids result from the fertilization of unreduced ovules by normal haploid pollen (Esen et al. 1979). This ploidy ratio of 5/3 between endosperm and zygotic embryo is generally considered by citrus breeders as the origin of precocious abortion of endosperm development and subsequent overdevelopment of embryos found in small citrus seeds. Precocious endosperm abortion should occur as well for monoembryonic cultivars and polyembryonic ones where the triploid sexual embryo is associated with diploid nucellar embryos (Wakana et al. 1981). However, another hypothesis should be considered: the Endosperm Balance Number (EBN) theory proposed in the early 1980s

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(Johnston and Hanneman 1980) to explain the basis of normal development of seeds in interspecific and interploid crosses in potato. According to this hypothesis, each species has a genome-specific effective ploidy (EBN) that can be different from the actual ploidy level, which must be in a 2:1 (maternal to paternal) ratio in the endosperm for normal development. This hypothesis initially, developed for potato, has also been applied in several other plants species, such as Trifolium, Lycopersicum, Avana, Datura, and Impatiens (Carputo et al. 1999). The frequency of duplication in the female gametes varies between less than 1% to more than 20% (Soost 1987; Iwamasa and Nito 1988), probably due to the abortion of the second meiotic division in the megaspore (Esen et al. 1979). This hypothesis has been confirmed for Clementine by molecular marker analysis showing that less than 50% of maternal heterozygosity is transmitted by the diploid ovules (Luro et al. 2000, 2004). However, recently Chen et al. (2007), proposed that 2n gametes of sweet orange result from first meiotic division restitution. The possibility of very rare events of formation of triploid hybrids by fertilization of a haploid egg cell by a diploid pollen has also been observed by Luro et al. (2000). 2. Tetraploid Formation. In the citrus literature, tetraploids arising from chromosome doubling (spontaneous in nucellar tissue, or induced by colchicine) are generally called autotetraploid while tetraploid somatic hybrids obtained by protoplast fusion are considered allotetraploid. We adopt this terminology here, keeping in mind that it is quite different from the general definition of auto- and allotetraploidy (Otto and Whitton 2000) and that it may not be related to disomic or tetrasomic chromosomal segregation. Tetraploidization seems to occur frequently in polyembryonic citrus lines. In Russia, Lapin (1937) found tetraploid seedlings among eight Citrus species (rate from less than 1 to 5.6%) and Poncirus (4%). Russo and Torrisi (1951a) detected tetraploid forms of C. aurantium and C. limon among nucellar seedlings. Cameron and Frost (1968) observed that 2.5% of 3,600 nucellar progenies from a broad range of citrus cultivars were tetraploid. Tetraploid seedlings occurred at a frequency of 3% from the rootstock ‘Troyer’ citrange and 2.5% from ‘Carrizo’ (Hutchison and Barrett 1981). Chromosome doubling in nucellar seedlings seems to be the general rule generating tetraploidy (Cameron and Frost 1968). Indeed, these tetraploid seedlings are homogenous and do not display traits of the pollen parents in controlled crosses. This has been proven by isoenzymatic studies of tetraploid seedlings of C. volkameriana (Ollitrault and


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Jacquemond 1995). It seems that this doubling occurs repeatedly in the ovule tissues because tetraploid embryos are found in seeds from fruit that contain seeds with mostly diploid embryos (Cameron and Frost 1968). Moreover, diploid and tetraploid seedlings can arise from the same polyembryonic seed (Hutchison and Barrett 1981). From a systematic search of autotetraploids in a wide range of taxa, Barrett and Hutchison (1978) postulated that the ability to produce such autotetraploid seedlings is a variable genetic trait present in polyembryonic Citrus and relatives. They also proposed that the rates of tetraploid seedlings are affected by environmental conditions. This assertion is clearly demonstrated by Hutchison and Barrett (1981), who show that the rate of tetraploid seedlings of citranges ‘Troyer’ and ‘Carrizo’ vary among years and position of the fruit on the tree. In a recent work, L. Navarro (pers. comm.) from IVIA (Spain) also found differences of tetraploid rates in seedlings of several rootstocks coming from different parts of the world that suggest that colder conditions favor spontaneous tetraploidization in nucellar tissues. The effect of cold on polyploidization events seems to be a general rule both in plants and animals (Ramsey and Schemske 1998; Otto and Whitton 2000). Chromosome doubling in somatic tissues has been observed by Raghuvanshi (1962). Chromosome doubling in meristems should lead to chimeric shoots and branches. However, very few tetraploid bud sports have been identified (Iwamasa and Nito 1988), but these authors suggest that it is a consequence of unfavorable competition between diploid and tetraploid cells in the meristem. The formation of fully developed tetraploid seeds from diploid female tetraploid male hybridization has been reported (Tachikawa et al. 1961; Cameron and Soost 1969; Esen and Soost 1972). Esen and Soost (1972) proposed that they originate from unreduced gametes fertilized by diploid pollen and leading to a favorable 3:2 ratio between endosperm and embryo. Considering the EBN theory, this leads to an adequate 2:1 EBN ratio in the endosperm leading to normal endosperm and seed development. In conclusion, two major basic mechanisms are involved in spontaneous polyploidization in citrus: (1) 2n gametes arising mostly from division restitution during meiosis of the megaspore that produce triploid hybrids in diploid diploid hybridization and allotetraploidy in diploid tetraploid hybridization; and (2) duplication of chromosomes without cell division in nucellar tissues that gives rise to autotetraploidy. Even if the rates of these natural polyploidization events in citrus are very high compared with those proposed as a general rule for plants by Ramsey and Schemske (1998) (autotetraploid plants form at a

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rate of about 105 per individual, per generation), it appears that polyploidy has played a minor role in citrus evolution. The lower vigor and fertility of most of the autotetraploids and their lack of appeal for human consumption due to thick peel and rough pulp may have resulted in a strong selection against autotetraploid nucellar plants. Moreover, the fact that autotetraploids are mostly polyembryonic strongly limits the potential for evolution of the natural tetraploid gene pool. Indeed tetraploid plants should principally contribute to the next generation as male parents pollinating monoembryonic diploid. Pollen competition between diploid and haploid pollens is generally unfavorable to the diploid one. Fertilization with diploid pollen produces generally abnormal seeds with low probability of germination in natural conditions. For the same reason, very few triploid hybrids arising from 2n ovules have a chance to germinate and multiply under natural conditions. B. Vegetative and Fruit Characteristics of Polyploids The increase of cell volume is certainly the most common and universal effect of polyploidization. This change in cell volume may alter metabolic processes, especially those that involve membranes because of variation in surface to volume ratio (Otto and Whitton 2000), and could explain the slower developmental rate generally observed in polyploids versus diploids. In citrus, autotetraploid nucellar lines allow an accurate evaluation of the effect of polyploidization itself because of the homogeneity of allelic constitution of these tetraploids and their parental diploids. Cameron and Frost (1968) have given a precise description of the autotetraploid plants cultivated at Riverside: Tetraploid leaves are considerably broader relative to their length than diploid ones. They are also thicker and darker. Seedlings are thornier than diploids and remain more thorny for a longer time than diploids. Growth is slower in tetraploids and the top is smaller and more compact. Tetraploids are slower than diploid to bloom and set fruit and are generally less fruitful. However, it appears that tetraploid grapefruits are much more vigorous and productive than most other autotetraploids. Tetraploid fruit are generally smaller and less elongate than diploid ones. In most cases, fruit shape is irregular and the rinds are thicker and rougher with more prominent oil glands. The juice proportion, relative to whole fruit weight, is much lower than for diploids but flavor, acid and soluble contents are little affected. Seed number is generally lower


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as well as the number of embryos per seed. There is an exception with a tetraploid ‘Lisbon’ lemon which produces more seeds and bigger fruits than the diploid parent. In conclusion tree and fruit characteristics of autotetraploids are not valuable for production but they constitute an interesting germplasm for triploid breeding. These observations are generally confirmed by more recent studies. Barrett (1992) described an autotetraploid of ‘Key’ lime having darker leaves and larger fruit with the same flavor as parental diploids. Autotetraploid lines of Poncirus trifoliata used as rootstock confer a reduced vigor to the tree (Jacquemond and Blondel 1986). Autotetraploid C. volkameriana displayed a very depressed growth compared with diploids (Ollitrault and Jacquemond 1995) while autotetraploid ‘Kinnow’ mandarin has shown moderately reduced growth and interesting morphological characters (Khan et al. 1992). This author suggested that leaf thickness, size, breadth, and the number and size of stomata should be good morphological markers to select spontaneous tetraploids in nucellar seedlings. An illustration of morphological differentiation between diploid and tetraploid seedlings of C. deliciosa is given in Plate 7.3. The identification of triploid plants by morphological traits appears much more difficult because they present high variability due to their zygotic origin (Cameron and Frost 1968). Starrantino (1992) observed that triploid hybrids arising from diploid female tetraploid male present generally good vigor and display similarities at the fruit level to the male parent providing the diploid gamete. For example, it appears that pigmentation of blood oranges used as male parent is highly heritable. Most of the triploid citrus hybrids are nearly seedless (Cameron and Frost 1968; Starrantino 1992). C. Meiosis and Fertility of Polyploids 1. Meiosis of Tetraploids. Gametic viability is generally lower in autotetraploid genotypes having multivalent chromosome association during meiosis than in allotetraploids that form bivalents leading to equilibrated disomic segregation. In citrus it has been shown that the degeneration of pollen mother cells is more frequent in autotetraploids than in their diploid parental genotypes (Frost and Soost 1968). These authors also observed a great variability in chromosome conjugation (quadrivalent, trivalent, bivalents, and univalents) during metaphase I and showed that one-third to one-half of sporads have more than the normal number of 4 microspores (generally 6 or 7). As a consequence, most autotetraploids generally produce few pollen

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grains with normal chromosome complement (Frost and Soost 1968) and have a lower pollen viability than diploid parental lines. However, the remaining fertility is sufficient to make controlled handpollinations. 2. Meiosis of Triploids. Triploids have generally been considered to be an evolutionary dead end because they have very low fertility and tend to produce aneuploid gametes, due to problems of chromosome pairing during meiosis. However, triploids can produce haploid, diploid, or triploid gametes at low rates, which can lead to diploid, triploid, and tetraploid progenies (Otto and Whitton 2000). In the case of citrus, early cytogenetic studies have described triploid meiosis. Trivalent pairing and some univalents were observed by Longley (1926) while Frost and Soost (1968) described a predominance of trivalents but also the presence of numerous bivalents and univalents as well as a great variation of the number of extra microspores in some genotypes. Abortion of megasporogenesis has been observed for ‘Oroblanco’ and LCNR46 C. limon C. sinenis triploid hybrid, from the first division of embryo sac to the stage of fertilized egg-cell (Fatta Del Bosco et al. 1992). In contrast, a low percentage of pollen abortion has been observed by the same authors. As a consequence of megasporogenesis abortion, most of the triploid hybrids obtained from diploid autotetraploid crosses are seedless (Cameron and Frost 1968; Starrantino 1992). However, some triploid seeds have been found in progenies of maternal triploids (Lapin 1937; Russo and Torrisi 1951b) while we found equal proportion of triploid hybrids and diploid hybrids among seedlings of ‘Oroblanco’ (unpublished data). In the same study, only diploid hybrids were found in progenies of Clementine fertilized by ‘Oroblanco’ pollen, suggesting an unfavorable competition of 2n pollen as compared to n pollen, or the absence of 2n pollen.

III. PLOIDY MANIPULATION FOR TRIPLOID SEEDLESS CULTIVAR BREEDING Several strategies have been developed for triploid citrus breeding. Some of them exploit natural events, such as 2n gametes, while the more recent strategies combine haplomethods, such as androgenesis or induced gynogenesis and somatic hybridization for a direct synthesis of triploid hybrids. For each strategy, we will discuss briefly their biological limitations and practical aspects as well as their implications for gene segregation and recombination.


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A. Selection of Spontaneous Triploids in 2x 2x Sexual Crosses 1. Biological Limitations and Practical Aspects. The selection of triploid hybrids arising from 2n megagametophytes was described in the 1970s (Esen and Soost 1971; Esen and Soost 1973; Geraci et al. 1975). Triploids were obtained from small seeds of monoembryonic cultivars with high rate of diploid megagametophytes, such as ‘Temple’, ‘Wilking’, and ‘Fortune’ (Esen and Soost 1977; Wakana et al. 1981; Soost 1987). However, this approach was limited by a relatively low efficiency and the difficulty of screening large populations of seedlings by classical cytogenetic methods of chromosome counting. More recently the use of in vitro embryo rescue and ploidy evaluation by flow cytometry (Ollitrault and Michaux-Ferrire 1992) resulted in much better efficiency (Ollitrault et al. 1996b). In this way it was possible to exploit low rates of spontaneous diploid megagametophyte such as that of ‘Clementine’ (1%). This strategy is routinely used by several teams in the Mediterranean Basin countries (France, Spain, Morocco) to select new ‘‘easy- peeler’’ cultivars (Plate 7.4). Wakana et al. (1981) showed that in polyembryonic cultivars triploid zygotic embryos were found with diploid nucellar embryos in small seeds. However, the practical possibility of selecting these triploid embryos is limited greatly by polyembryony (Geraci et al. 1977; Wakana et al. 1981). To avoid this problem, Geraci et al. (1977) proposed very early rescue of zygotic embryos harvested from immature fruits, but it appears that selection of spontaneous triploids from polyembryonic seedlings has not found real applications in citrus breeding. With this strategy, endosperm ploidy/embryo ploidy ratio and EBN are respectively 5/3 and 4. 2. Segregation and Recombination. Second meiotic division restitution (SDR) has been proposed for diploid megagametophyte development in Clementine (Luro et al. 2004) while Chen et al (2002) concluded for first meiotic division restitution (FDR) for sweet orange in both cases, only a part of the maternal heterozygosity is transmitted to the triploid hybrid. The rate of maternal heterozygosity transmission varies among the loci in relation with the rate of single crossing over between the centromere and the considered locus. It varies from 0 for loci very close to the centromere, to 1 in case of systematic single crossing over, while for FDR it varies between 1 to 0.5 from centromere to telomere (Park et al, 2007) . Thus, the homozygosity level of 2n gametes is higher for SDR then FDR (Carputo et al. 2003). Segregation and recombination occur in both the pollen and the seed parents. For a single locus A, if we consider SDR in the seed parent, in a

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cross between two heterozygous parents (A1A2 A3A4), the number of allelic combinations in the triploid progeny varies between 2 in case of systematic single crossing over between the locus and the centromere (1 heterozygous diploid female gametes A1A2) (2 haploid male gametes, A3 or A4) ¼ A1A2A3A1A2A4]; 4 for loci very close to the centromere [(2 homozygous diploid female gametes, A1A1 or A2A2) (2 haploid male gametes, A3 or A4) ¼ A1A1A3,A1,A1A4, A2A2A3, A2A2A4] to 6 [(2 homozygous diploid female gametes, A1A1, A2A2 or 1 heterozygous A1A2)] (2 haploid male gametes, A3 or A4) ¼ A1A1A3,A1,A1A4, A2A2A3, A2A2A4, A1A2A3, A1A2 A4] for genes with no systematic crossing over to the centromere. In case of FDR, genotype of 2n gamete will be as follows: A1A2 for locus close to the centromere and A1A1, A2A2 for other locus leading respectively to two (A1A2A3, A1A2A4) or six (A1A1A3, A1A1A4, A1A2A3, A1A2A4) triploid hybrid genotypes. Analysis of the origin of 2n gametes (FDR or SDR) for the different seed parents used for triploid breading will be necessary to predict the genetic structures of triploid populations. B. Sexual Crosses between Diploids and Autotetraploids (Doubled Diploids) 1. Biological Limitations and Practical Aspects Obtaining Autotetraploid Parents. Most of the autotetraploids used for triploid breeding are spontaneous nucellar tetraploids from polyembryonic cultivars and are themselves polyembryonic. Obtaining of such autotetraploid seedlings should be enhanced by selecting seeds from gigas sectors of chimeric fruits (Bowman et al., 1991). Relatively few studies have described chromosome duplication by colchicine treatments. Tachikawa (1971) and Barrett (1974) mentioned some autotetraploid as well as periclinal ploidy chimeras obtained by such treatments. To avoid chimera development, several authors have combined in vitro techniques and colchicine treatments. Gmitter and Ling (1991) obtained nonchimeric tetraploid plants of ‘Valencia’ sweet orange and ‘Orlando’ tangelo via somatic embryogenesis from culture of undeveloped ovules treated with colchicine. Gmitter et al. (1991) selected ‘Hamlin’ and ‘Ridge Pineapple’ sweet oranges from embryogenic cultures treated with colchicine. More recently Juarez et al. (2004) obtained tetraploid Clementine by in vitro colchicine treatment of shoot tips while Wakana et al. (2005) obtained tetraploid branches by top-grafting shoots with sprouting axial buds treated with colchicine. In this way these authors avoid the juvenility of seedlings and have been able to use these tetraploids as parents two to four years after colchicine treatments. Considering that autotetraploid selection from seedlings of polyembryonic genotypes is now very easy


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using flow cytometry (even for cultivars with low rates of chromosome duplication), colchicine treatments remain interesting, mostly with the objective of monoembryonic tetraploid creation or to avoid the juvenile phase inherent with the use of seedlings. Colchicine should be applied in budwood while flow cytometry will help greatly to select nonchimeric tetraploid shoots. Tetraploid plants have also been regenerated by somaclonal variation from C. limon embryogenic callus obtained from ovules culture on medium containing 2,4-D (Vardi and Spiegel-Roy 1982). Hybridization with Autotetraploid Parents. Crosses between 2x female 4x male and 4x 2x have been used by breeders to obtained triploid hybrids (Cameron and Soost 1969; Esen et al. 1978; Oiyama et al. 1981; Starrantino and Recupero 1982). It appears that 4x 2x crosses have been more successful in producing triploid embryos (Cameron and Frost 1968; Soost and Cameron 1975). It was proposed that the 5:3 ratio between endosperm and embryo ploidy obtained with these last crosses was more favorable than the 4:3 obtained in the reciprocal ones. Regarding the EBN theory, that would suggests that an EBN¼4 obtained with 4x 2x should be more favorable than the EBN¼1 in 2x 4x. However, most of the available tetraploid parents are polyembryonic. Thus, monoembryonic diploid female tetraploid male hybridizations have been exploited much more than the reciprocal ones. Precocious embryo rescue, three to four months after anthesis, was proposed by Starrantino and Recupero (1982) to enhance the production of triploid hybrids from 2x 4x crosses. ‘Oroblanco’ and ‘Melogold’ triploid pummelo grapefruit hybrids (Soost and Cameron 1980, 1985) resulted from such crosses as well as interesting tangor and mandarin triploid hybrids selected in Italy (Starrantino 1992). Tetraploid hybrids were found in addition to triploid hybrids in 2x 4x hybridization (Cameron and Soost 1969; Oiyama et al. 1981) while only triploids were obtained in reciprocal crosses. These tetraploids were found in normal seeds. This result is in agreement with the spontaneous production of diploid megagametophytes and a suitable endosperm/embryo ploidy ratio or EBN when diploid pollen fertilized unreduced megagametophyte. These tetraploid hybrids should be incorporated in the tetraploid gene pool for further triploid breeding. The occurrence of tetraploid hybrids in 2x 4x crosses implies that ploidy of seedlings hybrids should be checked before hybrid evaluation. 2. Segregation and Recombination. Segregation and recombination of tetraploids are complex (Wu et al. 2001a, b). They involve: (1) preferential pairing between homologous chromosomes that defines the proportion of

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bivalent and multivalent formation (Wu et al. 2001a); (2) the rate of crossover between the locus and the centromere and the type of centromere segregation (Marsden et al., 1987); and (3) double reduction frequency in case of quadrivalent formation (Mather 1936). The latter parameter is constant for a specific locus depending on the locus distance from the centromere (Wu et al. 2001a). Many economic Citrus species, such as sweet orange or grapefruit, are of interspecific origin (Barrett and Rhodes 1976; Nicolosi et al. 2000). Moreover, differences in nuclear genome size exist among the three ancestral taxa of cultivated citrus (Ollitrault and Michaux-Ferriere 1992; Ollitrault et al. 2002), which certainly contributes to structural heterozygosity in interspecific hybrids. It should be supposed that preferential pairing of duplicated chromosomes should occur for doubled interspecific Citrus hybrids. If we consider an autotetraploid line (AAaa) resulting from a parental diploid line (Aa), the transmission of the parental heterozygosity (Aa) to the triploid hybrid is a function of the (1) preferential pairing of the chromosome of the autotetraploid and (2) distance of the locus to the centromere. It should be 100% in the case of total preferential pairing between duplicated chromosomes leading to systematic bivalent formation and disomic segregation. It will be 66% regardless of the frequency of crossing over if random bivalents are formed (tetrasomic inheritance) while in case of tetravalent formation, the prediction of heterozygosity transmission rate is complicated by locus-centromere crossing over and the type of centromere segregation and tetravalent configuration (Marsden et al. 1987). For diploid doubled diploid hybridization, segregation and recombination occur in both the tetraploid pollen parent and the diploid seed parents. For a single locus A, consider a cross between two heterozygous parents (A1A2 A3A3A4A4). The number of allelic combinations in the triploid progeny varies, between 2 in the case of disomic segregation of the doubled diploid [(2 kinds of haploid female gametes, A1 or A2) (1 heterozygous diploid male gamete, A3A4)], to 6 [(2 kinds of haploid female gametes, A1 or A2) (3 kinds of diploid male gametes, A3A3, A3A4. A4A4)]. Equivalent segregations would occur for the reciprocal tetraploid female diploid male cross. C. Sexual Hybridization between Diploid Females and Tetraploid Somatic Hybrids 1. Biological Limitations and Practical Aspects Obtaining Allotetraploid Parents. Some allotetraploids used for triploid breeding have been selected from 2x 4x crosses (see Section III.B.1).


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A tetraploid hybrid from ‘Temple’ tangor 4n ‘Dancy’ mandarin was used as the female parent and pollinated with ‘Encore’ mandarin that gave rise to three patented triploid cultivars (Williams and Roose 2005). However, in the last 20 years, very many allotetraploids have been developed by somatic hybridization in various labs around the world. Since the first results obtained by Ohgawara et al. (1985), protoplast fusion has become an increasingly important component of citrus breeding programs (Grosser et al. 2000). Somatic hybridization is a very powerful tool for triploid breeding to expand the pool of tetraploid parents with new highly heterozygous allotetraploids. Moreover, this method allows using some sterile cultivars, such as ‘Satsuma’ or ‘Navel’ sweet orange as breeding parents. Establishment of embryonic nucellar callus lines is a prerequisite for an efficient citrus somatic hybridization program. Since leaf protoplasts do not have the capacity for plant regeneration, at least one of the parental protoplasts must be obtained from embryonic lines. Such lines are generally obtained by ovule culture of polyembryonic genotypes (Rangan et al. 1969; Ollitrault et al. 1994). Plants regenerated from such callus display little or no somaclonal variation (Starrantino and Russo 1983) even after protoplast isolation (Kobayashi 1987). Conversely, induction of embryogenic callus lines from ovules of monoembryonic cultivars is a difficult task (Kobayashi et al. 1982b) and generally results in a high level of variation (Navarro et al. 1985). Style and stigma culture (Carimi et al. 1995, 1999) or anther culture (Germana 2003) have also been tested with some success. However, even with these last methods, examples of efficient embryonic callus system for monoembryonic cultivars remain rare. Citrus somatic hybrids are generally produced from the fusion of protoplasts isolated from embryogenic callus or suspension cultures of one parent with leaf-derived protoplasts of the second parent. The embryogenic parent provides the capacity for plant regeneration from fusion products. Fusion of embryonic line protoplasts of the two parents has also been efficiently used to obtain somatic hybrids (Ollitrault et al. 1996c, 2000). Citrus protoplast fusion is induced either chemically using polyethylene glycol (Ohgawara et al. 1985; Grosser and Gmitter 1990) or electrically (Saito et al. 1991; Ling and Iwamasa 1994; Hidaka et al. 1995; Ollitrault et al. 1996b). More recently Olivares et al. (2005) have proposed a new method named electro-chemical protoplast fusion combining chemical protoplast aggregation and DC pulse promoted membrane fusion. After protoplast fusion, plant regeneration is accomplished without any selection pressure. However, in most of the published experiments, the majority of regenerated plants are allotetraploid somatic

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hybrids. The presence of some alloplasmic plants has also been demonstrated by molecular studies and ploidy analysis (Grosser et al. 2000). Cytoplasmic genome analysis has shown that chloroplasts have monoparental inheritance with random segregation (Ollitrault et al. 2000; Moreira et al. 2000; Liu et al. 2004). It has been demonstrated that in leaf þ callus protoplasts, mitochondrial DNA was inherited from the embryonic callus parent (Moreira et al. 2000; Cabasson et al. 2001). These results suggest the possible relation of mitochondria with regeneration ability (Grosser et al. 1996). Some cases of potential mitochondrial recombination have also been reported (Moreira et al. 2000; Liu et al. 2004). Somatic hybridization programs for triploid citrus improvement have been developed worldwide, and in some cases for many years. Countries supporting programs based in this technology include the United States, Brazil, Japan, China, Spain, Italy, and France (Grosser et al. 2000), and 94 combinations of allotetraploid parents (48 from Florida and 23 from France) have been listed by the same authors. New contributions have been published from New Zealand (Wu et al. 2005), Brazil (Calixto et al. 2004), Florida (Grosser and Chandler 2004; Guo et al. 2004), and China (Guo et al. 2000) with 23 additional allotetraploid parents. Hybridization with Allotetraploid Plants. Good levels of pollen fertility have been found in several allotetraploid somatic hybrids (Deng et al. 1995), and some of them display earlier flowering than autotetraploids. Endosperm/embryo ploidy ratios and EBN values are the same as the ones given for crosses between diploid and autotetraploid plants. Due to unbalanced ploidy ratio, embryo rescue is systematically used to recover triploid hybrids. Viloria et al. (2005) compared different culture media to optimize embryo rescue of acid citrus fruit derived from interploid hybridization. The first interploid hybridization with somatic hybrids was reported by Oiyama (1991) between diploid Clementine and tetraploid ‘Trovita’ sweet orange þ Poncirus trifoliata. Embryos of underdeveloped seeds at fruit maturity were rescued in vitro. They obtained mostly triploid hybrids and a few tetraploids (from normal seeds) resulting from the fertilization of unreduced ovules. Deng et al. (1996) observed 74.5% pollen fertility in hybrid ‘Hamlin’ sweet orange þ ‘Rough’ lemon and used it to fertilize diploid lines. Mostly abortive seeds were taken around 100 days after pollination for in vitro embryo rescue. Diploid, triploid, and tetraploid plants have been obtained, suggesting that perhaps the tetraploid somatic hybrid produced haploid and diploid pollen. Similar observations were done by Tusa et al. (1996) in sexual crosses between diploid ‘Feminello’ lemon and three allotetraploid somatic hybrids (‘Valencia’ sweet orange þ


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‘Feminello’ lemon; ‘Milam’ lemon þ ‘Feminello’ lemon; ‘Key’ lime þ ‘Valencia’ sweet orange). Indeed, they obtained an equivalent number of diploid, triploid, and tetraploid hybrids. These results have been correlated with cytogenetic studies of the microsporogenesis of the allotetraploid hybrid ‘Valencia’ sweet orange þ ‘Feminello’ lemon (Fatta del Bosco et al. 1999). The use of diploid pollen from allotetraploid hybrids has been systematized in Florida. Currently the UF-CREC breeding program has more than 13,000 interploid hybrids growing in the field. Most of these are mandarin-type hybrids (mandarins, tangelos, and tangors), with a lesser percentage of pummelo-grapefruit–type crosses and even fewer acid fruit types (F.G. Gmitter Jr., pers. comm.). Recently (Plate 7.5) we have started to pollinate monoembryonic mandarins with allotetraploid hybrids (C. reticulata þ C. sinensis and C.reticulata þ C. paradisi) to produce triploid tangor and tangelo cultivars (Froelicher et al. 2005). 2. Segregation and Recombination. Genetic structure of gametes of allotetraploid parents depends on the mode of chromosome association at meiosis and of the position of the locus relative to the centromere. The main difference between autotetraploid and allotetraploids is that tetraploid somatic hybrids can have four different alleles for the same locus. So diploid gametes from allotetraploids can display high heterozygosity. We consider an interspecific hybrid A1A2A3A4 obtained by somatic hybridization with A1A2 þ A3A4. If the chromosomal differentiation between the two parents of the somatic hybrid is high, leading to total preferential pairing of chromosomes from a same parent and disomic segregation, the diploid gametes will exclusively transmit interspecific heterozygosity (A1A3, A1A4, A2A3, A2A4). In other cases, diploid gametes should transmit either intraparental heterozygosity (A1A2 or A3A4) or interparental heterozygosity (A1A3,A2A4, A2A3, A2A4) or be homozygous (A1A1, A2A2, A3A3, or A4A4). Diploid hybrids of all or nearly all Citrus species have substantial levels of fertility that can only result from high levels of pairing at diploid level. So it is probable that there will be some preferential pairing in tetraploid somatic hybrids, but it is very unlikely to be 100%. Recombination and segregation occur both for the female and the male parents, and this strategy leads to the greatest diversity of the triploid progeny. For one locus, if we consider a cross between two totally heterozygous parents (diploid female A5A6 allotetraploid A1A2A3A4), the number of allelic combinations in the triploid progeny can vary, between 8 [(2 kinds of haploid female gametes, A5 and A6) (4 kinds of heterozygous diploid male gametes, A1A3, A1A4, A2A3 A2A4)]

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in the case of disomic segregation of the allotetraploid, to 20 [(2 kinds of haploid female gametes, A5 and A6) (10 kinds of diploid male gametes ¼ 6 heterozygous diploid male gametes, (A1A3, A1A4, A2A3 A2A4, A1A2, A3A4) þ 4 homozygotes (A1A1, A2A2A3A3, A4A4,)] if there are possibilities of quadrivalent formation and double reduction (Wu et al. 2001a). D. Endosperm Culture of 2x 2x Sexual Crosses 1. Biological Limitation and Practical Aspects. Endosperm culture could be a tool to overcome the barriers to sexual hybridization that result from nucellar embryony and can theoretically be applied to all germplasm with female fertility. Successful regeneration of triploid plantlets has been reported by Wang and Chang (1978) and Gmitter et al. (1990). However, the step of shoot or embryo regeneration from endosperm calli appeared critical (Jaskani et al. 1996). This technique is really of no value for triploid citrus breeding; because of the tremendous difficulties in achieving successful and efficient plant regeneration, it is virtually impossible to obtain a large recombinant population suitable for selection. 2. Segregation and Recombination. The triploid structure of the endosperm results from the fertilization of two haploid polar nuclei of the embryo sac by a haploid vegetative nucleus of pollen. There is no restitution of maternal heterozygosity because the two polar embryos arise from a same haploid cell by mitotic division. This lead to a high level of homozygosity (in terms of duplicated alleles at each locus) in the triploid hybrids. Recombination and segregation occur both for the male and the female parent. For one locus, if we consider a cross between two heterozygous parents (A1A2 A3A4), the progeny from endosperm culture would be constituted of four triploid genotypes: A1A1A3, A1A1A4, A2A2A3, A2A2A4. E. Diploid þ Haploid Protoplast Fusion 1. Biological Limitations and Practical Aspects Establishment of Haploid Lines. Haploidy induction by anther culture was first investigated in citrus by Drira and Benbadis (1975), but they obtained only callus formation with C. limon. Later, haploid embryo or plant recovery was reported for Poncirus (Hidaka et al. 1979), C. microcarpa (Chen et al. 1980), sweet orange ‘Trovita’ (Hidaka and Kajiura 1989), C. clementina (Germana 1992; Germana et al. 1992, 1994), C. limon (Germana et al. 1992), and tangelo (C. deliciosa


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C.paradisi) ‘Mapo’ (Germana and Reforgiato-Recupero 1997). Androgenesis methods were recently improved for C. clementina by Germana and Chiancone (2003). Haploid embryos and plants have also been obtained from gynogenesis in monoembryonic cultivars. About 2% haploid plants were obtained from fully developed seeds derived from pollination of diploid C. clementina and ‘Lee’ mandarin with triploid pollen (Oiyama and Kobayashi 1993), and some haploid plants have been obtained in diploid diploid crosses (Toolapong et al. 1996, for C. grandis and our unpublished data for C. clementina). Ollitrault et al. (1996a) demonstrated the efficiency of induced gynogenesis by irradiated pollen of monoembryonic cultivars (C. clementina and ‘Seedless’ C. maxima), and this method has been extended to several C. reticulata and tangor cultivars (Y. Froelicher et al., 2007). Germana and Chiancone (2001) induced gynogenesis in C. clementina by in vitro pollination with triploid pollen. Haploid plants obtained by androgenesis or gynogenesis are generally weak and need to be grafted to survive. Morphological and reproductive potential of a haploid pummelo has been recently described (Yahata et al. 2005). It had slightly fertile pollen grains and was able to produce diploid hybrid progenies when crossed with a diploid cultivar, suggesting that it produces unreduced pollen grains (n¼9). Somatic Hybridization between Haploid and Diploid Lines. The technique of haploid þ diploid somatic hybridization was developed simultaneously by Ollitrault et al. (1997, 1998b; Plate 7.6) and Kobayashi et al. (1997). It should be applied to polyembryonic or monoembryonic diploid cultivars if haploid embryogenic callus lines are available. For combination with leaf protoplasts of haploid plantlets, it is necessary to use diploid protoplasts from embryogenic callus lines arising mainly from polyembryonic cultivars. In the case of haploid callus lines, this method produces triploid hybrids, but also tetraploid and pentaploid ones, due to the ploidy instability of haploid calli (Ollitrault et al. 1998b). The great limitation of this strategy is the scarcity of haploid lines available in citrus. It should be overcome by the development of gametosomatic hybridization. This method, which combines haploid protoplasts from gametic cells (generally from pollen) and diploid somatic protoplasts, has been successfully applied in Nicotiana and Petunia (Pirrie and Power 1986; Lee and Power 1988). It was mentioned in citrus by Deng et al. (1992), who have regenerated only chimeric plants with 18 and 19 chromosomes. It is actually an important objective of the triploid citrus breeding program of CIRAD in France.

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2. Segregation and Recombination. This strategy allows a complete restitution of the diploid cultivar nuclear genome and its heterozygosity, without segregation and recombination. For each locus, if we consider the combination of a diploid heterozygote (A1A2) with a haploid (A3), all regenerated triploids will have the same genotype (A1A2A3). In case of gametosomatic hybridization, if we consider a combination of heterozygote diploid A1A2 with a segregating population of haploid gametes arising from A3A4, two triploid genotypes will be obtained (A1A2A3, A1A2A4). Segregation should occur at the level of cytoplasmic genomes. When fusions are done with embryogenic callus protoplast both for diploid and haploid lines, this should lead to four different kinds of nucleo cytoplasmic interaction. For perennial vegetatively propagated crops with long juvenile phase like citrus, breeding strategies are generally based on one cycle of induction of variability (hybridization, mutagenesis, biotechnologies) followed by a selection/validation process and clonal propagation of elite genotypes. In order to improve the effectiveness of such strategies, it appears essential to optimize the transfer of the genetic gain obtained by phenotypic selection at the parental level. Citrus cultivars being generally highly heterozygous, a good transmission of parental heterozygosity of one parent to the triploid progenies appears important to prevent a global breakage of the complex favorable multilocus genotypic structure selected at the parental level. As already mentioned, the transmission of parental heterozygosity varies according to the strategies of triploid breeding. Figure 7.1 summarizes the theoretical heterozygosity transmission for one locus and its schematized implication on the distribution for a quantitative trait in triploid progenies. In case of triploid obtaining by 2x 2x crosses, the heterozygosity transmission rate is function of the rate of single crossing over between the centromere and the locus. It varies between 0 and 100% for SDR and 100 to 50% for FDR (Park et al. 2007). In Solanum, corresponding average transmission values are respectivly 40% and 80% (Carputo et al. 2003). For ‘autotetraploid’ (double diploid) with tetrasomic inheritance, the transmission rate is 66% (all loci on chromosome with random bivalent association and locus close to the centromere for chromosome with tetravalent association; chromosome segregation, Muller 1914; Marsden et al. 1987). With systematic tetravalent formation, assuming that a single crossing-over occurs between each homologous chromosome pair and all types of centromere segregation take place with equal frequency, the rate will be of 5/9 (model of maximum equational chromatid segregation; Mather 1935; Marsden et al. 1987). Heterozygosity transmission


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Parental heterozygosity restitution Gametosomatic hybridization (2x + x) 2x × 4x disomic inheritance (bivalent with systematic preferencial pairing) 1 2n gametes FDR 0.75 0.66

2x × 4x tetrasomic inheritance (random bivalent formation)

0.5

2n gametes SDR

0

Single crossing over rate from centromere

1

Theoretical variability of quantitative traits in triploid progenies

Fig. 7.1 Implications of triploid breeding strategies on heterozygosity transmission rate and theoretical distribution of quantitative traits in progenies.

rate can reach 100% for 2x 4x progenies in case of disomic segregation of the doubled diploid (systematic chromosome preferential pairing). The same result is obtained for haploid þ diploid somatic hybridization. However, it is probable that chromosomal differentiations in Citrus genus are not sufficient to imply disomic segregation over the whole genome of double diploid. Thus haploid þ diploid somatic hybridization appears to be the most efficient strategy in terms of heterozygosity transmission. It is more interesting in terms of efficiency of multilocus selection at the diploid cultivar level to confer multi-locus desirable traits to the triploid hybrid. It should be noted that the worst heterozygosity transmission rate is given by albumen culture with 0% (not represented in Fig. 7.1). The efficiency of a breeding program is also based on the possibility of selecting, in a polymorphic population, new genomic associations that fit with the breeding objective. Gametosomatic hybridization should allow combining these two conditions with (1) total transmission of heterozygosity (and whole genome) of the preselected diploid variety and (2) polymorphism of the triploid population induced by recombination and segregation of the haploid component.

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IV. CONCLUSION Triploid citrus cultivars with notable commercial production remain relatively rare. The large-fruit lime such as ‘Tahiti’ and ‘Bears’ (C. latifolia) is the most consumed triploid and is produced all over the world. ‘Oroblanco’ (‘‘Sweetie’’) obtained from a cross between a diploid pummelo and an autotetraploid grapefruit (Soost and Cameron 1980), is undergoing increasing production and consumption. In the last 10 years several new triploid cultivars resulting mostly from the oldest breeding strategy (diploid autotetraploid sexual crosses) have been released in Italy, the United States, and Japan, and some of these cultivars are now being produced for the market. ‘Tacle’ (Starantinno 1999) is a very promising tangor obtained in Sicily (Istituto Sperimentale per l’Agrumicultura) by crossing diploid ‘Clementine’ with autotetraploid ‘Tarocco’ sweet orange. It possesses the blood-red flesh characteristic of its tetraploid parent. Other triploid tangor and mandarin hybrids are currently being released by the same group as well as some triploid lemon types (Russo et al. 2004). In California, recent releases of the UC Riverside Citrus Breeding Program (Williams and Roose 2005) are three mid- and late-season triploid mandarin hybrids resulting from the same cross [(‘Temple’ tangor tetraploid ‘Dancy’ mandarin) ‘Encore’ mandarin]: (1) ‘TDE2’ mandarin hybrid (Shasta Gold1 mandarin ), (2) ‘TDE3’ mandarin hybrid (Tahoe Gold1 mandarin), and (3) ‘TDE4’ mandarin hybrid (Yosemite Gold1 mandarin). In Japan, a triploid kumquat was released in 1999 by the Department of Citriculture (Okitsu), National Institute of Fruit Tree Science, from a cross between ‘Naja Kinkan’ and tetraploid ‘Ninpou Kinkan’ (Yoshida et al. 2003). More recently (Tokunaga et al. 2005) a triploid C. sudachi hybrid (Tokushima 3x n8 1), obtained from 4x 2x cross, has been submitted for registration by the Tokushima Research Center for Agriculture Forestry and Fisheries. In Spain, four triploid hybrids obtained from 2x 2x crosses with ‘Fortune’ mandarin as female parent have been registrated: ForEll 96–058 and ForEll 96-060 from ‘Fortune’mandarin ‘Ellendale’ tangor crosses, ForKar 96-037 from pollination with ‘Kara’ mandarin, and ForMur 96–081 from pollination with ‘Murcott’ tangor (Navarro et al. 2006). In the future, the ongoing programs of ploidy manipulation will result in a broad range of triploid seedless cultivars and will find promising applications to combine seedlessness and other traits, such as fruit quality, adaptation to various climatic conditions, biotic resistances, and a range of harvesting periods. The different strategies to obtain triploid hybrids can be applied in different kinds of cultivars (monoembryonic,


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polyembryonic, sterile) and can lead to populations that are very variable in terms of genetic segregation and recombination. The development of gametosomatic hybridization should enhance considerably the efficiency of triploid breeding in the next years. Overall, it is clear that field evaluation and market validation in close relation with different actors of the citrus industry are absolute necessities to provide an opportunity for new seedless hybrids to reach the market as popular cultivars. V. LITERATURE CITED Barrett, H.C. 1974. Colchicine-induced polyploidy in citrus. Bot. Gaz. 135:29–41. Barrett, H.C. 1992. An autotetraploid of the Key lime, Citrus aurantifolia. Fruit Var. J. 46:166–170. Barrett, H.C., and D.J. Hutchison. 1978. Spontaneous tetraploidy in apomictic seedlings of Citrus. Econ. Bot. 32:27–45. Barrett, H.C., and A.M. Rhodes. 1976. A numerical taxonomic study of affinity relationships in cultivated Citrus and its close relatives. Syst. Bot. 1:105–136. Bowman, K.D., F.G. Gmitter Jr., G.A. Moore, and R.L. Rouseff. 1991. Citrus fruit sector chimeras as a genetic resource for cultivar improvement. J. Am. Soc. Hort. Sci. 116(5):888–893. Cabasson, C.M., F. Luro, P. Ollitrault, and J.W. Grosser. 2001. Non-random inheritance of mitochondrial genomes in Citrus hybrids produced by protoplast fusion. Plant Cell Rep. 20(7):604–609. Calixto, M.C., F. de A.A. Mourao-Filho, B.M.J. Mendes, and M.L.C. Vieira. 2004. Somatic hybridization between Citrus sinensis (L.) Osbeck and C. grandis (L.) Osbeck. Pesquisa Agropecuaria Brasileira 39(7):721–724. Cameron, J.W., and H.B. Frost. 1968. Genetic, breeding and nucellar embryony. pp. 325– 370. In: W. Reuther, L.D. Batchelor, and H.J. Webber (eds.), The citrus industry. Vol. 1. Univ. California, Riverside. Cameron, J.W., and R.K. Soost. 1969. Characters of new populations of Citrus polyploids, and the relation between tetraploidy in the pollen parent and hybrid tetraploid progeny. Proc. First Int. Citrus Symp. 1:199–205. Carputo, D., L. Frusciante, and S.J. Peloquin. 2003. The role of 2n gametes and endosperm balance number in the origin and evolution of polyploids in the tuber-bearing Solanums. Genetics 163(1):287–294. Carputo, D., L. Monti, J.E. Werner, and L. Frusciante. 1999. Use and usefulness of endosperm balance number. Theor. Appl. Genet. 98:478–484. Carimi, F., F. de Pasquale, and F.G. Crescimanno. 1995. Somatic embryogenesis in Citrus from styles culture. Plant Sci. Limerick 105(1):81–86. Carimi, F., F. de Pasquale, and F.G. Crescimanno. 1999. Somatic embryogenesis and plant regeneration from pistil thin cell layers of Citrus. Plant Cell Rep. 18(11):935–940. Chandler, J.L., Z. Viloria, and J.W. Grosser. 2001. Acid citrus fruit cultivar improvement via interploid hybridization. Proc. Florida State Hort. Soc. 2000. 113:124–126. Chen, S., F. Gao, and J. Zhang. 1991. Studies on the seedless character of citrus induced by irradiation. Mutation Breed. Newslet. 39:8–9. Chen, Z.Q., M.Q. Wang, and L. Huihua. 1980. The induction of Citrus pollen plants in artificial media. Acta Genet. Sin. 7:189–191.

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8 Breeding Southern Highbush Blueberries Paul Lyrene Horticultural Sciences Department University Of Florida Gainesville, Florida 32611, USA

I. INTRODUCTION II. BIOLOGY OF THE HIGHBUSH BLUEBERRY A. Morphology B. Ecology and Physiology C. Reproductive Biology D. Effects of Vaccinium darrowi Genes on Southern Highbush Cultivars III. GENETIC RESOURCES FOR BREEDING SOUTHERN HIGHBUSH BLUEBERRY A. Primary Gene Pool B. Secondary Gene Pool 1. Tetraploid V. corymbosum from Georgia and Florida 2. Vaccinium darrowi 3. Vaccinium elliottii 4. Diploid Vaccinium fuscatum from Central and South Florida C. Tertiary Gene Pool IV. ORIGINS OF HIGHBUSH BLUEBERRY BREEDING A. Northern Highbush B. Southern Highbush V. METHODS A. Basic Procedures 1. Making Crosses and Growing Seedlings 2. Field Testing Procedures 3. Screening for Disease Resistance and Other Characteristics B. Population Improvement by Recurrent Selection C. Test Crosses and Proven Crosses D. Heritability of Characters E. Major Genes in Southern Highbush Blueberry F. Incorporating Additional Wild Germplasm VI. BREEDING OBJECTIVES A. Plant Characteristics


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1. Chilling Requirement 2. Cessation of Growth in the Fall 3. Flower Bud Initiation 4. Time of Flowering 5. Time of Ripening 6. Vigor and Carrying Capacity 7. Plant Architecture 8. Drought Tolerance and Tolerance of Higher Soil pH 9. Disease and Insect Resistance 10. Self-Fruitfulness 11. Yield 12. Mechanical Harvestability 13. Evergreen Production and Protected Culture B. Berry Characteristics 1. Size 2. Color 3. Picking Scar 4. Firmness 5. Flavor 6. Texture 7. Ability to Maintain Quality as a Ripe Berry on the Bush 8. Freedom from Miscellaneous Defects 9. Postharvest Life and Quality 10. Berry Components with Health Benefits VII. RECOMMENDING CULTIVARS FOR COMMERCIAL PLANTINGS A. Regions of Adaptation B. Old Proven Cultivars versus New Improved Cultivars C. Cultivar/Culture Interactions D. Cross-pollination Considerations E. Cost and Availability VIII. OUTLOOK AND ISSUES A. Current Situation B. Grafted Blueberries C. Germplasm Conservation IX. LITERATURE CITED

I. INTRODUCTION Worldwide, four Vaccinium taxa have considerable economic importance: blueberries (section Cyanococcus), cranberries (section Macrocarpon), lingonberries (section Vitis-idaea), and bilberries (section Myrtillus). Species in several other sections are gathered for local consumption. This review focuses on southern highbush blueberries, a group of hybrid cultivars based on V. corymbosum L., V. darrowi Camp, and other species of section Cyanococcus (Camp 1945; Vander Kloet 1983). Other commercially important blueberries, including northern

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highbush (V. corymbosum), lowbush (V. angustifolium Ait.), and rabbiteye blueberry (V. ashei Reade), are discussed only as they relate to southern highbush breeding. Previous reviews on highbush and rabbiteye blueberry breeding include Moore (1966), Galletta (1975), Lyrene (1987), and Lyrene and Moore (2006). Southern highbush blueberry cultivars are tetraploid (2n ¼ 4x ¼ 48) clones developed by interspecific hybridization for adaptation to areas with mild winters. They were bred by crossing northern highbush cultivars from Michigan and New Jersey with native blueberries from Florida and other southeastern states. The initial crosses were followed by several generations of backcrosses, intercrosses, and selection to bring together the desired traits: adaptation to mild winters, tolerance of summer heat and humidity, high yield, early ripening, and high berry quality. The name ‘‘southern highbush’’ seems to have originated with Camp (1945), who called plants of V. australe Small ‘‘southeastern highbush blueberry’’ and plants of V. corymbosum ‘‘northern highbush blueberry.’’ Most taxonomists now combine the two species as V. corymbosum, but the names northern highbush and southern highbush have been retained for high-chill and low-chill highbush cultivars. Sharpe and Sherman (1976a, b, c) called their first low-chill highbush cultivars, released in 1976 and 1977, ‘‘low-chill tetraploid hybrids.’’ Thereafter, ‘‘southern highbush’’ came to be used for tetraploid blueberry clones whose chilling requirement (hours below 78C per winter) was less than 800 hours (hr), which is approximately the chilling requirement of cultivars with no parentage from the southeastern United States. Chilling requirement in V. corymbosum hybrids can range from zero to more than 1,000 hr (hr between 08 and 78C per winter) depending on how much southern germplasm was used in their breeding, the particular genetic combination, and the test that was used to assign chilling requirement. Highbush blueberries are grown in irrigated fields, typically with 0.9 to 1.5 m between plants in rows and 3 m between rows. Plants are pruned to keep them 2 to 3 m tall. Cultivars are asexually propagated clones. Bees are brought into the fields for pollination, which is necessary for berry development where native bee populations are not adequate. Berries typically weigh 1 to 3 g each, are borne in clusters, and ripen during a four- to six-week period, 50 to 90 days after flowering, depending on climate and cultivar. About half of the annual highbush blueberry crop in the United States is marketed fresh. The other half is marketed as frozen berries or processed berry products. Most blueberries for processing are harvested by machine, cleaned, and then frozen.

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Frozen blueberries are used in pastries, yogurt, ice cream, and other processed products. Some processed blueberries are freeze-dried or used for juice/concentrate. Fresh-market blueberries are usually harvested by hand, but machine harvest of berries for the fresh market is increasing. The first commercial highbush blueberry plantings were made in New Jersey and Michigan during the 1930s and 1940s. In the 1950s and 1960s, improved highbush cultivars were planted in southeastern North Carolina, near the southern limit of where the New Jersey cultivars could be grown successfully. Today there is also important production in Washington, Oregon, California, and British Columbia, as well as in Chile, Poland, and other parts of Europe (Banãdos 2006; Strik 2006). Low-chill cultivars allowed the extension of highbush blueberry cultivation to Florida, Georgia, California, Spain, Argentina, and elsewhere. By 2003 there were over 30,000 ha of cultivated highbush blueberries worldwide (Banãdos 2006; Strik 2006).

II. BIOLOGY OF THE HIGHBUSH BLUEBERRY A. Morphology Camp (1945) described wild highbush blueberry plants as being 1 to 4 m tall, crown-forming, having several stems, or suckering to form a compact colony 0.5 to 1.0 m in diameter at the base. The leaves are deciduous (except in Florida, where native highbush blueberry plants are semideciduous), 2 to 3 cm wide and 4 to 8 cm long. Flowers have five petals, fused into a tubular corolla 6 to 10 mm long, white or pinktinged. The berries of wild plants are blue or black, 5 to 10 mm in diameter, usually of good to excellent flavor. The berries contain 5 to 50 small seeds. Berries that contain no viable seeds normally abort within 30 days after the flowers open. Flower buds are initiated in leaf axils during the fall as a result of shorter days and cooler nights. In the spring, each flower bud produces a short, leafless branch bearing 5 to 10 flowers. Following pollination, usually by bees, the berries mature in 50 to 100 days, depending on cultivar and temperature. B. Ecology and Physiology Wild highbush blueberry plants grow on coarse, acid soils of sand mixed with organic matter. These soils are moist but well drained. The plants lack drought tolerance but cannot long survive standing


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water or saturated, poorly aerated soils. Highbush blueberries grow poorly where soil pH is above 5.5. The root system is fibrous and usually quite shallow. Highbush blueberries are somewhat shade tolerant but yield most when grown in full sun. Blueberry plants grown from seed have a short juvenile period, normally producing some fruit in their second or third year. Plants from cuttings may flower even before they are rooted. Once established, highbush blueberries can live 50 years or more. In commercial fields, they are pruned annually to control bush size and shape and to stimulate growth of strong, new canes. Like many plant species that have an extensive north-south native range in eastern North America, highbush blueberries differ markedly in many adaptive features depending on the part of the range from which the plants are taken. Plants from the north are very cold hardy. They flower and produce new leaves late in the spring (mid-April to early May) and enter a photoperiod-induced dormancy early in the fall, by mid- to late September (Hall et al. 1963). Native V. corymbosum plants from the southern end of the natural range, in central and northern Florida, are partially evergreen, but normally lose most of their leaves by the end of winter. They flower in late February and early March and ripen in May and early June. They continue growing later into the fall than plants from the northern end of the range. If temperatures are maintained above freezing, they can be kept growing throughout the winter in greenhouses in north Florida under natural photoperiods. Once they have entered winter dormancy, V. corymbosum plants from the northern end of the range require about 1,000 hr of chilling (08 to 78C) to satisfy the dormancy requirement. Plants from the southern end of the range require only 100 to 300 hr of chilling. C. Reproductive Biology Highbush blueberry flowers are well suited to cross-pollination by bees. Most highbush that have not been altered by breeding have low selfcompatibility. When self-pollinated, they produce few berries, and the few berries that form contain few viable seeds. In a study of wild diploid, tetraploid, and hexaploid V. corymbosum plants collected from 26 sites from Florida to Nova Scotia, Vander Kloet and Lyrene (1985) found that self-pollination greatly reduced all fertility parameters. Crosses between full or half siblings produced fewer seedlings than crosses between unrelated seedlings, mainly because the number of plump seeds per berry was reduced from 21 for outcrosses to 13 for sibling crosses.

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Commercial blueberry cultivars have been bred and selected for increased self-fruitfulness. Some cultivars in some locations achieve full or nearly full yields when planted in solid blocks of one cultivar. However, most low-chill highbush cultivars require mixed blocks to promote cross-pollination. Some cultivars produce very low yields unless cross-pollinated. Blueberries also show inbreeding depression. Blueberry seedlings grown from self-pollination are usually much weaker than seedlings from cross-pollinated flowers. Under cultivation in Florida, southern highbush cultivars make three to five growth flushes per year. Flower buds are initiated when days are short enough and nights cool enough to meet the inductive threshold of the cultivar. By the end of harvest (mid-May in north Florida), flower buds often are already visible on the first growth flushes of the spring, having been induced by low temperatures and short days after the first spring flushes appeared. When flowers and young fruit are destroyed by freezes in February or early March in Florida, flower buds that were induced earlier on late-winter growth flushes may produce heavy flowering in late March and early April, but under normal conditions, flower buds induced on the first spring growth flushes remain dormant until the following February. Growth flushes produced from April through August in north Florida normally first show visual evidence of flower buds in late August and September. Axillary buds produced on summer growth flushes may either remain as vegetative buds or be converted to flower buds during the period from early August through late October. Buds that are transformed to flower buds become visually larger and more rounded during the fall, although they normally do not produce flowering branches until late winter, after chilling. D. Effects of Vaccinium darrowi Genes on Southern Highbush Cultivars Vaccinium darrowi, a diploid evergreen blueberry native to Florida, was the species most extensively used in adapting cultivated northern highbush blueberries to areas with warm winters and long, hot, rainy summers. Although V. darrowi and V. corymbosum are closely related and can make vigorous, fertile tetraploid hybrids and backcross seedlings, the two species are quite distinct in morphology and ecology. Vaccinium darrowi is evergreen, low-growing, and adapted to frequent forest fires. It forms large clonal colonies that spread by rhizomes in the understory of pines. The small evergreen leaves are coriaceous, persist for more than one year, and are typically 3 to 5 mm wide and 7 to 11 mm long (Vander Kloet 1988). Vaccinium darrowi has no chilling requirement, but


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it flowers in Florida about 20 days later than sympatric V. corymbosum (Lyrene and Sherman 1980). Vaccinium darrowi berries have a relatively long flowering-to-ripening period. The date when 50% of the berries are ripe in north-central Florida averages about 12 June for V. darrowi, 28 May for native tetraploid V. corymbosum, and 1 May for early-ripening southern highbush cultivars. Vaccinium darrowi is capable of producing very high flower numbers; even though the berries are small (about 0.2 g), berry yields can be quite high on potted plants. V. darrowi berries have fair eating quality, with medium to high percent soluble solids and low acidity. They are sometimes somewhat bitter. Seeds are small but numerous, and skins are similar to those of highbush blueberry in thickness. Historically, V. darrowi was gathered from the wild by rural Floridians for making pies and pastries. Because the two principal components of southern highbush blueberry—V. corymbosum and V. darrowi—differ so greatly in morphology and physiology, segregating populations following interspecific hybridization presents abundant opportunity for breeders to select useful combinations of characteristics. V. darrowi has contributed genes for low chilling requirement, heat tolerance, and improved berry quality to southern highbush cultivars (Luby et al. 1990; Ballington 2001). Although V. darrowi is highly variable in Florida (Lyrene 1986), almost all southern highbush cultivars trace back to a single V. darrowi clone, Florida 4b, collected by Ralph Sharpe at Winter Haven, Florida, in 1949 (Lyrene 1998).

III. GENETIC RESOURCES FOR BREEDING SOUTHERN HIGHBUSH BLUEBERRY Genetic resources available for breeding highbush blueberries can be divided into the primary, secondary, and tertiary gene pools. The primary gene pool consists of cultivars and breeding lines of northern highbush, southern highbush, and, to some extent, rabbiteye and lowbush blueberry, in which many horticultural characteristics have been improved by breeding. The secondary gene pool includes wild plants of all Vaccinium section Cyanococcus species, including V. corymbosum and V. darrowi. The tertiary gene pool consists of plants of Vaccinium species in sections other than Cyanococcus. A. Primary Gene Pool Many improved cultivars of blueberry have been developed and released during the past 100 years. These include more than 70 northern

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highbush cultivars, 50 southern highbush cultivars, 40 rabbiteye cultivars, and 10 cultivars each of northern lowbush and half-high cultivars bred from V. corymbosum V. angustifolium hybrids (Anon. 1997). Despite this large number of cultivars, the gene base of the cultivated tetraploid blueberries is narrow. Southern highbush cultivars are based largely on northern highbush genes and genes from V. darrowi Fla. 4b. In turn, the gene pool of cultivated northern highbush rests heavily on three early cultivars: ‘Brooks’ (23.5% of the genes), ‘Sooy’ (12.5%), and ‘Rubel’ (28.6%) (Hancock and Siefker 1982). The contribution of these three selections to more recent northern highbush cultivars remained high (Ehlenfeldt 1994). The gene base for highbush blueberries has expanded somewhat in the last 20 years with the release of some new tetraploid cultivars with complex ancestry. Except for rabbiteyes, which are hexaploid, all cultivars in the primary gene pool can be readily intercrossed. The resulting hybrids are tetraploid and fertile. Jelenkovic (1973), Chandler et al. (1985b), and Vorsa et al. (1987) showed that even the hexaploids are relatively easy to use in breeding tetraploids, because tetraploid hexaploid crosses are easy to make, the pentaploid hybrids are easy to backcross to tetraploids, and some of the progeny from the backcrosses are highly fertile tetraploids or near tetraploids. Even though some excellent rabbiteye (hexaploid) blueberry cultivars have been produced and are not hard to cross with tetraploid highbush, rabbiteyes have been used less in breeding southern highbush cultivars than might have been expected. The difference in chromosome numbers has not been the main problem; some of the hybrids obtained by backcrossing the pentaploids to highbush are quite fertile and near tetraploid. A more significant problem in using rabbiteye cultivars to improve southern highbush is berry color. Berry color of F1 hybrids and backcrosses is surprisingly dark, even when both the highbush and the rabbiteye parents have light-blue color. Another obstacle to practical hybridization is that earliness is usually important in southern highbush cultivars, and rabbiteye cultivars are mostly much later ripening than highbush. B. Secondary Gene Pool A vast secondary gene pool is available to breeders looking for novel traits or seeking the benefits of new heterotic combinations or a broader genetic base (Draper 1977). Evidence indicates that the chromosomes of all Cyanococcus species are structurally similar, and interspecific homologs pair well during meiosis.


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Section Cyanococcus is endemic to North America (Vander Kloet 1983). Vander Kloet suggested that the ancestral group had a probable South American origin and migrated through the Caribbean to Florida, where adaptive radiation produced the many Cyanococcus species that exist today. The section consists of about six diploid, five tetraploid, and three hexaploid species (Longley 1927; Darrow et al 1944). The exact number of species is subject to debate. Some species have restricted native ranges; others cover broad areas. Homoploid crosses involving different species in section Cyanococcus are usually easy to make in a greenhouse, and most give fully fertile hybrids (Coville 1927; Darrow 1960; Ballington and Galletta 1978; Vander Kloet 1983). The mechanisms that provide genetic isolation that allows these species to maintain their identities in the wild (and presumably allowed them to differentiate into recognizable species in the first place) include geographical and ecological disjunction, differences in time of flowering, and differences in ploidy level (Camp 1942; Darrow 1949). Some species tolerate frequent wildfires but little shade. Others are shade tolerant but do not persist where fire is frequent. Some species are limited to high elevations in the southern Appalachians, while others are restricted to the coastal plain. Darrow and Camp (1945) found from field studies that, in relatively undisturbed areas, it was rare to find different homoploid species occupying the same habitat. They believed that ‘‘it was extensive and repeated clearing and burning, the formation of fields and pastures, and often their later abandonment—which made it possible for the various homoploid blueberries to enlarge their ranges and thus make contact.’’ Despite Camp’s (1942, 1945) belief that the polyploid species in Vaccinium section Cyanococcus were mostly allotetraploids, all recent evidence shows that the tetraploid species are autotetraploid, that is, polysomic (Qu and Hancock 1997; Qu et al. 1998). All diploid species in the section produce fertile hybrids when crossed, an indication that the chromosomes of the section are not differentiated. The chromosomes in wild plants of the tetraploid species pair mainly as bivalents during meiosis. There appears to be little or no preferential pairing, and segregation ratios from crosses of the type AAaa AAaa (where A is the dominant allele of a marker gene and a the recessive) give 1/36th of the population with the recessive phenotype. This has been confirmed with both visible (Wenslaff and Lyrene 2003) and isoenzyme (Krebs and Hancock 1989) markers. Interspecific crosses between tetraploid Cyanococcus species produce fertile tetraploid hybrids, and seedlings from backcrosses of the F1 hybrids to either parent are typically fully fertile. If the chromosomes of different Cyanococcus species were structurally

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differentiated, diploid interspecific hybrids, and backcross populations involving tetraploid interspecific hybrids would be expected to have reduced fertility, but this is not the case. Interploidy crosses within section Cyanococcus give variable results, depending on the ploidy levels involved and the frequency of unreduced gametes in the parents. Most diploid species produce some unreduced gametes (Ballington and Galletta 1976; Cockerham and Galletta 1976; Ortiz et al. 1992). The frequency is variable within the species. Some clones produce 1% or more 2n gametes; others produce few or none. In blueberry, the four pollen grains resulting from each meiotic cell are shed as a tetrad of attached pollen grains. Thus, microscopic examination of pollen can reveal monads and diads that result from meiotic abnormalities, and these indicate that 2n gametes are being produced. Megalos and Ballington (1988) found that diploid plants that produced no diploid pollen, as determined by microscopic examination of pollen, produced no tetraploid hybrids when crossed with tetraploid highbush cultivars. Ortiz et al. (1992) studied unreduced pollen formation in 1,475 seedlings of 46 diploid populations of seven section Cyanococcus species. In most plants, they found no 2n gametes, but some plants in each of the seven species produced some 2n gametes. Unreduced pollen producers produced both reduced and unreduced pollen. Among plants that produced unreduced pollen, the percentage of the pollen that was unreduced ranged from 1% to 28.6%. Blueberry chromosome number can be doubled with colchicine (Aalders and Hall 1963; Moore et al. 1964; Perry and Lyrene 1984; Sanford 1983). Colchicine-derived tetraploids of diploid species such as V. elliottii have been easy to cross with tetraploid cultivars, but in most cases the easiest way to obtain diploid tetraploid hybrids has been through the use of diploid clones that make unreduced gametes (Lyrene et al. 2003). The triploid block is strong in Vaccinium section Cyanococcus. This eliminates most (but not all) triploid embryos and avoids the need to screen large seedling populations to find the few tetraploid hybrids produced from unreduced gametes. Sharpe and Darrow (1959) pollinated 651 flowers of diploid V. darrowi with pollen from tetraploid V. corymbosum and obtained only 9 hybrids. The reciprocal cross gave only 22 hybrids from 1,027 pollinated flowers. For comparison, homoploid crosses, either diploid or tetraploid, normally produce at least 10 seedlings per pollinated flower. A triploid clone of Vaccinium vitisidaea L. was reported from the wild by Ahokas (1971). The first triploids reported in Vaccinium section Cyanococcus resulted from crosses between tetraploid highbush cultivars and diploid V. elliottii (Lyrene


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and Sherman 1983). Seven thousand pollinated flowers gave 25 hybrids. Chromosome counts for 18 of the hybrids showed 2 triploids, 11 tetraploids, and 5 plants with higher chromosome numbers. Vorsa and Ballington (1991) studied the fertility of 11 triploid highbush blueberry plants when used as male and female parents in various crosses. From 4,021 triploid diploid pollinations involving 10 triploids and 21 diploid pollen parents, no seedlings were obtained. No seedlings were obtained in 705 pollinations representing 17 different crosses in which plants of diploid species were pollinated with pollen from 3 triploid hybrids. Triploid tetraploid crosses, in which 856 flowers were pollinated, gave 23 progeny. Thus, incorporation of genes from diploid species into cultivated tetraploids using triploids as bridges is possible but not easy. Diploid clones, which, because of unreduced gamete production, can produce tetraploid hybrids in crosses with tetraploid cultivars, have been much more useful in breeding tetraploid cultivars. Munoz and Lyrene (1985a) attempted to increase the recovery of triploids by embryo culture following V. elliottii tetraploid highbush crosses (and the reciprocal) but were unsuccessful. They found that cultured embryos from V. elliottii V. elliottii crosses taken as early as 15 days after pollination developed into seed and germinated at very low rates. The original plan of Sharpe and Darrow for producing low-chill highbush blueberries was to cross northern highbush cultivars with synthetic low-chill tetraploids, which they planned to create by crossing diploid V. darrowi with hexaploid V. ashei (rabbiteye) cultivars (Darrow et al. 1954; Sharpe and Sherman 1971). They pollinated 7,500 flowers in their attempt to cross V. darrowi with V. ashei and obtained 5 hybrids. The hybrids were intermediate in characteristics between the parents and were sufficiently fertile to produce large seedling populations when crossed with high-chill tetraploid northern highbush cultivars. Although presumed to be tetraploid, the V. darrowi V. ashei may actually have been pentaploid (Goldy and Lyrene 1984). Although difficult to produce, V. darrowi V. ashei hybrids are a potential bridge for incorporating genes from diploid and hexaploid Cyanococcus species into southern highbush at the tetraploid level. After it was found that tetraploid hybrids could easily be produced by directly crossing tetraploid highbush cultivars with diploid V. darrowi plants that produced unreduced gametes, little further work was done with V. ashei V. darrowi hybrids. What opportunities were missed by not incorporating more V. ashei genes into southern highbush cultivars is unknown. Darrow (1956) applied 1% naphthalene acetamide in lanolin paste to the ovaries of rabbiteye cultivars at the time of pollination with

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V. darrowi pollen. Although this greatly increased the number of berries that were retained to maturity, it did not increase the number of hybrid plants he obtained. The secondary gene pool has already been widely used in breeding highbush blueberries, but because there are many species in section Cyanococcus, and these could be combined in many ways, many breeding opportunities await investigation. Some taxa that have given the most interesting hybrids when crossed with southern highbush cultivars are discussed below. 1. Tetraploid V. corymbosum from Georgia and Florida. Tetraploid V. corymbosum is the principal species upon which all highbush cultivars are based. The principal foundation stock for both northern and southern highbush blueberry breeding consisted of high-chill plants selected from the woods of New Hampshire and southern New Jersey (Coville 1937). Tetraploid V. corymbosum plants occur in abundance as far south as Alachua County in the northern Florida peninsula (Lyrene and Sherman 1981). Vigorous, productive wild highbush plants with light-blue fruit color are widespread in southeast Georgia. The Florida plants have a chill requirement of less than 400 hours below 78C. Darrow wrote in 1942: ‘‘The highbush is native to north Florida, and varieties adapted to north Florida conditions can undoubtedly be derived from selected wild plants crossed with northern sorts.’’ Darrow et al. (1939) described a large planting of V. corymbosum at Brunswick, in southeastern Georgia, dug from the wild and set in rows. Of 1,420 of these wild bushes that were evaluated on 7 May 1938 for season of ripening, 284 were early, 713 midseason, and 323 late-ripening. Curiously, when Sharpe wrote to Darrow in 1948 for advice on which Florida blueberry species to use in developing low-chill highbush blueberries, Darrow recommended that Sharpe concentrate on V. myrsinites and possibly V. caeserense and V. darrowi, but he did not mention Florida’s native low-chill V. corymbosum (Lyrene 1998). Between 1978 and 1985, Ballington (1990) made extensive collections of elite blueberry germplasm in southern Georgia, southeastern and south-central North Carolina, and southern Michigan. He observed that genes for large fruit size apparently occur throughout the range of tetraploid V. corymbosum. In 1978 and 1979, seven tetraploid highbush plants were selected from the swamps and pinelands near Gainesville, Florida, based on large berries and vigorous bushes (Lyrene 1990). These were crossed in 1980 with seven advanced selections from the Florida breeding program, and 100 seedlings were evaluated per cross. The best seedlings,


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based mainly on berry size and quality, were intercrossed and backcrossed to the cultivated southern highbush gene pool. In 1987 and 1988, we crossed 18 additional tetraploid wild V. corymbosum selections with cultivars and advanced selections from the breeding program. Eleven of these wild selections came from wet pinelands east of Valdosta, Georgia, and 7 came from the Naylor, Georgia area. The wild parents were selected during fruit ripening from large populations of wild highbush growing in southeastern Georgia. In their native habitat, these plants flowered from mid-February to mid-March and ripened during May and early June. Many of the native selections had good color, scar, and firmness and were quite large for wild blueberries. All crosses appeared to be fully compatible. One hundred wild cultivated seedlings were grown and evaluated from each of the 18 crosses. The best seedlings were backcrossed with cultivars and selections from the breeding program, and the resulting seedlings were fully fertile. Of the wild taxa we have used in breeding at the University of Florida, tetraploid V. corymbosum from northeast Florida and southeast Georgia has given the fastest return to cultivar-quality selections. 2. Vaccinium darrowi. Only one clone of V. darrowi has been widely used in breeding southern highbush cultivars, selection Florida 4b from Winter Haven, Florida (Ballington 2001). V. darrowi is quite variable in Florida due to its tendency to cross with V. elliottii in northern and western Florida and with diploid V. fuscatum Ait. (a diploid form of V. corymbosum) in central and southern Florida (Lyrene 1986). Thus, use of a wider range of V. darrowi germplasm seems important. Undesirable traits that must be eliminated after crossing with V. darrowi include small berries, short stature with many twiggy branches, a tendency to produce too many flowers, late fruit ripening (Ballington et al. 1984), and a tendency in many of the hybrids to maintain leaves through the winter and drop them in early spring at the very time they are most needed to support the developing crop. Great opportunities for selection become available when highbush blueberries are crossed with V. darrowi, because the two taxa are morphologically and physiologically very different but completely compatible genetically. 3. Vaccinium elliottii. This diploid species grows in much-restricted colonies, 2 to 4 m tall (Camp 1945). The leaves are small, deciduous, and thin-textured. The berries are small but have good texture and flavor. The species is widespread and abundant in the southeastern U.S. from southeastern Virginia to north Florida and west to southeastern Texas and Arkansas. The attractive features of V. elliottii for breeding include

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early flowering and ripening, desirable plant habit, and better adaptation to upland sites than V. corymbosum. In west Florida, wild plants of V. elliottii and V. ashei (the rabbiteye blueberry) frequently occur together on upland sites, whereas V. corymbosum is confined to river bottoms, seepage slopes, and the margins of swamps. Lyrene and Sherman (1983) crossed V. elliottii from north Florida and southwest Alabama with tetraploid highbush selections from the Florida breeding program and obtained several fertile, tetraploid hybrids. These were backcrossed to cultivated types. One backcross selection, FL 89–119, when backcrossed to the cultivated gene pool, produced a number of very early-ripening selections, including the cultivar ‘Snowchaser’, which, in years when the early flowers are not killed by freezes, ripens three weeks earlier than the major southern highbush cultivars grown in north Florida. Vaccinium elliottii has also been widely used in breeding southern highbush blueberries in North Carolina (Ballington et al. 2006). 4. Diploid Vaccinium fuscatum from Central and South Florida. Native tetraploid highbush blueberries have not been reported from Florida south of Gainesville in the north Florida peninsula, but a diploid form of highbush, called V. fuscatum by Camp (1945), continues down the peninsula in bayheads and along streams and rivers almost as far as the north end of Lake Okeechobee (Vander Kloet 1977). These plants are 2 to 4 m tall, have small, black berries, and make vigorous, fertile hybrids with V. darrowi. When crossed with improved tetraploid selections, diploid V. fuscatum produces only a few hybrids, due to the ploidy difference, but some of these are highly vigorous, upright, and have a very low chilling requirement. They have a desirable bush habit compared with hybrids of V. darrowi, which are normally low-growing and twiggy. C. Tertiary Gene Pool A large tertiary gene pool, consisting of Vaccinium species outside of section Cyanococcus, is available for potential use in breeding southern highbush, but so far, little of it has been tapped (Lyrene and Ballington 1986). All sections of Vaccinium have a base chromosome number x ¼ 12, and several sections have polyploid series that contain diploid, tetraploid, and hexaploid species. Hall and Galletta (1971) studied the chromosome structure of representative diploid species of three Vaccinium sections—Cyanococcus, Herpothamnus, and Oxycoccus—and found that length of the chromosome complement and the ratio of the longest to the shortest chromosome for the various species were quite


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similar. The sections had surprising karyotypic stability in view of the broad variability in morphology and adaptation of the species. Many intersectional crosses in Vaccinium probably cannot be made by conventional techniques. Some of the crosses that have been made have given weak hybrids, for example, V. elliottii or V. darrowi (section Cyanococus) V. stamineum (section Polycodium). Since the review of Lyrene and Ballington (1986), Vorsa (1997) has crossed two diploid species from section Cyanococcus (V. darrowi and V. tenellum) with diploid V. vitis-idaea (section Vitis-idaea). Ballington (2001) has crossed V. darrowi with diploid plants from three other sections— V. ovatum (section Pyxothamnus), V. arboreum (section Batodendron), and V. stamineum (section Polycodium). Such diploid hybrids, if vigorous, may be crossable with highbush tetraploids via 2n gametes. Diploid intersectional hybrids in Vaccinium have very low fertility, probably due to poor chromosome pairing during meiosis. In Florida, genes from V. arboreum (section Batodendron) have been moved into cultivated tetraploid southern highbush using V. darrowi as a bridge (Lyrene 1991; Brooks and Lyrene 1998a, b). The range of Vaccinium arboreum extends from North Carolina to central Florida and westward to Kentucky, Indiana, and through the Ozarks to the Edwards Plateau in Texas (Camp 1945). Vaccinium arboreum can reach the size of a small tree, in extreme cases up to 10 m tall, with 35 cm diameter at breast height (Vander Kloet 1988). An interesting feature of V. arboreum is its adaptation to xeric woodlands and dry sites such as granite outcroppings, sand dunes, pine scrub, and barren siliceous soils (Vander Kloet 1988). On well-drained soil, the root system of V. arboreum can be very deep, in contrast to the relatively shallow, netted root system that is typical of species in section Cyanococcus. V. arboreum can be planted on sites with well-drained soil that are very low in organic matter, and, once established, the plants are much more drought tolerant on such sites than highbush blueberries. There is also evidence that V. arboreum can tolerate a somewhat higher soil pH than section Cyanococcus species (Stockton 1976). The ability to grow blueberries on drier sites would simplify water management in the fields and would reduce problems with diseases of wet soils, such as phytophthora root rot. Despite much effort, Brooks and Lyrene (unpublished) were unable to make direct crosses between diploid V. arboreum and tetraploid southern highbush cultivars. However, crosses between V. arboreum and diploid V. darrowi gave large numbers of seedlings that had medium to high vigor (Brooks and Lyrene 1995). Starting several years after they were established in the field, many of the hybrids flowered copiously each year, and it was possible to grow several hundred seedlings from

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open-pollinated seed. Some of these were vigorous and moderately fertile. The most fertile were found to be tetraploid hybrids with southern highbush cultivars that had been growing in the same field. A few Florida selections that have 1/8 to 1/16 of their genes from V. arboreum have fruit quality good enough to be commercialized and retain some traits of interest from V. arboreum, such as open fruit clusters, upright bush habit, and better survival on dry sites. The fact that V. corymbosum is a polysomic tetraploid (autotetraploid) presents the possibility of developing fertile amphidiploid hybrids by crossing tetraploid V. corymbosum with polysomic tetraploids from other sections of Vaccinium. In the tetraploid hybrids, the two sets of chromosomes from V. corymbosum could pair with each other, as could the two sets from the other section. This might enable the wide hybrid to produce balanced gametes, which could confer medium to high fertility. Backcrosses of these amphidiploid hybrids to tetraploid highbush cultivars would give hybrids with three sets of highbush chromosomes and one set from the other section. Such plants might not be fully fertile, due to chromosome pairing problems, but could be used to introduce exotic genes into the highbush breeding pool. Crosses between highbush blueberry cultivars and Vaccinium uliginosum in Finland provide an interesting example of what might be achieved by crossing highbush blueberry cultivars with tetraploid species in other sections. Vaccinium uliginosum, in section Vaccinium, is a circumboreal or arctic shrub with diploid, tetraploid, and hexaploid races (Vander Kloet 1988). Rousi (1963) crossed tetraploid Finnish selections of V. uliginosum with the tetraploid highbush cultivars Rancocas and Pemberton. The hybrids showed spectacular hybrid vigor, and chromosome pairing in the F1 hybrids was surprisingly regular, 24 bivalents being the most common situation at metaphase I (Rousi 1966). Segregation of chromosomes at anaphase I was in most cases a normal 24 þ 24. The percentage of pollen grains that could be stained with acetic orcein averaged 85.7, compared to 87.3% for the V. uliginosum parent and 91.5% for the V. corymbosum parent. When the F1 hybrid was backcrossed to highbush, 199 seeds were obtained from 69 berries, and the seed germinated well. Rousi gave two possible explanations for the near-perfect pairing of chromosomes in the tetraploid F1 hybrids. Either the chromosomes of the two parent species are largely homologous or, alternatively, autosyndesis occurs in the hybrid, with V. uliginosum chromosomes pairing with one another and Cyanococcus chromosomes doing the same. The second explanation seems more probable in view of the sterility of diploid intersectional Vaccinium hybrids.


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In further studies of these intersectional tetraploid hybrids, Hiirsalmi (1977) found that despite the unexpectedly regular meiosis in the F1 hybrids, many of the berries had few or no seeds, and germination of the seeds was rather poor. When the F1 plants were backcrossed to highbush varieties, the unfavorable gene combinations were partly eliminated. In the backcross progenies, phenotypic variation was greater than in the F1 population (Hiirsalmi 1985). A selection from the backcross-1 generation was released in Finland as ‘Aron’ (Hiirsalmi 1985). Czesnik (1985) collected V. uliginosum ecotypes from various parts of Poland and crossed them reciprocally with 10 tetraploid V. corymbosum cultivars. From 16,000 pollinated flowers, he obtained 31,000 seeds and 20,000 seedlings. The V. uliginosum crosses are significant because they show how the autotetraploid makeup of highbush cultivars and the apparent ability of their chromosomes to pair autosyndetically might enable the production of fertile tetraploid intersectional hybrids that could be used to transfer genes from other sections into the highbush gene pool.

IV. ORIGINS OF HIGHBUSH BLUEBERRY BREEDING A. Northern Highbush The germplasm base in highbush blueberry is very narrow, with the nuclear genome being derived primarily from three native selections, ‘Brooks’, ‘Sooy’, and ‘Rubel’ (Hancock and Siefker, 1982; Luby et al., 1990). The breeding of northern highbush blueberries began in 1911, when Fredrick Coville of the U.S. Department of Agriculture (USDA) crossed two blueberry plants selected from the wild in New Hampshire (Coville 1937; Hancock and Draper 1989). One plant, called ‘Brooks’, was V. corymbosum. It was high-yielding and produced berries that were unusually large (up to 12 mm in diameter), and unusually light blue in color. The other selection, which he called ‘Russell’, was lowbush (V. angustifolium). It produced large berries (up to 14 mm in diameter) that were also light blue in color. ‘Brooks’, ‘Russell’, and five highbush blueberry plants selected from the wild in southern New Jersey are the only plants in the pedigrees of the 14 cultivars released by Coville up to his death in 1937 (Coville 1937). Seventeen additional highbush cultivars had been released by the USDA or USDA-North Carolina State University breeding programs by 1966, but the only additional wild selection that appeared in the pedigrees was ‘Crabbe 4’, a clone selected from the wild in North Carolina (Darrow and Scott 1966; Varney and

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Stretch 1966). Thus, through 1966, improved northern highbush cultivars were based almost entirely on seven wild highbush selections and one wild lowbush. Northern highbush cultivars released since 1966 have involved few if any additional wild gene sources. B. Southern Highbush Northern highbush cultivars from New Jersey were tested in northeast Florida during the 1930s and were found unsuited for cultivation because they received insufficient cold to break the rest period of the plant (Anon. 1941). Two northern cultivars, ‘Pioneer’ and ‘Concord’, were fairly vigorous and productive in northeast Florida, but harvest was not complete until the last week in July (Anon, 1941). Early work to develop highbush blueberries adapted to the southeastern U.S. began in North Carolina (North Carolina State University), in the USDA (in conjunction with experiment stations in Georgia, Florida, and Mississippi), and at the University of Florida. Northern highbush plantings in southeastern North Carolina during the 1930s indicated that most cultivars received enough chilling to do well, and the berries ripened about four weeks earlier than in New Jersey (Mainland 1987). By 1950, nearly 400 ha of commercial highbush blueberries had been planted on the coastal plain in North Carolina, and crop area was rapidly increasing (Eck and Childers 1966). Stem canker, caused by the fungus Botryosphaeria corticis (Demaree and Wilcox) Arx and Muller, was first observed in a cultivated field in North Carolina in 1938 (Varney and Stretch 1966). All major varieties of northern highbush were more or less susceptible to the fungus, and over a period of years were usually killed (Moore 1966). By 1950, cane canker and other diseases were devastating fields of northern highbush in North Carolina (Moore 1966). Resistance to cane canker disease was introduced into highbush blueberries in North Carolina using a wild highbush selection from North Carolina, ‘Crabbe 4’. In 1934 a cross was made between F6 (‘Wolcott’ ‘Crabbe 4’) and the northern highbush cultivar ‘Stanley’ (Anon. 1997). This cross produced what can be considered the first four southern highbush cultivars, ‘Murphy’ and ‘Wolcott’ in 1950, ‘Angola’ in 1952, and ‘Croatan’ in 1954 (Darrow 1960). ‘Croatan’ remained the most important blueberry cultivar in North Carolina for more than 40 years. The North Carolina southern highbush breeding program has remained very active. Cultivars ‘O’Neal’ and ‘Reveille’ have been important in low-chill production areas throughout the world. Numerous V. corymbosum selections from North Carolina have been used in the breeding


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program, along with many interspecific hybrid selections (Ballington 2001; Ballington et al. 2006). Rabbiteye blueberries, based on hexaploid V. ashei, are native and well adapted in the southeastern U.S., and improved rabbiteye selections from the wild were being cultivated in Georgia and north Florida by 1940 (Lyrene 1987). It was recognized that the late ripening of rabbiteye cultivars was a major disadvantage, because it meant that the berries were harvested at the same time as northern highbush berries from North Carolina and New Jersey. Rainy, high-humidity weather in Florida and southeast Georgia during the rabbiteye harvest season from June through August is another problem with late-ripening berries. The potential advantages of having low-chill highbush blueberries that could be grown farther south motivated Darrow of the USDA to produce synthetic lowchill tetraploid hybrids that could be crossed with northern highbush cultivars to be used in breeding low-chill highbush (Darrow et al. 1949). Darrow’s plan was to cross diploid low-chill species with hexaploid rabbiteye selections to obtain tetraploids. Over the years, a number of such hybrids were produced and used in breeding with tetraploid highbush. Some of the hexaploid diploid hybrids were later found to be pentaploid rather than tetraploid (Goldy and Lyrene 1984), but the pentaploids produced numerous seedlings when crossed with tetraploid highbush. ‘Hildebrant’, a selection of southern highbush from southeastern Georgia, and other V. australe selections from the southeastern United States were crossed with northern highbush cultivars and selections were made at Tifton, Georgia (Brightwell et al. 1955). Some of these selections were later used as parents in the Florida breeding program. Southern highbush blueberry breeding began in Florida about 1950 with the goal of developing early-ripening, low-chill varieties (Sharpe and Darrow 1959; Lyrene 1998). The first Florida blueberry breeder, Ralph Sharpe, noted that some of the highbush blueberry cultivars that had been bred in New Jersey ripened 60 days after flowering, compared to 100 days or more for the rabbiteye blueberry seedlings then being grown in Florida (Sharpe and Darrow 1959). It was known from earlier work at the USDA in Washington, D.C. that crosses could easily be made between Florida’s tetraploid lowbush blueberry, V. myrsinites L., and cultivated northern highbush blueberry (Darrow et al. 1952; Darrow 1960). Hybrid seedlings of this type, evaluated at Ivanhoe, North Carolina, and at Beltsville, Maryland, were highly vigorous, but the berries were small, dark, and late ripening. Similar highbush cultivar V. myrsinities hybrids were evaluated at Tifton, Georgia, in the late 1940s and at Gainesville, Florida, in 1952. The seedlings were not promising at either location (Sharpe 1954). In Georgia they produced

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small dark fruit on low-growing bushes (Brightwell et al. 1955). When the F1 hybrids were backcrossed to northern highbush cultivars, the chilling requirement was increased so much that most of the seedlings showed delayed foliation in average seasons at Tifton. In Florida, the fruit were dull black and unattractive, and growth was badly delayed, not starting until after 15 April, while adapted wild blueberries were in growth by 15 February (Sharpe and Darrow 1959). Later crosses between cultivated highbush and V. myrsinites were made and studied by Draper (1997). He found that seedling populations were productive and well adapted to southern environments, but they were too short to be easily machine harvested, and seedling populations did not segregate for height. In 1951, Sharpe collected 31 clones of diploid V. darrowi from Winter Haven, Florida (Sharpe and Darrow 1959). Darrow et al. had already shown that tetraploid hybrids compatible with northern highbush cultivars could be produced by crossing low-chill diploid species with the low-chill hexaploid, V. ashei (Darrow et al. 1949, 1954). Thirty-three tetraploid hybrids had been produced using the diploid V. tenellum and 70 using diploid V. atrococcum (a wild highbush closely related to V. corymbosum). V. darrowi proved to be much harder to cross with V. ashei. From over 7,500 pollinations, only five hybrids were obtained (Sharpe and Darrow 1959). Although two of the hybrids were male sterile, all five had excellent fruit and seed set when crossed as females with northern highbush. The fruit of the hybrids required 119 days from bloom to maturity in the greenhouse, very similar to the parents. Fruit size was intermediate, ranging from 11 to 13 mm. Fruit color was dark to medium blue. Direct crosses between diploid V. darrowi and tetraploid northern highbush were not attempted at first because Sharpe and Darrow expected such crosses to produce only sterile triploid hybrids. When such crosses were tried, they proved much easier than the V. darrowi V. ashei cross, and 31 tetraploid hybrids were obtained by pollinating 1,600 flowers, apparently as a result of unreduced gametes from the diploid parent (Sharpe and Sherman 1976c ). All V. darrowi germplasm used in breeding southern highbush up to 2001 was collected by Sharpe (Ballington 2001), and almost all of the crosses that eventually led to cultivars involved the one selection, V. darrowi Florida 4b. In Florida, initial interspecific hybrids involving V. darrowi were followed by multiple generations of backcrosses and intercrosses. Selection was concentrated on low chilling requirement, early ripening, large berries, high berry quality, high yield, and good survival of the bushes in the field. By 1970, Sharpe and Sherman had fruited over 120,000 tetraploids seedlings in Florida. The first southern highbush cultivars from Florida


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were ‘Sharpblue’ and ‘Flordablue’. introduced in 1976 (Sharpe and Sherman 1976 a, b). In breeding southern highbush blueberries, Sharpe practiced rapid-cycle phenotypic recurrent selection. ‘Flordablue’, released in 1976 and ‘Avonblue’, released in 1977, were already four generations removed from their most recent V. darrowi parents (Sharpe and Sherman 1976a; Sherman and Sharpe 1977). In 1971–72, A. D. Draper of the USDA at Beltsville, Maryland, crossed V. darrowi Florida 4b with northern highbush cultivars and obtained the hybrid selections US 74 and US 75 (Draper 1997; Ehlenfeldt et al. 1995). These were backcrossed to northern highbush selections. From the backcross populations were selected ‘‘Blue Ridge’ (from US 74) and ‘Cooper’. ‘Gulfcoast’, ‘Georgiagem’, ‘‘Cape Fear’’, ‘Sierra’, and ‘Legacy’ (from US 75). The breeding program at North Carolina State University has produced a large number of tetraploid selections derived from crosses of cultivated highbush with a wide diversity of wild diploid selections, most important of which were V. darrowi and V. elliottii (Ballington et al. 2006). Although North Carolina receives enough chilling to grow northern highbush blueberries, most of the effort in the North Carolina breeding program in recent years has been with southern highbush improvement, due to the superior adaptation and fruit quality southern highbush has shown in North Carolina. Vaccinium darrowi, V. tenellum, and V. elliottii have been used extensively in breeding southern highbush in North Carolina (Ballington et al. 2006). Of eight cultivars originated by the North Carolina program since 1977, five are BC2 from V. darrowi V. corymbosum to V. corymbosum, one resulted from an intercross of a BC3 with a BC1 and involves V. darrowi, V. corymbosum, V. tenellum, and V. virgatum, one is a BC1 from V. elliottii V. corymbosum to V. corymbosum, and one is a pentaploid derived from (V. corymbosum V. myrtilloides) V. virgatum (Ballington et al. 2006). Two new southern highbush cultivars, ‘Camellia’ and ‘Rebel’, have recently been released by the University of Georgia (NeSmith 2006). These are adapted to the 400- to 500-hr chill zone. The USDAbreeding program at Poplarville, Mississippi, has also recently released two new southern highbush cultivars, ‘Dixieblue’ and ‘Gupton’ (Stringer et al. 2006).

V. METHODS A. Basic Procedures 1. Making Crosses and Growing Seedlings. Southern highbush blueberries root readily from softwood or hardwood cuttings, can be

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maintained for years in pots, and are precocious, flowering when they are one to two years old from seed. The flowers are easy to emasculate and give 60 to 90% fruit set after compatible hand pollinations in the greenhouse. Hand-pollinated berries produce 20 to 40 viable seeds per pollinated flower. Seeds germinate readily, and seedlings are easy to grow in trays and survive at high rates if transplanted to field nurseries. Flowering times can be synchronized by moving plants between cold boxes and greenhouses. Dried seeds can be stored for at least 20 years in a home refrigerator with little loss of viability, and pollen remains viable for at least a year if dried and frozen. All of these factors make blueberries a cooperative plant to breed and account in part for its rapid transition from a wild forest species to a major world crop. The need to asexually propagate superior seedlings for advanced testing slows the evaluation process, but the potential rate of multiplication by vegetative propagation is high. Starting with one 12-cm softwood cutting in early May, it should be possible to generate at least 10,000 rooted cuttings by September, 2.5 years later. In vitro micropropagation is often used for commercial propagation of released cultivars, but is not much used by breeders, who need to propagate large numbers of clones for testing but only a few plants per clone. Details on methods for collecting and storing pollen, emasculating and pollinating flowers, extracting and germinating seeds, transplanting and field evaluation of seedlings have been reviewed by Moore (1965, 1966), Galletta (1975), and Lyrene and Moore (2006). Making crosses in an insect-proof greenhouse eliminates the need to remove flowers that are not pollinated.Although many cultivars are partially self-compatible, any berries that ripen from unpollinated flowers in a greenhouse from which bees have been excluded ripen much later than hand-pollinated flowers and usually contain very few seeds. 2. Field Testing Procedures. Evaluation of seedlings from crosses has two objectives: identifying superior parents for the next round of crosses and finding superior clones that can be used for berry production in commercial plantings. Although the two goals have much in common, they do not coincide exactly. Seedlings that look extremely good in field plots in their first or second fruiting season may be used as parents, but many additional years of testing are needed before they can be trusted on thousands of commercial hectares. Test selections having many desirable features but no cultivar potential, because of one or two serious flaws, may be useful parents. When cultivars are crossed with wild germplasm, there is little expectation that cultivars will be found in the first generation, but parents can be selected for further crosses.


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For most breeders, initial roguing of seedling populations is based mainly on fruit size, color, and quality. Initial roguing is sometimes done on potted seedlings, but more commonly in field plots. At the University of Florida, a high-density system is used for first evaluation of seedlings, in which 10,000 to 15,000 seedlings from seed sown the previous November in a greenhouse are transplanted in May to a 0.2-ha field nursery (Sherman et al. 1973). One year after being transplanted, the seedlings fruit for the first time, and all but 1,000 are dug and discarded, mostly because of undesirable berry characteristics. The 1,000 retained seedlings (now called the Stage II test) are left in place for three more years. Each year they are examined during fruit ripening for possible advance to a third level of testing (Stage III). Approximately 300 selections per year are advanced to the Stage III test, which consists of one 15-plant plot for each clone. Plants used to establish these plots are propagated by softwood cuttings from the mother Stage II plant. The Stage III plots, which are managed as recommended for commercial plantations in Florida, are maintained for approximately 10 years. They are observed throughout the year, but particularly during flowering and ripening. During the 10-year life of a Stage III planting, approximately 15 clones are selected for planting at multiple locations in larger plots (Stage IV), and from the Stage IV plots, cultivars are selected for release, on average, about one cultivar from each Stage IV test. During the first years of observation, when evaluations are based on a single plant, berry characteristics are the principal basis for selection, although plant health, vigor, and adaptation are important to the extent that they affect berry quality. As plots become larger and plants older in advanced testing stages, the focus of selection becomes plant vigor and survival, disease resistance, plant architecture, reliability of yield, time of harvest, picking speed, and postharvest properties of the berries. Few breeding programs use the high-density screening nurseries described above. More commonly, seedlings are planted at wider spacing, which permits initial evaluation of larger plants that have more berries and give a better indication of plant architecture and yield potential. Breeders use various methods for identifying the few best seedlings from their large seedling populations, but all use some type of sequential testing scheme, in which seedlings with clear defects are quickly eliminated and the best seedlings are planted in replicated plots observed over multiple fruiting seasons. 3. Screening for Disease Resistance and Other Characteristics. Since large segregating populations of blueberry seedlings can be produced easily, the ability to screen seedlings at an early age for adaptation and

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resistance to pests and diseases would be a great advantage. Chandler et al. (1985a) used one highbush blueberry clone and three interspecific hybrid clones in a diallel set of crosses and screened the resulting seedlings for their ability to grow on a fine sandy loam soil low in organic matter. They found that V. ashei, V. atrococcum, and V. darrowi contributed to upland adaptation. They also found that it was very hard to eliminate soil variations, even within a small test field. In North Carolina, blueberry selections have been screened in pots for resistance to cane canker and stem blight (Botryosphaeria dothidea (Moug.:Fr.) Ces. & De Not). With cane canker, the usefulness of screening was reduced by the existence of many races of the pathogen and by the poor correlation between greenhouse results and field survival. Mummy berry susceptibility was evaluated after inoculation of blueberry flowers in New Jersey (Ehlenfeldt and Stretch 2002). B. Population Improvement by Recurrent Selection Because blueberry cultivars are clones, all that is needed to produce a new cultivar is a single superior seedling. However, to get the best cultivars, the quality of seedlings in the segregating populations needs to be as high as possible. To get better seedlings, it is necessary to breed better parents. Inbreeding depression is strong in blueberries. Seedlings from self-pollination of highbush cultivars are quite weak compared to outcrossed seedlings. The best way to develop better parents is through long-term recurrent selection in which the frequency of beneficial alleles for the numerous traits important in blueberry are systematically increased generation after generation, while a broad genetic base is maintained and inbreeding is minimized (Lyrene 1981). Shortening the time required to complete each cycle of selection will maximize the rate of progress. In the Florida program, approximately one-third of the parents used in each generation of crossing are three years old from seed. An additional one-third are four to six years old from seed. To minimize inbreeding depression and genetic drift, a large number of parents is used in each generation. In Florida, approximately 150 different clones are used each year in crosses. In selecting parents, traits with high heritability, high economic importance to the grower, and high variability in the population are emphasized. C. Test Crosses and Proven Crosses Specific combining ability is high in blueberries with respect to some important traits. Some cross combinations produce high frequencies of


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weak or dwarf seedlings. Large populations of seedlings from some crosses are uniformly rejected because of bad picking scar or stems that remain attached to the berries after picking. In some crosses, all seedlings produce dark fruit. When large numbers of crosses are made using new parental combinations, some combinations will be found to produce exceptionally good seedlings. In sugarcane, these are called ‘‘proven crosses.’’ Breeders debate whether it is better to make numerous crosses with few seedlings grown per cross or fewer crosses with more seedlings grown per cross. The ‘‘proven cross’’ method involves some of both. A small number of seedlings is grown and evaluated from each of a large number of new test combinations. Based on the performance of these seedlings, the best crosses are identified and repeated on a much larger scale. Good seedlings from both test crosses and proven crosses are further evaluated for cultivar potential. How many seedlings are needed to exploit the possibilities of a good pair of parents? The answer probably depends on the cross. Seedlings from some crosses are much more variable than those from others. From a good cross, 1,000 seedlings or more could profitably be evaluated without exhausting the possible combinations. Seedlings that look quite similar in small plots may later show large differences in subtle traits such as yield potential or resistance to various diseases. D. Heritability of Characters Broad-sense heritability, or repeatability, quantifies the relative importance of genetic variation versus environmental plus error variation for various traits. High repeatability over years and locations makes a trait easier to improve by breeding. Repeatability can be increased by taking into account or minimizing various nongenetic causes of variation, because some characteristics change in predictable ways when the environment changes. Berries become softer and scars are more likely to tear or bleed juice as the berries get riper. Berry acidity falls and percent soluble solids rises as ripe berries are allowed to hang longer on the plant. Ripe berries are softer on hot days than on cool days. Berry size is reduced by heavy crops, poor pollination, and drought stress. Berry color becomes darker and percent soluble solids lower if rain is frequent just before and during ripening. By taking into account environmental factors that affect fruit characteristics, breeders can more accurately identify plants with superior genetics. In a study of character repeatability over two years in a population of 200 southern highbush seedlings in Florida, Edwards et al. (1974) found that fruit size, picking scar, and plant vigor were more repeatable over the two years than fruit color and firmness.

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Edwards et al. (1974) calculated narrow-sense heritability in low-chill highbush blueberries in Florida by regressing progeny means on midparent phenotype. Berry size had the highest heritability, followed by fruit color, fruit firmness, and fruit scar, all of which had heritabilities between 0.29 and 0.58. Plant vigor was not heritable in their study. In a study of the inheritance of fruit quality components in a partial diallel mating scheme involving 17 V. corymbosum, V. angustifolium, and half-high (V. corymbosum V. angustifolium hybrids) parents, Finn and Luby (1992) found that general combining ability mean squares were significant for all traits (berry color, firmness, size, and pedicel attachment scar) but specific combining ability was not significant. In highbush blueberry crosses in which one or both parents had light-blue fruit, many progeny had light-blue fruit. For date of 50% bloom, date of 50% ripe, length of the fruit development interval, and berry weight, additive genetic variance was more important than nonadditive genetic variance, based on general combining ability variance components. The authors stated that in these populations, parental phenotype should be indicative of relative progeny performance. Heritability estimates for these traits were between 0.44 and 0.78. Finn et al. (1993) grew 33 seedling populations from crosses involving V. corymbosum, V. angustifolium, and half-high parents on three types of soil (pH 5.0, 5.5, and 6.5) and found that seedlings from some crosses grew significantly better than others, but there was no population soil pH interaction. Draper and Scott (1969) found that fruit size in the progeny of three crosses of large-fruited large-fruited parents averaged significantly larger than in the progeny from large-fruited small fruited parents. Mean fruit size of the progeny from crosses between large fruited small fruited parents was equal to the fruit size of the small-fruited parent, indicating that small fruit size was dominant. Breeder experience indicates that several important berry traits in blueberry have high heritability. Berry size, color, scar, firmness, and flavor all show strong parental influences when seedlings from many crosses are compared. This indicates that southern highbush breeding populations retain high genetic variability for berry characteristics, and it predicts continued progress if breeding work continues. E. Major Genes in Southern Highbush Blueberry Known genes having two or more alleles whose phenotypes are so distinct that plants in a segregating population can be divided into discrete categories are few in highbush blueberry. This is probably so


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because blueberry breeding has been done by so few workers for such a short time and because inbreeding, which might reveal rare recessive alleles, is avoided in blueberry cultivar development. Crosses that segregate albino seedlings are not uncommon in northern (Draper and Scott 1971) and southern highbush. Profoundly dwarf plants, with large numbers of short canes, segregate from many southern highbush crosses (Draper et al. 1984; Baquerizo 2005). Highbush plants with albino fruit were reported by Coville (1937). A gene that profoundly alters leaf, stem, and bud anthocyanin was found in the diploid species V. elliottii and transferred to tetraploid southern highbush populations (Lyrene 1988; Wenslaff and Lyrene 2003). As more breeding is done with southern highbush and as levels of inbreeding increase, additional major genes will undoubtedly be found. To date, the intentional use of classical Mendelian genes has not been very important in blueberry breeding. F. Incorporating Additional Wild Germplasm A wealth of germplasm with potential value in breeding blueberries is present in the wild blueberries of Vaccinium section Cyanococcus in eastern North America. This germplasm will be needed in the future to broaden the genetic base of the cultivated blueberry, to solve new problems that arise in production, and to take advantage of new marketing opportunities and production methods. Methods have been developed for using this germplasm, including ways of transferring genes among diploid, tetraploid, and hexaploid species. The greatest problem in using wild material is that after crossing cultivars with unimproved plants, several generations of backcrosses and selection are needed to obtain plants that can compete as commercial cultivars. As cultivars get better and better, the path from the forest to the farm gets longer and longer. Ballington et al. (2006) summarized the results of many years of interspecific hybridizing to produce plants with attributes desired in North Carolina. Species used for breeding southern highbush included V. darrowi, V. corymbosum, V. virgatum, V. elliottii, V. myrsinites, V. tenellum, V. pallidum, V. simulatum, V. hirsutum, and V. angustifolium. Major objectives in using the wild material included resistance to stem blight, cane canker, and root rot (Phytophthora cinnamoni Rands); increased tolerance to low-organic soils; improved heat tolerance; improved berry quality (light-blue color, increased firmness, increased acidity); better plant architecture; and adaptation to mechanical harvest. In Michigan and North Carolina, wild cultivated highbush crosses have produced firm, small-fruited genotypes that yield well and are

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adapted to mechanical harvest (Ballington 2001). In the North Carolina State University breeding program, native V. corymbosum from southern Michigan and central Pennsylvania has been a particularly valuable source of genes for late flowering combined with early fruit maturity. Hybrids of highbush cultivars with V. elliottii in North Carolina have been extremely vigorous, with broad soil adaptation, but generally lack sufficient fruit quality to be cultivars (Ballington 2001). The F1 hybrids flower very early. Backcross-1 hybrids in North Carolina retain broad soil tolerance and produce midsize, high-quality fruit and are adapted to machine harvesting. V. elliottii has also proved a southern source of genes for resistance to stem blight (Ballington 2001).

VI. BREEDING OBJECTIVES A. Plant Characteristics I will divide breeding goals into plant characteristics and berry characteristics, although the division is arbitrary, because plant characteristics such as vigor, crop load, disease resistance, and ability to leaf well in the spring affect berry characteristics such as size, color, flavor, and time of ripening. 1. Chilling Requirement. Once highbush blueberries enter winter dormancy, a period of low temperatures is required to release the buds from dormancy (Spiers et al. 2006). This was first shown by Coville (1916), who kept a northern highbush blueberry plant in a warm greenhouse through the winter in Washington, D.C., except for one branch that he extended through a hole in the greenhouse to the outside. In the spring, the branch that had been exposed to cold through the winter flowered and produced new leaves normally, whereas the part of the plant that was inside the greenhouse remained dormant late into the spring, and flowered and leafed unevenly and over a long period of time. Blueberry clones differ in chilling requirement from zero to over 1,000 hr (08 to 78C). Native highbush blueberry plants collected from the forest from North Carolina to Canada have a high chilling requirement. Darrow (1942) found that 13 northern highbush cultivars he tested varied somewhat in chilling requirement. Although they grew better after 800 hr of chilling, ‘Weymouth’, ‘Rancocas’, ‘Wareham’, and ‘Cabot’ produced many new shoots after only 650 hours of chilling. Wild highbush blueberry plants collected from south of latitude 28 in the Florida peninsula have little or no chilling requirement. Clones from


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intermediate zones have chilling requirements that best serve plants growing in those areas. Quantifying the chilling requirement of a blueberry clone is difficult. In general, the longer a blueberry plant is chilled at 08 to 78C, the faster and more completely the buds will come out of dormancy when the plant is placed in a warm greenhouse. The chilling requirement of a clone has been satisfied when additional hours of chilling produce little additional improvement in the speed and completeness of bud break. Blueberry cultivars vary greatly in the number of heat units required to induce full bloom after chilling has been satisfied. Vaccinium ashei cultivars such as ‘Brightwell’ and ‘Powderblue’ consistently flower quite late in north Florida. This lateness could not be due to a high chilling requirement, because these cultivars fruit heavily and reliably in north Florida, even after the mildest winters. Thus, determining the relative chilling requirement of clones by noting the sequence of flowering in the spring, a method used in peaches, does not work well with blueberries, where flowering time is influenced both by chilling requirement and heat unit requirement (Lyrene 2005). A functional definition of the chilling requirement of a clone is given by answering the question: ‘‘What is the mean winter temperature (December, January, and February in the northern hemisphere) of the warmest area where the clone is commercially successful in the field?’’ This can be determined only by field testing over a number of years, and the results often differ markedly from laboratory tests of chill requirement. The reaction to insufficient chilling varies greatly for different clones. When grown in fields in north Florida (where the average winter provides about 400 chill hr), well-adapted clones have a short period of flowering, with all flower buds opening during a week or two, and strong vegetative bud break at or near the time of flowering. After winters with less chilling than normal, some clones flower normally but are slow to initiate vegetative growth. Others leaf fully at the normal time but abort all or many of their flower buds. A few clones are much delayed in both leafing and flowering, but eventually flower and leaf vigorously and produce normal crops, but late. Northern highbush cultivars, when grown in pots outside in north Florida, normally abort all flower buds and begin vegetative growth two to three months after the flowering of locally adapted wild highbush. By late summer, however, the plants are normally growing well and producing healthy flower buds for the next year. Mainland (1985) described situations in southeastern North Carolina in which certain highbush blueberry cultivars in some years leaf poorly

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and sporadically even after receiving as much as 1,294 hr of chilling. Inadequate leaf surface caused by little or no bud break on a fruiting cane is sometimes a problem in North Carolina, even in years when chilling is adequate (Mainland 1985). Drought, inadequate pruning, and a heavy bloom and fruit set appear to make the problem worse, but temperatures during bud break seem to be the critical factor. Warm temperatures during bud break cause flower bud development to begin before leaf bud development. In some years, in both North Carolina and Florida, some highbush cultivars approach full bloom with only scattered leaf development. In some years, blueberry gall midge (Dasineura oxycoccana Johnson) exacerbates the problem by killing the apical meristems of newly emerging shoots (Lyrene and Payne 1995). Fruit that mature on poorly leafed bushes tends to be small and late (Mainland 1985). Cultivars vary greatly in their susceptibility to spring leafing problems. In areas with mild winters, such as Florida and southeastern Georgia, dormancy-breaking compounds such as hydrogen cyanamide may be sprayed on southern highbush blueberry plants during early to midwinter to improve flower bud break and leafing in late winter (Williamson et al. 2001, 2002). Some clones respond to these sprays with greatly improved bud break, whereas other clones show much less response. Furthermore, the flower buds of some clones are easily damaged by hydrogen cyanamide, whereas buds of other cultivars are far more resistant. Where chemical sprays are used to improve bud break, breeding for clones that respond favorably is important. Chilling requirement is highly heritable. High-chill parents strongly tend to produce high-chill progeny. Low chilling requirement appears to be partially dominant in the F1 generation, and intercrosses between high-chill low-chill F1 clones gives strong segregation for chilling requirement. 2. Cessation of Growth in the Fall. Northern highbush blueberries, when grown in Florida, stop producing new vegetative flushes and enter dormancy much earlier in the fall than native Florida blueberries. In order to take full advantage of the long growing season at low latitudes, cultivars need to continue growing at least through September. Plants that stop growing and initiate flower buds too early in the fall tend to lose vigor over a number of years. On the other hand, plants that grow too late into the fall are subject to winter injury. To some extent, this problem can be minimized by reducing nitrogen fertilization earlier in the fall. However, lack of winter hardiness limits the utility of low-chill highbush cultivars in some areas (Hancock 1998, 2006).


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3. Flower Bud Initiation. Flower bud initiation in southern highbush blueberry is promoted by short days and cool nights (Banãdos and Strik 2006). The number of flower buds initiated depends on the cultivar as well as the environment (Darnell and Williamson 1997). Genotypes that readily make large numbers of flower buds are needed for areas where fall temperatures are warm and autumn days are long relative to higher latitudes. Selections that are well adapted in south Florida commonly produce too many flower buds when planted in north Florida. They require extensive winter pruning to prevent over fruiting if grown in the north. Clones that produce the optimum number of flower buds in north Florida frequently produce too few buds in south Florida. Leaf diseases that prematurely defoliate shoots in the fall reduce flower bud formation. Genetic resistance and cultural methods of control are necessary to ensure enough flower buds for heavy crops. 4. Time of Flowering. It is important that blueberries flower at the right time of the year. In some locations, such as on the central coast of California, southern highbush blueberries flower excessively in the fall. Fall flowers may freeze during the winter or encounter unfavorable conditions for pollination and berry development. In north Florida, some low-chill blueberry cultivars are prone to heavy flowering in December and early January if good chilling conditions in November are followed by above-normal temperatures in December and January. Hard killing freezes can occur in north Florida throughout January and February. Florida growers want to harvest during April and early May, so they plant early-flowering cultivars. Overhead irrigation is used to protect flowers and berries from freezes after 1 February, but January flowering is highly undesirable. Although the timing of blueberry flower bud swell in winter and early spring is highly influenced by the timing and extent of chilling and subsequent warm-up, it is possible to select low-chill clones that resist premature flowering. That this is possible is indicated by the fact that Vaccinium darrowi, the main source of low chilling in southern highbush blueberry, normally does not flower in the wild in Florida until mid- to late February. Efforts have been made to select low-chill, late-flowering clones that avoid spring freezes. This can be done, as evidenced by rabbiteye cultivars such as ‘Powderblue’ and ‘Brightwell’, which flower quite late in north Florida but consistently produce heavy crops. With highbush blueberries, the cultivar ‘Legacy’ comes closest to this goal in north Florida. In the field, selecting for late-flowering after unusually cold winters tends to give clones with a higher chilling requirement. These clones may do poorly after warmer winters.

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5. Time of Ripening. Time of ripening in blueberry depends on time of flowering and on the many factors that determine the length of the flowering-to-ripening interval. The flowering-to-ripening interval is very sensitive to temperature, being shortest when nightly minimum temperatures during the fruit development period are 208C or above and daily maximums are about 328C. Other factors that shorten the fruit development period include high seed content in the berries, light crop load, and strong, early leafing. Mainland and Bland (1998) compared blossom to ripe-berry intervals for eight highbush cultivars over three years in southeastern North Carolina. ‘O‘Neal’, an early cultivar, had a blossom to ripe-berry interval of 50 days in 1995 and 62 days in 1996. For ‘Elliott’, a late-ripening cultivar, the values were 73 days in 1995 and 63 days in 1996. Most low-chill highbush cultivars have been selected for early ripening and are intended to ripen before high-chill cultivars grown in colder areas. Biologically, there is no reason why late-ripening lowchill cultivars could not be developed to extend the harvest season in low-chill growing areas. 6. Vigor and Carrying Capacity. Some blueberry clones grow well, survive well, and produce high-quality berries only if a high percentage of the flower buds are pruned off each winter, substantially reducing the yield potential. Other clones can carry to maturity large numbers of berries without damaging the bush, but their berries are much smaller when the crop is heavy. The best clones can produce large numbers of berries without weakening the plant or greatly reducing berry size or quality. Winter pruning, which reduces flower bud number, is used in blueberry to adjust the crop load to the carrying capacity of the cultivar. Cultivars that perform well only with light berry loads are of little value. 7. Plant Architecture. Two contrasting plant architectures are in the genetic background of southern highbush blueberry. One, represented by V. darrowi and by northern lowbush blueberry (V. angustifolium) (whose influence came into southern highbush via northern highbush), is lowgrowing and colonial. Individual stems are typically less than 30 cm tall. Individual seedlings spread by rhizomes, forming colonies consisting of hundreds of shoots, sometimes covering up to 0.1 ha. The second plant type is that of V. corymbosum, which is upright, has only a few major canes, and reaches a height up to 4 m. Various combinations of genes from these two plant types create a wide range of plant architectures in segregating populations. The V. corymbosum plant type has been favored in selection of southern highbush cultivars, because the plants are easier to prune, less likely to sprawl under the influence of a heavy crop, and


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tend to have fewer, larger berries. Mechanical harvest is easier when plants have a narrow base and relatively few canes. A major gene that produces vigorous, dwarf plants is present in both northern and southern highbush blueberry (Draper et al. 1984). More than 10% of the Florida crosses segregate vigorous dwarfs at the rate of 10 to 50% (Baquerizo 2005). Open flower clusters result from long pedicels and peduncles. Open clusters are desired because large berries in tight clusters are hard to pick. Berries may become deformed if the cluster is so tight that berries press together as they develop. In tight clusters, it may be hard to remove ripe berries without also removing immature berries. Tight clusters are not conducive to mechanical harvest. Vaccinium arboreum has been a good source of open clusters, but there is also much variation in cluster tightness in the primary gene pool. Response to pruning is an important and highly variable trait. Southern highbush are often pruned twice per year. Summer pruning is done immediately after harvest is complete, often by mechanically hedging the tops and the sides. Winter pruning involves removal by hand of some of the older, larger canes at the base and removal of weak internal branches bearing large numbers of small flower buds. Annual or alternate-year summer pruning is possible with southern highbush because harvest is complete early in the growing season. Nearly six months remain between the end of harvest and the first frost. This leaves plenty of time for the plant to grow a new canopy above the hedging level, for the wood to mature, and for flower buds to form that will open after the following winter. The purpose of hedging is to keep the plants from becoming too tall for easy harvest, to reduce cane diseases that may become established in senescent wood that has fruited, and to help control pests such as blueberry bud mite (Aceria vaccinii Keifer). The height of hedging is 120 to 150 cm aboveground. Some culitvars are slow to regrow after summer hedging, whereas others regrow rapidly and profusely. In some cultivars, hedging stimulates an abundance of new shoots from the base of the plant. In others, most of the regrowth is from the tops of the stumps, just below the cuts. 8. Drought Tolerance and Tolerance to Higher Soil pH. Wild highbush blueberries are adapted to moist, acid, sandy soils with excellent drainage and high organic matter content. These conditions are hard to reproduce in areas where they do not naturally occur. In some areas, restrictions on draining wetlands prevent new plantings of blueberries on ‘‘natural’’ highbush soil. Planting highbush blueberries on upland locations usually requires either the incorporation of organic matter into

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the soil or the use of organic mulches. Irrigation, drainage, and soil pH control require careful management on upland soils, and added organic matter oxidizes over time and may need to be renewed. Various sources of drought resistance have been used in breeding southern highbush cultivars. Of these, V. elliottii and V. ashei have been important. Vaccinium darrowi, which survives well on some dry upland sites, owes its drought resistance to having very small leaves and a growth habit that maintains most of its biomass underground. The extent to which V. darrowi genes can contribute to better drought tolerance in southern highbush is unknown, but may be slight. The most drought-tolerant Vaccinium species in the southeastern U.S. is V. stamineum, which produces large plants with large leaves and large fruit on forested sites with excessively drained deep sandy soils. Unfortunately, hybrids between V. stamineum and species in section Cyanococcus have not been vigorous. V. arboreum is perhaps a more promising source of improved drought tolerance. Its hybrids with V. darrowi can be highly vigorous, and using V. darrowi as a bridge, genes from V. arboreum have been transferred to southern highbush tetraploids (Lyrene 1991; Brooks and Lyrene 1998a, b). The root system of V. arboreum is quite different from that of section Cyanococcus blueberries. On deep, well-drained soils, it produces a thick, deep taproot, in contrast to the spreading, netted root system of section Cyanococcus blueberries. In selecting for improved drought tolerance, it is important that the genotypes being evaluated be grown on deep, welldrained soils, so deep roots can develop on clones that have the genetic potential to make them. Highbush blueberries typically grow best when soil pH is below 5.5. Micronutrient availability, soil texture, and soil organic matter content will determine whether or not blueberries can be grown successfully when soil pH is marginally high. In some areas, such as the central valley of California, the pH of high-pH sandy soils can be lowered before planting using sulfuric acid, and southern highbush blueberries have grown well on such sites if the pH of soil and irrigation water is kept low using acid injection systems. Good tolerance of high soil pH has not been found in any of the Cyanococcus blueberry species. Soil samples from sites where V. arboreum grows wild indicate that it can sometimes grow well where soil pH is as high as 6.0 (Stockton 1976). Scheerens et al. (1999a, b) compared V. corymbosum cultivars and a wide diversity of interspecific hybrids for their ability to grow on Wooster Silt Loam soil in Ohio. They developed a ‘‘mineral soil adaptation index,’’ based on plant vigor, nutrient deficiency symptoms, and the ability to balance fruit load with vegetative growth. They compared


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13 groups of clones, the groups defined according to which Vaccinium section Cyanococcus species were predominant in their pedigrees. The group that consisted of the V. corymbosum cultivars ‘Bluecrop’, ‘Jersey’, and ‘Spartan’ had a median value: six groups averaged higher scores and six lower. The high variation among individual clones, both within and among groups, indicated that progress for upland adaptation could be made by recurrent selection. Soils that do not support vigorous growth of highbush blueberry constitute a diverse group. For some, the pH is too high. Some are too low in organic matter, some are too wet, some too dry. Some are fine textured and do not provide the excellent root aeration required by V. corymbosum. Some are infested with soil pathogens, such as Phytophthora cinnamomi. In breeding to broaden soil adaptation in highbush blueberry, the goal should be chosen carefully, since it is unreasonable to expect to obtain tolerance to all soil problems in one clone. 9. Disease and Insect Resistance. In the southeastern United States, southern highbush blueberries are subject to many disease and insect problems, some severe (Caruso and Ramsdell 1995; Cline and Schilder 2006). The fact that this area is a center of origin for both the crop and its pests and pathogens contributes to the problem. Additionally, southern highbush blueberries are frequently planted on soils where they would not normally be found. The breeding of cultivars with high yield potential (chiefly by tripling and quadrupling berry size compared to what is normal for wild highbush blueberry) puts a further strain on the plants. The most serious diseases of southern highbush blueberry are different from those of northern highbush blueberry. This may have more to do with the soils and climate where they are grown than with the genetics of the plant material (Cline et al. 2006). One of the most serious diseases of highbush blueberries in the northern United States is mummy berry, caused by Monolinia vaccinii-corymbosi (Reade) Honey (Ehlenfeldt and Stretch 2002). Although mummy berry has occasionally been seen on wild blueberries in extreme north Florida after the coldest winters, it has never appeared in Florida in cultivated fields. Blueberry virus diseases have been important in northern production areas but have not been seen in Florida. However, fungal diseases, such as stem blight, cane canker, leaf rust, and leaf spots caused by various fungi, have been severe and widespread on southern highbush blueberries in the southeastern United States, and at least some level of resistance to each of these diseases is essential in useful cultivars.

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Cane canker was first observed in a cultivated field in North Carolina in 1938 (Varney and Stretch 1966). Infections were subsequently found in most fields of highbush blueberry in North Carolina and of rabbiteye blueberry throughout the southeastern United States. In North Carolina, cane canker eliminated ‘Weymouth’, an important cultivar that had been brought to the state from New Jersey (Darrow and Scott 1966). The North Carolina blueberry industry was saved by a breeding program that used a wild North Carolina highbush selection, ‘Crabbe-4’’, to develop the resistant varieties ‘Wolcott’, ‘Murphy’, ‘Angola’, and ‘Croatan’. These were released between 1950 and 1954. The resistance of ‘Wolcott’ subsequently broke down, but ‘Croatan’ has remained a major North Carolina variety up to the present. ‘Crabbe-4’ was involved in the breeding of all 10 of the canker-resistant cultivars released in North Carolina up to 1997, including ‘Croatan’, ‘Reveille’, and ‘Bladen’ (Ballington et al. 1997). Research in North Carolina indicated that there are races of the cane canker fungus, defined according to the cultivars on which they are virulent (Ballington et al. 1993). Nonetheless, resistance has proved quite durable in a number of cultivars in North Carolina and Florida. The pathogen is widespread in the southeastern United States on wild blueberry plants of various species, but most wild clones of most species have high levels of resistance (Lyrene and Sherman 1980). Stem blight, caused by the fungus Botryosphaeria dothidea (Demaree & Wilcox) Arx & Muller, is currently the most serious disease of highbush blueberry in Florida and probably in other southeastern states. Some clones are highly susceptible, and a high fraction of the plants of unselected genotypes will die of the disease in field plots before they are three years old. Pathogenic races of the pathogen have not been identified on blueberries, but cultivars vary in resistance, and isolates of the fungus vary greatly in pathogenicity (Cline and Milholland 1990; Ballington et al. 1993; Cline et al. 1993). The southern highbush cultivars most widely grown in Florida—‘Star’, ‘Emerald’, ‘Jewel’, ‘Windsor’, and ‘Springhigh’-appear to be more resistant than 80% of the 3,000 advanced selections that have been field tested in 15-plant plots in Florida during the past 20 years. The disease is so important and cultural methods of control so limited that selection for acceptable levels of resistance to the disease eliminates over half of the otherwise-acceptable test clones in the Florida program. Stem blight has been much less severe on the same genetic material in southern Spain and in the central valley of California, where summers are dry, but have been severe in eastern Australia, which has rainy hot summers like Florida. Phytophthora root rot, caused by Phytophthora cinnamomi, is a serious disease on southern highbush blueberry (Smith 2002, 2006).


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It is worst in warm, poorly drained soils. Southern highbush blueberries are not very drought tolerant and require moist soils. Soils that are fine-textured and have poor internal drainage favor phytophthora development, even if the plants are established on raised beds. Southern highbush cultivars show a wide range in susceptibility to phytophthora. After Draper et al. (1972) found resistance to root rot in a diploid plant of V. atrococcum Heller, they used colchicine to produce a tetraploid form of the plant, which they crossed with the tetraploid cultivar ‘Earliblue’. They obtained 85 seedlings. These were fertile but had small, dark fruit. They also found phytophthora resistance in a lowbush tetraploid, ‘Michigan Lowbush 1’ (V. angustifolium). In addition to the killing diseases—cane canker, stem blight, and root rot—many other diseases can cause serious crop damage when southern highbush blueberry is grown where summers are warm and wet. Leaf diseases, caused by various fungi, are normally controlled with fungicides, but partial resistance to the major pathogens makes control easier. Premature defoliation of highbush blueberry plants in the autumn due to various leaf diseases reduces flower bud number and the potential for a large crop the following spring (Cline 2002; Williamson and Miller 2002). Botrytis (Botrytis cinerea) can blight the flowers, especially if rain and fog are frequent during flowering or if overhead irrigation is used for freeze protection during flowering (Smith 1998). Various fungi can cause blueberry fruit decay during storage. For most blueberry diseases, selection for resistance is done by testing advanced selections in replicated plots for multiple years in commercial fields that receive standard management practices. Clones that have poor survival, low yield, or poor fruit quality are eliminated regardless of which disease caused their poor performance. Insects and mites can cause problems on southern highbush blueberry, the pests varying from place to place. In Florida, flower thrips (Franklinellia spp.) damage flower clusters and flowers before, during, and after anthesis. When numerous, thrips can destroy flowers, prevent pollination, and, if present during berry ripening, can cause soft spots on the berries. Late-flowering varieties are more subject to damage in Florida since thrips populations tend to increase during the blueberry flowering season. Blueberry gall midge (Dasineura oxycoccana Johnson) can destroy flower buds as they swell and can interfere with vegetative growth during the critical weeks after flowering by killing apical meristems. Cultivars differ with respect to flower bud susceptibility to gall midge, with most having medium to high resistance. Red banded thrips (Selenothrips rubrocinctus Giard) sometimes defoliate southern highbush in

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late summer and autumn, and these have a strong preference for certain cultivars. Blueberry bud mite can infest flower buds during the fall and summer and can result in aborted flower buds. The severity of this problem depends strongly on genotype, and genetic resistance is very stable over time, although the mechanism of resistance is unknown. Selecting resistant cultivars is very effective in reducing the problem. 10. Self-Fruitfulness. Most wild highbush blueberry plants show great reductions in yield, berry size, and earliness if self-pollinated. Highbush cultivars tend to have increased self-fruitfulness compared to wild material, but most cultivars also show reduced fruit set and berry size and later ripening if planted in solid blocks (Morrow 1943; Meader and Darrow 1947; El-Agamy et al. 1981; Lyrene 1989; Ehlenfeldt 2001). Gupton and Spiers (1994) compared the results of self- and cross-pollination by hand in a greenhouse using seven southern highbush cultivars. Cross-pollination increased berry weight and reduced fruit development period but did not increase percent fruit set. In commercial production, fields planted with a single clone offer important advantages in management and harvest. These advantages are so great that solid-block plantings are often used even with cultivars that give higher yields in mixed plantings. Thus, selection for increased self-fruitfulness is important. Ehlenfeldt (2006) reported heritable parthenocarpy in northern highbush blueberry. Parthenocarpic cultivars could someday offer another way of insuring good fruit set in commercial fields. An observation made by Eck (1977) is an important caution to blueberry breeders and evaluators: One of the most perplexing problems to the blueberry industry today is the disappointing productivity of many of the cultivars that were developed and released with what appeared to be a consistent productivity character. Many theories abound as to why these productivity problems developed, but the underlying problem appears to be a failure of fruit set to occur. . . . The failure of pollination appears to be the prime reason for the poor fruit set that has occurred fairly generally throughout the industry. Cultivar trials on breeding stations normally have two characteristics that increase fruit set compared to what the clones later show in commercial fields. First, research plantings tend to be small. In small plantings, highly efficient native pollinators, such as Bombus spp. and


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Habropoda laboriosa Fabricius, are normally sufficiently abundant to pollinate all the flowers. By contrast, most large growers depend on managed honeybees to pollinate their fields. Second, research plantings often have a large number of small plots, each with a different genotype, which increases cross-pollination. It is hard to predict from small experimental plantings the potential yields clones would have if planted in large, solid blocks, because so many variables affect fruit set, such as quantity of pollen shed, the ease with which pollen flows from the anthers, the attractiveness of the flower to various potential pollinating insects, level of genetic self-compatibility, and the ability of berries to develop normally despite having few or no seeds. It is easy to determine fruit set and seed production after hand-pollinating potted plants in a greenhouse, and this is a valuable indication of whether a clone has a chance of yielding well in a solid-block field planting. The tetraploid evergreen lowbush blueberry of the southeastern United States, V. myrsinites, may be a source of increased self-fruitfulness. It crosses readily with highbush cultivars, and the one plant studied by Meader and Darrow (1944) had a fruit set of 79.1% from 239 selfpollinated flowers and produced an average of nine fully developed seeds per berry. 11. Yield. Yield is a complex trait with many components. One important component is the carrying capacity of the bush. Plants sometimes set more berries than they can support, resulting in fruit that are smaller, later-ripening, and lower in quality. Overcropped plants may grow poorly or die. Vigorous plants that leaf well early in the spring are needed to get high yields of berries that ripen early. Because early ripening is usually desired in low-chill cultivars, clones with a short fruit development period are usually selected. Early and concentrated fruit ripening (another desirable characteristic) may tend to limit the carrying capacity of the plant compared to clones that ripen later in the season and over a long period of time. This has been seen with peaches, where early-ripening cultivars achieve much lower yields than late-ripening cultivars (W.B. Sherman, pers. comm.) If the plant is not overcropped and can support all the fruit that were set, several other yield components contribute to high yield. Plant architecture that results in numerous canes and many stout terminal branches promotes high yield. Thin-diameter shoots and excessively twiggy growth result in numerous small flowers that produce small berries. Plants that produce few canes and few lateral branches may not produce enough flowers to attain high yields. The number of flower buds produced per shoot is under genetic and

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environmental control. Premature defoliation in the fall, which may result from leaf diseases, can severely reduce flower bud number. Even when the leaves remain healthy late into winter, some genotypes produce only 1 to 3 flower buds per twig, whereas others produce10 or more flower buds on strong terminal shoots. The number of flowers produced per flower bud ranges from 1 to 10. The number undoubtedly has a genetic component, but this variation has not been studied. Under favorable conditions, most highbush blueberry flowers will set fruit, the level approaching 100% for some cultivars under ideal conditions. Reduced percent fruit set is a common cause of yield loss in blueberry. Many factors affect fruit set. Most important are plant genotype, opportunities for cross-pollination, bee populations in the field, and weather during pollination (which affects flower diseases, the ease of pollen shed, and the activity of pollinating bees). Blueberries have poricidally dehiscent anthers. At the time of pollen shed, the flower is oriented such that the open end of the corolla tube is nearest the ground. Pollen exits through a small pore at the lower end of the mature anther. Experience from greenhouse crosses indicates that pollen sheds readily when the relative humidity is low but is difficult to remove when the humidity is high. When the humidity is high, clones differ greatly in how readily they shed pollen (Lyrene and Williamson 2003). Presumably these same differences occur in the field. The most efficient native pollinators of blueberries in eastern North America are sonicating bees, such as Habropoda laboriosa and various species of Bombus. Honeybees, which do not sonicate, are inefficient pollinators for blueberry but are the bee most widely used by blueberry growers, since they are most available and easiest to manage. Honeybees have difficulty extracting pollen from blueberries, especially during humid or rainy weather, and readily abandon blueberry fields if other flowers are available. Pollen taken from pollen traps of honeybees that are presumably working blueberry fields in Florida and Australia show a high percentage of nonblueberry pollen. Marucci and Moulter (1977) found that honeybees have strong preferences for the flowers of some highbush cultivars over others and that honeybee preference for certain cultivars varies little from one year to another. Bee preference was not related to sugar concentration in the nectar, but cultivars that are attractive to bees may make larger volumes of nectar. Pritts (1997) listed cultivar factors that might influence bee preferences including flower shape (short, wide corollas may be preferred by honeybees), UV reflectance pattern, floral display, and quality of nectar. Under field conditions, blueberry fruit normally require at least one viable seed if the berry is to be retained on the plant and develop


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normally. In most cultivars, berry size increases with seed number, up to at least eight seeds per berry. Clones vary in their response to low seed number; some set and size fruit well with low seed number, but others produce smaller, later-ripening fruit if they have few seeds. Wild highbush blueberries have very low levels of self-compatibility and parthenocarpy. Some northern highbush cultivars are sufficiently self-fruitful to allow large, single-clone plantings (Ehlenfeldt 2001). Solid-block plantings offer many advantages in field operations. Many southern highbush cultivars have low self-fertility, and the interplanting of two or more cultivars that flower synchronously is necessary for high yields. Self-fruitfulness is highly advantageous, and will undoubtedly be a strong selection criterion as other breeding objectives are attained and require less attention. In large commercial plantings of southern highbush blueberries in Florida, in years when pollination weather is poor, cultivars vary greatly in fruit set and development. Some clones that set well even when pollination conditions are poor are neither self-compatible nor parthenocarpic. What makes these clones easy to pollinate is not known. Berry weight is an important yield component in blueberry. Wild highbush blueberries, when taken into cultivation, average 0.2 to 0.5 g per berry. Most cultivars of southern highbush, when carrying a medium to heavy (but not excessive) crop, average 1.5 to 2.5 g per berry. Thus, increased berry weight alone may have resulted in a fivefold increase in yield of blueberry cultivars. Consumers sometimes prefer larger berries, and larger berries increase the kg/hr rate of hand harvest. Cultivars with berries that are twice as large require only half as many pollinated flowers to produce the same tonnage per hectare. In addition to pollination and fruit-set problems that reduce yield, harvest factors can also reduce packable yield per hectare. Ripe berries of some cultivars detach too readily and may be lost on windy days or during harvest. Prolonged rain when the berries are ripening can split the skin on berries that are ripe or near ripe, resulting in unmarketable fruit. Cultivars vary in susceptibility to rain cracking. Some cultivars with high fruit firmness are quite susceptible. Berries of some cultivars are susceptible to sunburn, which produces soft spots on the berry and makes them unsuitable for the fresh market. Because it is very hard to separate sunburned berries from undamaged berries, sunburn susceptibility is very undesirable. High harvest efficiency contributes to high yield. Hand harvest is fastest for cultivars that produce large berries that are borne in loose clusters, ripen synchronously within clusters, detach readily when ripe, and are displayed on the outside of the bush and not hidden among

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the leaves. With hand harvest, cultivars that produce variable berry size may result in the smaller berries not being harvested. Berries with uneven pigmentation may appear ripe on one side and green on the other. Many of these berries are picked and then graded out. Ballington (1980) noted that BC1 populations from crossing F1V. darrowi northern highbush cultivar hybrids back to northern highbush cultivars were more precocious than northern highbush seedlings, coming into commercial production at least one year earlier. Southern highbush cultivars planted in pine bark in north Florida using one-yearold plants in January can reach full production (about 8,000 kg/ha per year) between three and four years later. It is not clear how much of this precocity is due to the long growing season, how much to pine bark culture, and how much to precosity in the cultivars. Probably all three factors are important. 12. Mechanical Harvestability. Over-the-row blueberry harvesters became commercially available in 1966, and by 1971 more than 100 were being used in the United States (Mainland and Rohrbach 1971). Mechanical harvest presents several important problems (Galletta and Mainland 1971): reduced tonnage of recovered, packable berries; darker berries due to abrasion of the surface wax (bloom); reduced postharvest life of fresh berries; a delay in harvest date (berries are not mechanically harvested until a large volume are ripe); the need to prune the plants to fit the machine; and a restriction on which cultivars can be used, since mechanical harvest of some cultivars gives poor results, for one or more of several possible reasons. Nonetheless, mechanical harvest is rapidly adopted when reliable, affordable harvest labor becomes unavailable, or when reduced harvest costs spell the difference between profit and loss on a farm. At present, most berries intended for the fresh market are still harvested by hand. Most blueberries for processing are mechanically harvested. Where harvest is by hand, harvest costs constitute a high percentage of the annual cost of producing blueberries, in some cases up to 50% (Eleveld et al 2005). The future availability of migrant labor for blueberry harvest in North America and Europe is uncertain. Thus, mechanical harvest of blueberries for the fresh market is increasing (Strik and Yarborough 2005). A number of plant and berry characteristics are needed in a cultivar intended for mechanical harvest (Galletta 1975; Ballington 1990). The plant should be moderately vigorous and narrowly upright, with a moderate number of canes. Resistance to cane diseases is important, because wounds made during harvest allow pathogens to enter. Fruit


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should be very firm with a perfect scar, not too large, and produced in a loose cluster. The berries should have concentrated ripening. Ripe berries should be able to remain in good condition on the bush for at least 10 days, reducing the number of times the harvester must pass through the field. The detachment force required to remove ripe berries should be intermediate. The skin should be moderately tough and elastic. Two North Carolina cultivars, ‘Reveille’ and ‘Bladen’, were the first southern highbush cultivars released specifically for adaptation to mechanical harvesting for fresh fruit markets (Ballington et al. 1997). 13. Evergreen Production and Protected Cultivation. Southern highbush blueberries are hybrids between a highly evergreen species (V. darrowi) and a highly deciduous taxon (northern highbush blueberry cultivars). Thus, if the seedlings are grown where winters are not very cold, it is possible to select, from segregating hybrid populations, plants that range from fully deciduous to fully evergreen. In the southeastern United States, including Florida, southern highbush blueberries are normally managed as a deciduous crop. The plants enter a brief period of dormancy during late fall, are defoliated in early winter, and produce new leaves and flowers in February and early March. At latitudes below 358, many southern highbush cultivars can be kept growing throughout the winter without losing their leaves and without entering dormancy or receiving any chilling. This requires that leaf diseases are controlled, nighttime temperatures are kept above freezing, and temperatures during the day are warm enough to promote growth. The plants retain their old leaves and produce new leaves throughout the winter. From November through March, flowers form and open continuously on the new growth. If pollinated, these flowers produce berries that can be harvested throughout the winter and spring (Wright 1993). In a few production areas, cultivars produce a fall and a spring crop with little production in midwinter due to lack of heat units to ripen berries. Using tunnels or greenhouses, winter and early spring production can be extended to areas where unprotected winter production is infeasible. The harvest season in high-chill areas can be moved forward using low-chill cultivars and protected culture (Ozeki and Tamada 2006; Tamada and Ozeki 2006). Not all southern highbush cultivars do well under evergreen production. Some cultivars tend to go dormant, even if temperatures are kept warm. Some cultivars have a large picking scar when grown as evergreens, even though the same cultivars may have a small, dry scar when plants are managed by traditional deciduous methods.

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B. Berry Characteristics 1. Size. Larger berries have several advantages and a few possible disadvantages. Berry size is a major yield component. The tripling (or more) of berry size in cultivars compared to their ancestors accounts for most of the yield increase in the cultivars. When berries are harvested by hand, large berries increase the number of kilograms of berries that can be harvested per hour (Strik and Buller 2004). In most fresh markets, consumers prefer large berries. Disadvantages of large berries include possible lower tolerance for mechanical harvest and problems packing very large berries into small clamshells using automated clamshell fillers. Uniformity of berry size is important. Some genotypes produce large berries on stronger twigs and small berries on finer twigs. Some genotypes produce large berries at the start of the season and small berries at the end. Sometimes the small late berries are due to poor pollination; berries with few seeds tend to ripen late and small. High variability in berry size produces a less attractive pack and implies a yield loss compared to what would have been obtained had all berries been large. Genotypes are quite variable in both berry size and size uniformity. Berry size seems to have high narrowsense heritability, as observed from parent-offspring resemblance. 2. Color. Fruit color in blueberry involves two factors: anthocyanin pigmentation in the skin (Ballinger et al. 1970) and the amount, type, and durability of wax that coats the outside of the berry (Albrigo et al. 1980). Some Vaccinium species in section Myrtillus, such as bilberry (Vaccinium myrtillus L.) and V. membranaceum L., produce berries that are pigmented both externally and internally (Finn and Young 2002; Beccaro et al. 2006). In the Cyanococcus species, anthocyanin pigmentation is confined to the skin, although freezing or cooking the berries frees the water-soluble anthocyanins, which then permeate the berry. Berries with internal anthocyanin would be desirable because of the reported health benefits of anthocyanin consumption. So far, no sustained effort has been made to obtain internal pigmentation in highbush blueberry. Coville (1937) selected two albino-fruited cultivars of northern highbush, ‘Redskin’ and ‘Catawba’, from the second generation of a cross between the wild highbush blueberry ‘Brooks’ and the wild lowbush blueberry ‘Russell’. Albino fruited blueberries have not yet been successfully commercialized. Albino berries tend to be red on top and white on the bottom, and any bruises quickly turn brown. A pink-fruit gene transferred to southern highbush tetraploids from diploid V. elliottii was inherited 35 normal:1 albino in the F2 generation of crosses between homozygous parents (Wenslaff 2003).


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Low anthocyanin is normally undesirable in commercial blueberry cultivars. Berries with low anthocyanin may appear black (ripe) on the side exposed to the sun and green, white, or olive-colored on the other. Problems with weak anthocyanin development are exacerbated by low light intensity, hot weather during ripening, poor leafing, and excessive crop loads. Cultivars are needed that maintain full coloration when subjected to these environmental stresses. The blue color of blueberries results from the combination of waxes, produced by berry epidermal cells, and anthocyanins in the skin of the ripe berry. Shiny black blueberries are lustrous and attractive on the plant, but they quickly become dull in appearance after harvest due to loss of berry turgor. By contrast, berries that appear frosty blue on the plant can maintain this color even after several weeks of storage. The amount and type of wax on the outside of the blueberry vary. Even berries that are shiny black have epicuticular waxes (Albrigo and Lyrene 1980). The waxes that give the powdery blue color are only one component of the blueberry fruit wax. Cultivars vary greatly in color due to wax, and they also vary greatly in how well the powdery blue appearance is maintained when the berries are handled. In some cultivars, berries become dark after only slight handling; in the best selections, they retain a light-blue color even after being picked, packed, and shipped. Berry color due to wax has medium to high heritability. Crossing two parents, both having black fruit, normally produces seedlings with uniformly dark fruit. Crossing two parents that have powdery blue fruit usually produces some seedllings with powdery blue berries and others with darker berries. The wax genes in rabbiteye and highbush apparently differ. When blue-fruited rabbiteye is crossed with blue-fruited highbush, most seedlings have shiny black fruit. Wild populations of several Cyanococcus species are polymorphic with respect to fruit color. Vaccinium darrowi, V. elliottii, V. ashei, and tetraploid V. corymbosum have some plants with black fruit and others with light-blue fruit in single, cross-pollinating populations. Seedlings from southern highbush crosses occasionally appear in which the berries are decorated by distinct alternating stripes of dark and light wax. We have propagated several of these by cuttings, but the character is unstable. Some ramets produce no sectored berries; others produce sectored berries on some branches but not others. 3. Picking Scar. In section Cyanococcus, ripe berries, if unharvested, may hang on the plant for a month or more. They decay if the weather is wet but will dry on the plant if the weather is dry. Berries of V. arboreum (section Batodendron) ripen in north Florida from mid-September to

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mid-October and, if not eaten by birds, can hang on the tree through February with little change in flavor or physical appearance. Berries of V. stamineum (section Polycodium) fall from the plant shortly after they ripen, with the pedicel attached to the berry. During harvest of highbush blueberries, the pedicel normally remains attached to the peduncle, which remains on the bush. In a few cultivars, the pedicel often detaches with the berry. This slows harvest and packing, since pedicels must be eliminated from the packs. The area on the berry where the pedicel was attached is called the scar. A small dry scar is needed in fresh-market blueberries. Small dry scars allow berries to be harvested without juice loss or damage to the skin. Wet picking scars are unacceptable, because bleeding of juice darkens the berries and favors growth of fungi and bacteria. Blueberry breeders have been selecting for small, dry scars for 100 years, and it remains an important selection criterion (Darrow et al. 1939). Cultivars with high berry firmness and improved picking scar have made possible the long postharvest life and international shipping of fresh blueberries. Poor scar and firmness explain why lowbush blueberries are seldom seen in the fresh market. In some crosses, all seedlings have small dry scars. In others, few or no seedlings meet the minimum scar requirements for a modern cultivar. Some of the first important southern highbush cultivars, such as ‘Sharpblue’, ‘Gulf Coast’, and ‘Windsor’, have problems with pedicel attachment or scar. These berries can be shipped fresh only if they are hand-harvested shortly after they ripen. In the future, cultivars with a poor scar will probably not be competitive. 4. Firmness. Firm berries contribute to ease of harvest and long postharvest life, and consumers prefer firm berries over mushy berries. Berry firmness is a continuous character in blueberry, probably affected by many genes. Ehlenfeldt (2002) studied firmness in 87 highbush and species-introgressed blueberry cultivars. Southern highbush cultivars tended to have higher firmness than northern highbush. Genes from V. darrowi and V. ashei were thought to contribute to greater berry firmness. However, many of the northern highbush cultivars tested were more than 40 years old, and the greater firmness of southern highbush could be due to recent progress in breeding. During the past 20 years, several southern highbush cultivars with very high berry firmness have been released, including ‘Reveille’, ‘Bluecrisp’, and ‘Sweetcrisp’. In some crosses, the extra crispness of these clones segregates in a way that suggests the involvement of one or more major genes for crisp texture. In Florida crosses, high crispness is


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associated with low berry acidity and possibly with increased susceptibility to cracking in the rain. Where harvest weather is favorable, crisptextured cultivars could permit harvest of the entire crop in one pass, as much as five weeks after the first berries ripen. 5. Flavor. Blueberry flavor is determined by the amounts and kinds of sugars, organic acids (Ehlenfeldt et al. 1994), and aromatic compounds in the berries (Darrow and Camp 1945). Subjectively, berries from different clones may be tart, sweet, or bland. The combination of high percent soluble solids and high acid produces a flavor most people like. High solids and low acid gives a sweet flavor that is preferred by some. Low solids and low acid makes a bland or insipid berry. Aromatic compounds can produce intriguing flavors, but these are often ephemeral and may be present only at certain stages of berry ripening or in berries freshly harvested. Blueberries tend to be quite high in solids compared to strawberries and blackberries. Remberg et al. (2006) found that soluble solids percentage averaged over six years ranged from 13.0 to 14.9 for northern highbush cultivars grown in Norway. In New Jersey, eight northern highbush cultivars averaged 11.1% in soluble solids. Some tests selections were as high as 16.1% (Ehlenfeldt 1998). Solids percentage varies greatly with degree of ripeness (Ehlenfeldt 1998). Berries can continue to increase in solids and weight if left on the bush for a week or more after they turn blue. Berry flavor is subject to various environmental influences. Berries that seem too tart when they first turn blue usually seem less tart if allowed to hang on the plant for several days after they first turn blue. Cloudy, rainy weather or heavy irrigation just before harvest can reduce berry flavor. Plants that carry too many berries or branches that have leafed poorly produce berries with less flavor than is typical for the variety. Temperatures during the harvest season affect flavors. Warm nights during ripening improve flavor for most rabbiteye blueberry cultivars but make the berries of some highbush blueberries bland (Darrow and Camp 1945). Ballington (1980) and Ehlenfeldt et al. (1995) noted that cultivars with V. darrowi ancestry generally have outstanding flavor and can retain ripe fruit in good condition under hot weather conditions. Hancock (1998) noted that hybrids between V. darrowi and northern highbush cultivars often have surprisingly pleasant and complex flavors. Vaccinium darrowi itself does not produce particularly good-flavored berries; they tend to be low in acidity and are sometimes slightly bitter or astringent. The inheritance of flavor in blueberry has not been studied. However, when environmental differences are taken into account, genotypes

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differ considerably in flavor, and seedlings from different crosses may tend to be sweet, tart, bland, and so on. Genotypes that have berries with poor flavors are eliminated at early stages of selection. To maintain and improve the popularity of blueberries with consumers, it is important that breeders not release cultivars with below-average flavor. Growers will plant these cultivars if they are available and have market or production advantages. Because blueberries taste best if eaten fresh from the bush and can lose desirable flavors or acquire off-flavors during the days or weeks between harvest and consumption, it is useful to evaluate berries of advanced selections after several weeks of storage. To fill gaps in the fresh-market blueberry calendar, marketers sometimes store ‘‘fresh’’ blueberries for a month or more under controlled atmosphere or modified atmosphere. Unless long-term storage is done very carefully, it can greatly reduce berry quality in terms of texture, flavor, firmness, and appearance. It is hoped that increased production from areas that fill market gaps will reduce the use of controlled atmosphere storage in the future. 6. Texture. Highbush blueberries normally have a desirable texture, with thin skins, small seeds, few or no sclerids (stone cells), and good firmness. Among the gene sources that have been used to breed southern highbush cultivars, rabbiteye blueberry and V. arboreum are potential sources of sclerids and larger seeds. The skins of rabbiteye blueberries tend to become tough and leathery when the berries are frozen and stored. To date, southern highbush have been grown principally for the fresh market, and quality problems that might arise in frozen berries have not been noted. 7. Ability to Maintain Quality as a Ripe Berry on the Bush. Ideally, all berries on a bush would ripen the same day and could be picked in one pass. The best that has been achieved in southern highbush is cultivars that ripen 90% of their berries during a three- to four-week window. To minimize the number of times the plant must be harvested and to lessen problems that arise when harvest is delayed by weather or other factors, ripe berries should be able to hang on the bush for a week or more without loss of quality. The extent to which this is possible depends on the cultivar and on the weather during fruit maturation. Firm berries with small dry scars tend to store best on the bush. In cultivar evaluation, it is useful to examine field plantings at the end of the ripening season from which no berries have been picked. Cultivars with no soft or decayed berries are of special value. Rain cracking in blueberries is not nearly the problem it is in sweet cherries, but extended periods of rain during berry ripening can crack or


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rupture ripe blueberries. The problem is worst if berries are continuously wet for 24 hours or more. Resistance to rain cracking varies considerably among cultivars. 8. Freedom from Miscellaneous Defects. If enough seedlings are examined, many types of genetically determined berry defects can be found. Some berries crack with little or no rain. Some have excessively large calyx lobes, calyx lobes that poke out too far from the berry surface, or a deep calyx aperture in which spiders and insects can hide. Some berries are highly attractive to flower thrips, which scar and soften the berries. Berries on some cultivars are highly susceptible to sunburn, which can cook and soften the berries suddenly on hot days. On some seedlings, the pedicel or the dried corolla tends to remain attached to mature berries. Cultivars vary in berry shape from flat to oblong. A range of shapes is acceptable, but extremely oblong or extremely flat berries are undesirable. 9. Postharvest Life and Quality. Compared to other small fruits, such as raspberries, blackberries, and strawberries, blueberries can be stored for a long time as a fresh berry. This ability is sometimes abused by marketers who hold berries to get higher prices or to fill market windows that could be filled by fresh-picked berries from other production areas. Cultivars differ in the rate at which their berries lose quality during storage (Ehlenfeldt 2002). Softening, shriveling, darkening, microbial decay (Schilder et al. 2002), and loss of flavor are limiting factors in long-term storage of blueberries. Although southern highbush blueberries are usually destined for early markets and are not normally stored for long periods, good postharvest life is important in international shipment of fresh berries on container ships and in maintaining high berry quality in retailing. 10. Berry Components with Health Benefits. Blueberries have a good reputation as a healthy food (Kalt 2006). They are low in fat and relatively low in calories. Blueberries in the diet, like cranberries (Vaccinium macrocarpon), can reduce the incidence of urinary tract infection (Vorsa et al. 2000). Anthocanins, phenolic compounds, and pectins act as antioxidants, and are associated with vascular and coronary health and with the maintenance of good eyesight. Some evidence indicates that regular consumption of blueberries and other fruits and vegetables can slow aging-related loss of memory and other mental skills. Blueberry species and cultivars vary in concentration of various phytonutrients (Clark et al. 2002; Giongo et al. 2006), and levels of particular chemical constituents may become a selection criterion if the proposed health benefits of blueberry consumption continue to be confirmed and extended.

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VII. RECOMMENDING CULTIVARS FOR COMMERCIAL PLANTINGS Breeders of all crops face the question of when it is time to stop testing a cultivar and make it available to growers. The dilemma is especially acute with long-lived crops that are expensive to test. Good cultivars must maintain health and vigor for several decades, continuing to give high yields year after year. These attributes can be only imperfectly evaluated in trials that last only 10 to 15 years. Release of cultivars that have serious defects not revealed during testing can cost growers much money. However, delaying the release of superior cultivars can be even more expensive and can put growers in one region at a competitive disadvantage compared to growers elsewhere who have better cultivars (Hancock 2006). One way to reduce the risk to growers is to make new cultivars available while advising growers not to plant large quantities until a cultivar has proven itself commercially. During the first 5 to 10 years after release of a new blueberry cultivar, the breeder can provide continuous updates to growers regarding the performance of the original test plots at the experiment station along with information from commercial plantings of the cultivar. Breeders are often in a good position to advise growers on the relative strengths and weaknesses of all cultivars available for planting in an area. A. Regions of Adaptation Different cultivars are needed in different production areas. Chilling requirement of the clone must be no higher than what a farm site provides. Low-chill cultivars can sometimes be grown in high-chill zones if winter cold damage and spring frost are not normally problems. Early- or late-ripening cultivars may be chosen to exploit market windows or seasons when harvest weather is good. Some cultivars may cause trouble in areas where the climate favors insects and diseases to which they lack resistance. Some clones that do well where summers are dry may die of stem blight if planted in Florida where summers are rainy. B. Old Proven Cultivars versus New Improved Cultivars In planting, growers must choose between old cultivars of proven value, whose cultivation, harvest, and marketing have been optimized, and new, relatively unknown cultivars that are thought to be better in some important way. Sometimes growers have little choice, as in North Carolina during the 1950s when cane canker was destroying


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new plantings of ‘Wolcott’ before they became productive and all resistant cultivars were new and relatively untested. More commonly, growers can plant most of their acreage to proven cultivars while testing all available new cultivars that have potential adaptation in their area. C. Cultivar/Culture Interactions Irrigation, fertilization, pruning, soil-amendment practices, and disease control methods are developed over time to maximize performance of the principal cultivars in each production area. New cultivars may require different management practices to reach their full potential. Methods of pruning and pest control are particularly cultivar-dependent. High price or low availability of labor in an area may make it infeasible to grow cultivars that require special attention, such as detailed hand pruning or hand-harvest directly into clamshells. D. Cross-Pollination Considerations. Plantings are easier to manage if only one cultivar is planted in a field. Unfortunately, most highbush cultivars yield more, ripen earlier, and have larger berries if cross-pollinated. Whether the advantages of solidblock planting are greater than the disadvantages will depend on the cultivar. If cross-pollination is required, finding compatible cultivar groupings is important. E. Cost and Availability Plants of new cultivars are often more expensive than plants of older cultivars. Many growers propagate their own plants, but cuttings to propagate new cultivars may be expensive. Plants of new cultivars can be expensive when supplies are limited. Most new blueberry cultivars are patented by the developer in order to fund the breeding program. The patent holder may issue exclusive licenses for use of some cultivars. These may delay the availability and raise the cost of plants to growers.

VIII. OUTLOOK AND ISSUES A. Current Situation Blueberries have been bred for 100 years, although the personnel and money devoted to blueberry breeding was very small for the first 50

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years. In hindsight, it is easy to wish that a much wider genetic base had been used to start the program—for example, 50 or 100 wild northern highbush plants instead of 5 or 10. Progress in highbush blueberry breeding has been rapid with respect to increased berry size, higher berry firmness, and improved picking scar. With southern highbush, low-chilling requirement and resistance to diseases of warm, wet summers have made commercial highbush blueberry production possible in the southeastern United States. The date of first harvest in north Florida has been moved back about one month (from 1 May to 1 April) compared to the first southern highbush cultivars, but this early harvest has required early-flowering cultivars and large irrigation systems to protect flowers from freezes after 20 January. There is no evidence that progress per generation of selection is decreasing for such important factors as berry size and firmness. Berry color (wax intensity and durability) has not received as much attention, and it should be possible to develop cultivars with berries that are far more attractive in the market. Good plant survival has been a challenge in the southeastern United States. The main causes of plant decline are stem blight, cane canker, and root rot. Genetic variation exists to alleviate each of these problems, but selection for good plant survival is much slower than selection for berry traits, which can often be judged quite accurately from the first few berries a seedling produces. Transition to mechanical harvest seems inevitable during the next 10 to 20 years, at least in the United States (Strik and Yarborough 2005). This will require a sea change in selection criteria. The breeding of southern highbush cultivars suitable for mechanical harvest has progressed farthest in North Carolina. The pioneering ‘Reveille’ shows what can be done, but it also suggests that it may be hard to obtain top yields per hectare with mechanically harvested cultivars. B. Grafted Blueberries Coville (1921) described methods for budding blueberry and stated that budding was the best known means of quickly producing a large quantity of cutting wood from a valuable selected blueberry hybrid. He also stated that budding is not suitable for permanent commercial plantations because budded bushes continually send up new shoots from the rootstock. Highbush blueberries are relatively easy to graft on various rootstocks, including highbush, rabbiteye, and V. arboreum (Galletta and Fish 1971; Ballington et al. 1990). If grafted plants could be made practical in commercial blueberry plantations, the potential advantages are great. Grafting would allow the


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breeder to separate the breeding program into two parts: breeding clones that make good rootstocks and breeding other clones that make good scion cultivars. Grafting would allow the exploitation of the deep root system and drought tolerance of V. arboreum without having to overcome the berry quality problems of that species (Ballington 1998). Use of a V. arboreum rootstock could allow highbush blueberries to be grown on upland sites that are low in organic matter. On some upland sites, frost is less common and water management is much simplified. Highbush plants on a V. arboreum rootstock could be pruned to a single upright trunk to a height of 30 or 40 cm, facilitating mechanical harvest. C. Germplasm Conservation Soaring human populations and increasing per capita consumption are eliminating wild habitat and their animal and plant populations faster than at any previous time in human history. Worldwide, the last wild populations of many plant and animal species are now disappearing. Global warming and the current melting of glacial ice that has been present for at least 100,000 years will increase sea levels by an unknown extent, with unknown ecological consequences. Vaccinium section Cyanococcus is widespread and diverse and will not soon become extinct, although some of its species and ecological races are already threatened. The population of wild V. corymbosum in Florida is probably less than half of what it was 100 years ago, due to land clearing for agriculture and development. Races and species of Vaccinium whose ranges are confined to the highest peaks of the southern Appalachian mountains (e.g. V. constablaei) may disappear as the climate warms. Little is known about the security of wild populations of the hundreds of Vaccinium species scattered throughout the world. Methods of preserving Vaccinium genetic resources, listed in order of their potential impact, include habitat preservation and management, seed banks, and clonal repositories. In the United States, state and national forests and state parks are places where wild blueberry populations can be maintained. Unfortunately, many of these public lands are not being managed to preserve native plant diversity. In some, invasive exotic species are replacing blueberries. In the southeastern United States, Chinese privet (Ligustrum sinensis Lour.), Chinese tallow tree (Sapium sebiferum (L.) Roxb., Japanese climbing fern (Lygodium japonicum [Thunb.]), Brazilian pepper (Schinus terebinthifolius Raddi), and scores of other aggressively invasive introduced plant species are overrunning millions of hectares. Some Vaccinium species require fire for their maintenance, and cannot persist in the shade of broadleaf trees

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that become dominant in the absence of fire. Fire exclusion and the use of herbicides in commercial pine plantations of the Southeast have eliminated millions of hectares of former blueberry habitat. Seeds of Vaccinium section Cyanococcus species can be stored in coin envelopes for at least 30 years at 58C with little loss in viability. Under best storage practices, seeds could probably maintain viability for 100 years or more. Because blueberry seeds are very small, a few liters of seed could store far more genetic variability than is present in all the Vaccinium clonal storage facilities of the world. Collection of large, genetically diverse seed samples of each species and development of genetically sound strategies for reconstituting seed supplies every 50 years should be given high priority.

IX. LITERATURE CITED Aalders, L.E., and I.V. Hall. 1963. Note on aeration of colchicine solution in the treatment of germinating blueberry seeds to induce polyploidy. Can. J. Plant Sci. 43:107. Ahokas., H. 1971. Notes on polyploidy and hybridity in Vaccinium species. Ann. Bot. Fennici 8:254–256. Albrigo, L.G., P.M. Lyrene, and B. Freeman. 1980. Waxes and other surface characteristics of fruit and leaves of native Vaccinium elliottii Chapm. J. Am. Soc. Hort. Sci. 105:230– 235. Anon., 1941. Blueberries, with special reference to Florida culture. Bul. 33, New Series. Aug. State of Florida Department of Agriculture, Tallahassee. Anon., 1997. The Brooks and Olmo register of fruit and nut varieties. ASHS Press, Alexandria, VA. Ballinger, W.E., E.P. Maness, and L.J. Kushman. 1970. Anthocyanins in ripe fruit of the highbush blueberry, Vaccinium corymbosum L. J. Am. Soc. Hort. Sci. 95:283–285. Ballington, J.R. 1980. Blueberry improvement through interspecific hybridization: Recent progress and potential in North Carolina. pp. 35–39. In: J.N. Moore (ed.); Proc. 4th North Am. Blueberry Res, Workers Conf., Univ. Arkansas, Fayetteville. Ballington, J.R. 1990. Germplasm resources available to meet future needs for blueberry cultivar improvement. Fruit Var. J. 44(2):54–62. Ballington, J.R. 1998. Performance of own-rooted ‘Premier’ rabbiteye blueberry (Vaccinium ashei Reade) compared to ‘Premier’ grafted on Vaccinium arboreum Marsh (sparkleberry) over four harvest seasons. pp. 178–181. In: W.O. Cline and J.R. Ballington (eds.); Proc. 8th North Am. Blueberry Res. Ext. Workers Conf., North Carolina State Univ., Raleigh. Ballington, J.R. 2001. Collection, utilization, and preservation of genetic resources in Vaccinium. HortScience 36:213–220. Ballington, J.R., W.E. Ballinger, C.M. Mainland, W.H. Swallow, E.P. Maness, G.J. Galletta, and L.J. Kushman. 1984. Ripening period of Vaccinium species in southeastern North Carolina. J. Am. Soc. Hort. Sci. 109:392–396. Ballington, J.R., B.W. Foushee, and F. Williams-Rutkosky. 1990. Potential of chip-budding, stub-grafting or hot-callusing following saddle-grafting on the production of


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grafted blueberry plants. pp. 114–120. In: C.A. Brun (ed.), Proc. 6th North Am. Blueberry Res. Ext.Workers Conf., Washington State Univ., Pullman. Ballington, J.R., and G.J. Galletta. 1976. Potential fertility levels in four diploid Vaccinium species. J. Am. Soc. Hort. Sci. 101:507–509. Ballington, J.R., and G.J. Galletta. 1978. Comparative crossability of 4 diploid Vaccinium species. J. Am. Soc. Hort. Sci. 103:554–560. Ballington, J.R., S.D. Rooks, W.T. Bland, and A.D. Draper. 2006. The role of interspecific hybridization in the North Carolina State University blueberry breeding program. pp. 6–13. In: D. S. NeSmith, (ed.); Proc. 10th North Am. Blueberry Res. Ext. Workers’ Conf., Univ. Georgia, Tifton. Ballington, J.R., S.D. Rooks, W.O. Cline, J.R. Meyer, and R.D. Milholland. 1997. The North Carolina State University breeding program—toward V. covilleanum? Acta Hort. 446:243–247. Ballington, J.R., S.D. Rooks, R.D. Milholland, W.O. Cline, and J.R. Meyers. 1993. Breeding blueberries for pest resistance in North Carolina. Acta Hort. 346:87–94. Banãdos, M.P. 2006. Blueberry production in South America. Acta Hort. 715:165–172. Banãdos, M. P., and B. C. Strik. 2006. Manipulation of the annual growth cycle of blueberry using photoperiod. Acta Hort. 715:65–71. Baquerizo, D. 2005. The inheritance of dwarf seedings in southern highbush blueberries. Ph.D. diss., Univ. Florida, Gainesville. Beccaro, G., M. Mellano, R. Botta, V. Chiabrando, and G. Bounous. 2006. Phenolic and anthocyanin content and antioxidant activity in fruits of bilberry (Vaccinium myrtillus L.) and of highbush blueberry (V. corymbosum L) cultivars in northwestsern Italy. Acta Hort. 715:553–557. Brightwell, W.T., O. Woodard, G.M. Darrow, and D.H. Scott. 1955. Observations on breeding blueberies for the southeast. Proc. Am. Soc. Hort. Sci. 65:274–278. Brooks, S.J., and P.M. Lyrene. 1995. Characteristics of sparkleberry blueberry hybrids. Proc. Fla. State Hort. Soc. 108:337–339. Brooks, S.J., and P.M. Lyrene. 1998a. Derivatives of Vaccinium arboreum Vaccinium Section Cyanococcus: I. Morphological characteristics. J. Am. Soc. Hort. Sci. 123:273– 277. Brooks, S.J., and P.M. Lyrene. 1998b. Derivatives of Vaccinium arboreum Vaccinium Section Cyanococcus: II. Fertility and fertility parameters. J. Am. Soc. Hort. Sci. 123:997–1003. Camp, W.H. 1942. On the structure of populations in the genus Vaccinium. Brittonia 4:189–204. Camp, W.H. 1945. The North American blueberries with notes on other groups of Vacciniaceae. Brittonia 5:203–275. Caruso, F.L., and D.C. Ramsdell (eds.). 1995. Compendium of blueberry and cranberry diseases. APS Press, St. Paul, MN. Chandler, C.K., A.D. Draper, and G. J. Galletta. 1985a. Combining ability of blueberrry interspecific hybrids for growth on upland soil. HortScience 20:257–258. Chandler, C.K., A.D. Draper, and G.J. Galletta. 1985b. Crossability of a diverse group of polyploid blueberry interspecific hybrids. J. Am. Soc. Hort. Sci. 110:878–881. Clark, J.R., and C.E. Finn. 2006. Register of new fruit and nut cultivars. List 43. HortScience 41:1101–1133. Clark, J.R., L. Howard, and S. Talcott. 2002. Variation in phytochemical composition of blueberry cultivars and breeding selections. Acta Hort. 574:203–207. Cline, W.O. 2002. Blueberry bud set and yield following the use of fungicides for leaf spot control in North Carolina. Acta Hort. 574:71–74.

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9 Coffee Germplasm Resources, Genomics, and Breeding Fernando E. Vega Sustainable Perennial Crops Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Building 011A, BARC-W, Beltsville, Maryland 20705 USA Andreas W. Ebert Plant Genetic Resources and Biotechnology Unit, Department of Agriculture and Agroforestry, Tropical Agricultural Research and Higher Education Center (CATIE) CATIE 7170 Turrialba, Costa Rica Ray Ming Department of Plant Biology, 288 ERML, MC-051, 1201 W. Gregory Drive, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 USA

I. INTRODUCTION II. GERMPLASM RESOURCES A. Wild and Cultivated Coffea B. Collecting Wild Species C. Strategies for Conservation III. GENOMICS A. Developing Linkage Maps B. Mapping Genes and QTLs Controlling Biotic Stress and Economic Traits C. Construction of Large Insert Genomic Libraries D. Coffee Expressed Sequence Tags IV. BREEDING A. Historical and New Trends B. Exploiting Genetic Diversity 1. Disease Resistance 2. Nematode Resistance 3. Other Types of Resistance or Tolerance 4. Low Caffeine Content Plant Breeding Reviews, Volume 30. Edited by Jules Janick Copyright © 2008 John Wiley & Sons, Inc.

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C. Propagation Systems 1. Seed Propagation 2. Clonal Propagation 3. F1 Hybrids D. Future Based on Biotechnology V. LITERATURE CITED

I. INTRODUCTION Coffee is the second largest export commodity in the world after petroleum products with an estimated annual retail sales value of US $70 billion in 2003 (Lewin et al. 2004). Over 10 million hectares of coffee were harvested in 2005 (http://faostat.fao.org/) in more than 50 developing countries, and about 125 million people, equivalent to 17 to 20 million families, depend on coffee for their subsistence in Latin America, Africa, and Asia (Osorio 2002; Lewin et al. 2004). Coffee is the most important source of foreign currency for over 80 developing countries (Gole et al. 2002). The genus Coffea (Rubiaceae) comprises about 100 different species (Chevalier 1947; Bridson and Verdcourt 1988; Stoffelen 1998; Anthony and Lashermes 2005; Davis et al. 2006, 2007), and new taxa are still being discovered (Davis and Rakotonasolo 2001; Davis and Mvungi 2004). Only two species are of economic importance: C. arabica L., called arabica coffee and endemic to Ethiopia, and C. canephora Pierre ex A. Froehner, also known as robusta coffee and endemic to the Congo basin (Wintgens 2004; Illy and Viani 2005). C. arabica accounted for approximately 65% of the total coffee production in 2002–2003 (Lewin et al. 2004). Dozens of C. arabica cultivars are grown (e.g., ‘Typica’, ‘Bourbon’, ‘Catuai’, ‘Caturra’, ‘Maragogipe’, ‘Mundo Novo’, ‘Pacas’), but their genetic base is small due to a narrow gene pool from which they originated and the fact that C. arabica is the only tetraploid species in the genus (2n ¼ 4x ¼ 44) and is self-pollinated (Carvalho et al. 1991). In contrast, C. canephora is a diploid (2n ¼ 2x ¼ 22) and is crosspollinated. Evidence for a gametophytic system of incompatibility that is controlled by one gene with multiple alleles has been reported by Berthaud (1980). It is estimated that the genetic variation in C. canephora is 10 times that in C. arabica (Lashermes et al. 2000). Data from Berthou et al. (1980, 1983) and Lashermes et al. (1999) has revealed that C. arabica contains two genomes resulting from hybridization between the diploids C. eugenioides Moore as maternal donor and C. canephora

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as paternal donor (Anthony and Lashermes 2005). In contrast, Raina et al. (1998) reported C. congensis and C. eugenioides as the progenitors of C. arabica. Vega et al. (2003) have made a call for the establishment of an international coffee research development program that could coordinate coffee research efforts throughout the world, in an effort to avoid duplication and to better manage scarce research funds. Such an effort would serve to coordinate all coffee research, including pest and disease management, breeding, and molecular biology endeavors. To address the issue of coffee molecular biology, a meeting was held in 2004 at the United States Department of Agriculture (USDA) with scientists from the Centre de Coope´ration Internationale en Recherche Agronomique pour le De´veloppement (CIRAD, France), the Centro Agrono´mico Tropical de Investigacioń y Ensenãnza (CATIE, Costa Rica), and USDA, together with Ted Lingle, Chief Executive Officer of the Specialty Coffee Association of America (currently the Director of the Coffee Quality Institute). The purpose of the meeting was to address the needs within the coffee industry for a better understanding of how molecular biology tools could help the industry. Subsequently and completely independent of the USDA meeting, a call to establish an International Coffee Genomics Network (ICGN) was made by scientists from over 13 countries at the 2004 Association Scientifique International du Cafe´ (ASIC) conference held in Bangalore, India. The ultimate goal of the ICGN would be ‘‘to decipher the genetic and molecular bases of important biological traits in coffee tree species that are relevant to the crop.’’ Thus, it is clear that there is an international interest in developing a coordinated effort targeted at coffee molecular biology. The speed at which DNA is being sequenced is staggering, with one laboratory reporting that the genomes of 15 microorganisms were sequenced in one month (Preuss 2000). So far, over 180 organisms have been sequenced (Genome News Network 2006). The genome for C. arabica consists of approximately 1,176 million base pairs (Mbp) compared with approximately 386 Mbp for cacao, 482 Mbp for mango, and 965 Mbp for tomato (Kiehn 1986; Bennett and Leitch 1995). In 2003 the Minister of Agriculture of Brazil announced that Brazilian scientists had decoded the coffee genome (Hay 2004), and a recent paper by Vieira et al. (2006) reports approximately 33,000 genes. Obviously there is a strong interest among the rest of the coffee producers in understanding how the decoding of the coffee genome could help them. What would a coordinated effort on coffee molecular biology provide? This article is aimed at answering this question.

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II. GERMPLASM RESOURCES A. Wild and Cultivated Coffea Natural habitats of wild coffee trees are found in the understory of tropical forests in Africa comprising a wide geographical range from Guinea in West Africa through Central to eastern Africa (Charrier and Berthaud 1985; Davis et al. 2006). Other centers of diversity include Madagascar and the Comoros and Mascarene islands in the Indian Ocean (Davis et al. 2006). Centers of origin and primary diversity of the commercially most important species, C. arabica, are located in the highlands of southwestern Ethiopia, the Boma Plateau in southeastern Sudan, and on Mount Marsabit in Kenya (Thomas 1942; Sylvain 1955, 1958; von Strenge 1956; Meyer et al. 1968; Anthony et al. 1987). The tributaries of the Congo River represent a rich center of genetic diversity for C. canephora as well as many other diploid coffee species, such as C. congensis and C. liberica (Dulloo et al. 2001; Davis et al. 2006). Another center of genetic diversity of wild coffee species in East Africa is located in the Eastern Arc Mountain of Tanzania, where Davis and Mvungi (2004) recently described two new endangered species of Coffea: C. bridsoniae and C. kihansiensis. This brings the total number of naturally occurring wild Coffea species in Tanzania to 16 (Davis and Mvungi 2004), surpassed only by Madagascar with 59 species (Davis et al. 2006). Chevalier’s (1947) infrageneric classification of Coffea into four sections (Argocoffea, Eucoffea, Paracoffea, and Mascarocoffea) had many weaknesses and is no longer accepted. The current classification divides the genus in two subgenera: Coffea subgen. Coffea and Coffea subgen. Baracoffea (Davis et al. 2006 and references therein). The narrow gene pool of cultivated C. arabica is based mostly on how it disseminated throughout the world. Historical narratives have traced the movement of coffee from southwestern Ethiopia to Yemen; then to India; and from there to Ceylon and Java; and in about 1710 to Amsterdam; from there to Paris; and finally, in about 1718, from Paris a plant was taken to Martinique by the French captain Matthieu de Clieu. It is believed that progeny from this plant, later on referred to as ‘Typica’, gave origin to plantations throughout South America. Plants were also taken from Yemen to the island of La Reúnion in about 1715 and gave rise to a widely used cultivar known as ‘Bourbon’ (Anthony et al. 2002). Thus, there is a clear genetic bottleneck that gave rise to commercially grown coffee outside its center of origin. Molecular studies have provided evidence for reduced genetic variability among cultivated C. arabica varieties except those in Ethiopia.


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Steiger et al. (2002) compared 61 coffee accessions comprising the C. arabica cultivars ‘Bourbon’, ‘Catimor’, ‘Catuai’, ‘Caturra’, ‘Typica’, and ‘Tall Mokka’ (previously known as ‘Mokka Hybrid’) plus C. canephora and C. liberica. Using a technique known as amplified fragment length polmorphisms (AFLP), which results in specific DNA markers, they found an average genetic similarity of 0.93 (1 being completely identical) among C. arabica cultivars based on simple matching coefficient. Their results also indicated that C. arabica shares an average similarity of 0.54 with C. canephora, in contrast to 0.41 with C. liberica, thus providing evidence that C. arabica is more closely related to C. canephora than to C. liberica. The reduced genetic variability in C. arabica cultivars was also evident in a study by Moncada and McCouch (2004), where they used a different molecular technique known as simple sequence repeats (SSRs) on 30 Coffea accessions from the germplasm collection at the Centro Nacional de Investigaciones de Cafe´ (Cenicafe´) in Colombia, and in a study by Anthony et al. (2002) using both AFLP and SSRs with 26 Coffea accessions. Based on AFLP results, Lashermes et al. (2000) reported that the self-incompatible C. canephora is 10 times more polymorphic than C. arabica. In accessions collected from occasional escapes outside cultivated areas in the center of origin and primary diversity of C. arabica (i.e., Ethiopia), polymorphism was much higher than in cultivated accessions (Anthony et al. 2002). The use of inter-simple sequence repeat (ISSR) markers revealed high genetic variation between populations from different regions of Ethiopia, while genetic differentiation within the same population or among populations from the same region was much less pronounced (Aga et al. 2005). Greater genetic variation was detected in C. arabica germplasm from Africa than in germplasm from America and India using SSRs (G. Graziosi, pers. comm.). These and previous studies raised the awareness that successful C. arabica breeding programs depend to a large extent on the availability of genetic diversity from its center of origin. This fact has led to major collecting missions during the past decades (Table 9.1). B. Collecting Wild Species For coffee, there have been no alternatives so far to ex situ field collections for long-term germplasm conservation. Coffee seeds are recalcitrant, and conventional methods of seed storage cannot extend viability beyond two to three years. Important C. arabica field gene banks are located in Africa (Cameroon, Ethiopia, Ivory Coast, Kenya, and Tanzania), Madagascar, India, and the Americas (Brazil, Colombia, and Costa

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Table 9.1. Principal collecting missions for Coffea arabica.

Year

Country Explored OrganizationZ Collector

1964–65 Ethiopia FAO

Ethiopia, Costa Rica, India, Peru, Tanzania

1966

Ethiopia, Cameroon, Ivory Coast, Madagascar Kenya, Ivory Coast

1977

1989

F.G. Meyer, L.C. Monaco, R.L. Narasimhaswamy, L.M. Fernie, and D.J. Greathead Ethiopia ORSTOM J.L Guillaumet and F. Halle´ Kenya ORSTOM, J. Berthaud, CIRAD J.L. Guillaumet, and M. Lourd Yemen IPGRI, CIRAD A.B. Eskes

Countries Maintaining Germplasm

Yemen, Costa Rica, Brazil

z FAO ¼ Food and Agriculture Organization of the United Nations ORSTOM ¼ Office de la Recherche Scientifique et Technique Outre-Mer; renamed Institute de Recherche pour le De´veloppement (IRD) in 1998 CIRAD ¼ Centre de Coope´ration Internationale en Recherche Agronomique pour le De´veloppement IPGRI ¼ International Plant Genetic Resources Institute

Source: Adapted from Anthony et al. 1999; Charrier and Berthaud 1985; Eskes, personal communication.

Rica) (Anthony et al. 1999). C. canephora is well represented in the field collections of Cameroon, India, Ivory Coast, and Madagascar. The latter also holds a unique collection of approximately 50 species of the section Mascarocoffea. Approximately 30 species of diploid African coffee are mainly conserved in Ivory Coast, with, unfortunately, zero accessibility at present due to the ongoing civil war. Exploration missions for wild C. arabica were carried out in its center of origin (Ethiopia and Kenya) and in the secondary center of diversity, Yemen. During 1964–65, a Food and Agriculture Organization of the United Nations (FAO) mission collected coffee germplasm in different locations in Ethiopia (Meyer et al. 1968), followed by an ORSTOM (Office de la Recherche Scientifique et Technique Outre-Mer; renamed Institute de Recherche pour le De´veloppement [IRD] in 1998) mission in 1966 collecting germplasm from 70 different origins (Table 9.1). While some of these materials were obtained from the understory of tropical forests, the majority was collected from cultivated coffee (Anthony et al. 1999). An ORSTOM/CIRAD mission to Kenya collected 80 different materials of C. arabica at Mount Marsabit, as well as samples of C. eugenioides, C. zanguebariae and C. fadenii (Table 9.2). Finally, a 1989 International

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Central African Republic

Ivory Coast

Kenya

Tanzania

Cameroon

Congo

Guinea

1975

1975–1980

1977

1982

1983, 1985, 1987

1986

1987

IPGRI (1983), ORSTOM (1985), CIRAD (1987) IPGRI, IRD, CIRAD IRD, CIRAD

ORSTOM, CIRAD

ORSTOM, CIRAD

ORSTOM

J. Berthaud, F. Anthony, and M. Lourd F. Anthony, E. Couturon, and C. de Namur C. De Namur, E. Couturon, P. Sita, and F. Anthony D. Le Pierre`s, P. Charmetant, A. Yapo, T. Leroy, E. Couturon, S. Bontems, and H. Tehe

J. Berthaud, J.L. Guillaumet, and M. Lourd

J. Berthaud

C. congensis, C.liberica var. dewevrei, C. canephora, C. liberica, Cafeíer de la Nana C. liberica, C. stenophylla, C. canephora, C. humilis, Paracoffea sp., Psilanthus sp. C. arabica, C. eugenioides, C. fadenii, C. pseudozanguebariae, C. fadenii, C. sessiliflora C. costatifructa, C. pseudozanguebariae, C. mufindiensis, C. sessiliflora, C. sp. C. brevipes, C. canephora, C. congensis, C. liberica, 4 species of C. spp.,Psilanthus spp. C. canephora, C. congensis, C. liberica, 5 species of Coffea spp.,Psilanthus spp. C. canephora, C. humilis, C. liberica, C. stenophylla, Psilanthus spp.

50 species

J.F. Leroy, R. Porte`res, A. Vianney-Liaud, J.L. Guillaumet, and A. Charrier J.L. Guillaumet and J. Berthaud

Museum of Paris, CIRAD, ORSTOM

ORSTOM, CIRAD

Coffee Species

Collector

OrganizationZ

Congo, Ivory Coast Guinea, Ivory Coast

Tanzania, Ivory Coast Cameroon, Ivory Coast

Kenya, Ivory Coast

Ivory Coast, Central African Republic Ivory Coast

Madagascar

Country Maintaining Germplasm

Source: Adapted from Anthony et al.1999; Charrier and Berthaud 1985; P. Charmetant, personal communication.

CIRAD ¼ Centre de Coope´ration Internationale en Recherche Agronomique pour le De´veloppement ORSTOM ¼ Office de la Recherche Scientifique et Technique de Outre-Mer; renamed Institute de Recherche pour le De´veloppement (IRD) in 1998. IPGRI ¼ International Plant Genetic Resources Institute

z

Madagascar, Mauritius, Reunion Island, Comoro Islands

1960–1974

Years

Country Explored

Table 9.2. Principal collecting missions for diploid coffee species.

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Plant Genetic Resources Institute1 (IPGRI)/CIRAD mission concentrated on Yemen, where samples were collected from coffee plantations from 22 different origins. The mission identified six morphologically different types of coffee plants. Collecting missions for other Coffea species were motivated by the rapid destruction of the tropical forest ecosystems in Africa, Madagascar, and the Comoros and Mascarene islands. These missions (Table 9.3) resulted in the collection of material from 20,000 wild coffee trees representing more than 70 species and in the identification of 300 wild coffee populations (Anthony et al. 1999). CATIE’s International Coffee Germplasm Center at its headquarters in Turrialba, Costa Rica, dates back to the late 1940s and is being maintained on an area of nine hectares under the shade of the leguminous tree species Erythrina poeppigiana (Walp.) O. F. Cook (known as poro´ in Spanish) and, to a lesser extent, Eucalyptus deglupta Blume. CATIES’s collection is the third largest coffee field gene bank in the world, after those at the Ivory Coast and Cameroon (Monge and Guevara 2000) and comprises to a large extent the entire genetic diversity of C. arabica

Table 9.3. Description of genetic material conserved at CATIE’s International Coffee Germplasm Center for the Western Hemisphere.

Type of Material

No. Introductions

No. Individuals

1. Wild and Semi-wild Genotypes Collected by FAO in Ethiopia Collected by ORSTOM in Ethiopia Collected by IPGRI and CIRAD in Yemen Diploid species SUBTOTAL 1

433 148 11 288 880 (44.3%)

1,650 420 20 592 2,682

2. Cultivars, Mutants, and Selections Cultivars from Ethiopia Cultivars of ‘Typica’ and ‘Bourbon’ Introgressed varieties from C. canephora Mutants and other selections Unclassified varieties SUBTOTAL 2

191 293 312 84 43 923 (46.5%)

950 1,810 1,804 650 250 5,464

3. Hybrids Interspecific hybrids Intraspecific hybrids SUBTOTAL 3 TOTAL (1þ2þ3)

19 165 184 (9.3%) 1,987

90 820 910 9,056


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(Anthony et al. 1999) with a total of 1987 accessions and over 9,000 coffee trees (Table 9.3). The genetic diversity of C. canephora (68 introductions) and C. liberica (24 introductions) is represented to a lesser extent. CATIE’s International Coffee Germplasm Center forms part of the registry of base collections, which were established by the International Board for Plant Genetic Resources (IBPGR) in the 1970s. Since May 2004, all major germplasm collections held in trust at CATIE form part of the International Network of Ex-Situ Collections of the Consultative Group on International Agricultural Research (CGIAR) under the auspices of FAO and the International Treaty on Plant Genetic Resources for Food and Agriculture. With the designation of its collections to FAO, CATIE started to use the FAO standard Material Transfer Agreement (MTA) for the exchange of germplasm. Under this MTA, the recipient of the germplasm agrees not to claim ownership over the material, nor to seek intellectual property rights over the same or its genetic parts or components, in the form received. This applies also to related information received. The recipient further agrees to ensure that any subsequent person or institution to which he/she may make samples available is bound by the same provisions. The recipient of germplasm is encouraged to share the benefits accruing from its use, including commercial use, through such mechanisms as exchange of information, access to and transfer of technology, capacity building, and sharing of benefits arising from commercialization. Within CATIE’s International Coffee Germplasm Center, these types of genetic material are conserved (Table 9.3): 1. Wild and semi-domesticated genotypes obtained from the primary and secondary center of coffee origin and diversity, Ethiopia and Yemen, respectively. They comprise a total of 880 accessions or 44.3% of the entire collection and were collected by FAO in 1964 and 1965 (433 accessions) and ORSTOM in 1966 (148 accessions) in Ethiopia. Eleven accessions were obtained from a collecting mission conducted by IPGRI and CIRAD in 1989 in Yemen. In addition, this group of genotypes comprises 288 accessions of various diploid species. 2. The other major part of the collection (923 accessions or 46.5%) is composed of cultivars, mutants, and selections. Some were obtained from Ethiopia, others were derived from the base populations ‘Typica’ and ‘Bourbon’; some represent introgressed lines from C. canephora as well as unclassified varieties.

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3. Inter- and intraspecific hybrids comprising a total of 184 accessions. 4. Research material (not included in Table 9.3). C. Strategies for Conservation The future of coffee improvement in terms of quality, yield, and host resistance depends on the availability and use of the largely untapped genes found in the wild, in farmers’ fields and in ex situ germplasm collections. The high economic value of coffee genetic resources for breeding purposes was demonstrated by Hein and Gatzweiler (2006). Based on a 30-year discounting period, their cost/benefit calculations resulted in a net present value of genetic resources from Ethiopian highland forests of US $1,458 and $429 million, at discount rates of 5% and 10%, respectively. Part of the genetic diversity existing in the wild and on farms has been collected in the centers of origin and diversity and is being maintained in ex situ gene banks with varying success. Five case studies of coffee gene banks were presented by Dulloo et al. (2001): (1) International Coffee Germplasm Center at CATIE, Costa Rica; (2) Coffee Research Center of Kianjavato and Ilaka Est, Madagascar; (3) coffee gene bank in Jimma, Ethiopia; (4) Coffee Research Foundation, Kenya; and (5) coffee collections at Divo and Mont Tonkoui, Ivory Coast. Among these gene banks, CATIE’s International Coffee Germplasm Center is the only one in the public domain due to its designation to the International Network of Ex Situ Collections under the auspices of FAO. CATIE’s International Coffee Germplasm Center is suffering from three main problems: (1) the age of the trees, most of which were introduced before 1970 and are now some 35 years old; (2) the suboptimal climatic conditions at an elevation of 602 meters above sea level combined with excessive precipitation; and (3) the cultivation method, which is suited for cultivated accessions but is lacking adaptation for the wild genotypes, which clearly differ in their needs in terms of shade, pruning, and fertilizer application, among others. Substantial losses of coffee plants have occurred over the years, resulting in the loss of entire accessions. Especially among the wild genotypes, a number of accessions are represented by only one or two individuals and require immediate attention. Given the public domain status and the high value of CATIE’s coffee collection for future breeding programs in Latin America, the renovation and relocation of this international coffee germplasm collection to a


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new site is of utmost importance. For the rejuvenation, a restructuring of the collection into a base collection, mainly composed of wild and semi-domesticated genotypes from Ethiopia and Yemen, and an active collection, composed of selections, landraces, and varieties, has been proposed. The base and active collections will be established at distinct sites to be able to implement adequate collection management, adapted to the needs of the individuals in each category. Dense and almost permanent shade will be needed for the wild genotypes from Ethiopia, while full sunshine is required for the species from East Africa. The rejuvenation and multiplication of the highly valuable and endangered wild genotypes will be of highest priority and will be carried out first. A rejuvenated and restructured international coffee collection with a functional and easily accessible database of related up-to-date information will serve the entire region as strategic asset for future regional breeding efforts, aiming at the improvement of the competitiveness and sustainability of coffee plantations in Latin America. Since coffee field collections are very costly to maintain and the conserved material is constantly exposed to biotic and abiotic stress, research staff at CATIE developed a methodology for cryopreservation of coffee seeds in liquid nitrogen (196oC or 320oF) aiming at longterm storage. In C. arabica, seed tolerance to the exposure of liquid nitrogen varies greatly, ranging from zero to 74% seed survival after cryopreservation (Va´squez et al. 2005). This susceptibility of coffee seeds seems to be largely due to damages inflicted on the endosperm, as separate experiments have shown that the viability of cryopreserved embryos always remained high. Careful post-thaw rehydration of seeds over a six-week period using an osmo-conditioning treatment led to a dramatic increase in the viability of frozen seeds. Currently, a core subset of 63 accessions of the coffee germplasm collection at CATIE is being cryopreserved. Further studies are necessary to verify whether plantlets derived from long-term cryopreserved seeds are true-to-type in the field. Besides these mainly curational issues, it is urgent to further evaluate the large gene pool. While vegetative growth parameters of the coffee plants have been studied in the range of 20 to 50% of all the accessions in the collection, bean size has only been assessed in 16.9%, leaf rust resistance in 27.3%, and nematode resistance in 3.9% of all accessions. Molecular characterization has been carried out in 7.6% of the accessions. Future research efforts will focus on evaluating the coffee germplasm for distinctive quality flavors and sources of host resistance to diseases and pests.

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III. GENOMICS A. Developing Linkage Maps The development of various types of DNA markers has led to the construction of genetic maps in economically important plant species for crop improvement. Linkage mapping is the first step toward molecular dissection of complex traits such as yield components and quality followed by the cloning of genes or quantitative trait loci (QTLs) controlling these traits, and ultimately, the manipulation of genes to increase productivity and improve quality. Linkage mapping in coffee requires more effort and is more costly than in annual crops due to the longer generation time, a low polymorphism rate in C. arabica with the exception of Ethiopian cultivars and germplasm, and the absence of a large collection of DNA markers and genomic sequences. Genetic maps were first constructed in the diploid C. canephora. The first one was developed using a doubled haploid (DH) population derived from female gametes and 147 restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers. This map consisted of 15 linkage groups with a total length of 1402 cM (Paillard et al. 1996). A second C. canephora genetic map was constructed using a test cross (TC) population derived from the male gametes of the same clone that was used to generate the DH population. This second map consisted of 11 linkage groups spanning 1041 cM with 160 RFLP, RAPD, AFLP, and microsatellite markers, including more than 40 sequence-tagged site (STS), single-copy RFLP, and microsatellite markers (Lashermes et al. 2001). These two linkage maps of C. canephora revealed a substantial amount of genetic information and provided a set of low-copy DNA markers to be used in coffee genetic research and breeding programs. Segregation distortion has been observed in both the DH and TC populations, but distortion was more severe in the DH population (44% of the markers) than in the TC population (13% of the markers). The most significant finding from comparative analysis of these C. canephora maps was that the recombination rates were not distinguishable between the two populations, suggesting similar recombination rates in male and female gametes of C. canephora. Linkage maps were constructed from interspecific crosses that revealed insights on genome structure and the level of heterozygosity among diploid species. A linkage map was constructed from a backcross population between the wild species C. pseudozanguebariae (PSE) and a once-cultivated species C. liberica var. dewevrei (DEW) as the


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backcross parent (Ky et al. 2000). This map consists of 167 AFLP and 13 RFLP markers on 14 linkage groups with a total length of 1,144 cM. Segregation distortion was detected on 30% of the markers mapped with 26 loci distorted in favor of PSE and 21 loci in favor of DEW. The entire linkage group (LG) C consisted of distorted markers in favor of PSE while LG H consisted of distorted markers all in favor of DEW. Reduced recombination rate led to the reduction of map size in this interspecific mapping population compared with the linkage map constructed from intraspecific populations of C. canephora (Paillard et al. 1996; Lashermes et al. 2001). This reduction of map size is likely due to the lower degree of homozygous regions between these two genomes as observed in potato inter- and intraspecific maps (Bonierbale et al. 1988; Gebhardt et al. 1991). A second interspecific map was constructed from a backcross population derived from C. heterocalyx (HET) and C. canephora (CAN) with CAN as the recurrent parent (Coulibaly et al. 2003b). This map consists of 190 AFLP and SSR markers on 15 linkage groups. AFLP markers appeared to be randomly distributed on this interspecific map, independent of the AT/GC content of the selective nucleotides. Similar phenomena were observed on a high-density linkage map of the papaya genome except for the male-specific region of the Y chromosome (Ma et al. 2004), but correlations between AT/GC content of the selective nucleotides and the number of AFLP bands have been reported in soybean (Keim et al. 1997), Alstroemeria (Han et al. 1999), and Pinyon pine (Travis et al. 1998). Another interesting observation from this second interspecific map was that a higher number of total AFLP bands were amplified from the smaller CAN genome than the larger HET genome. This is likely due to the higher level of genome heterozygosity in the self-incompatible CAN than the self-fertile HET (Coulibaly et al. 2003a). Linkage mapping of the C. arabica genome is hindered by the extremely narrow genetic base and its reproductive nature of selfpollination. A preliminary linkage map was constructed using AFLP markers on a pseudo-F2 population derived from a cross between the cultivars ‘Tall Mokka’ and ‘Catimor’ (Pearl et al. 2004). A total of 456 dominant markers and 8 co-dominant markers were generated from 288 AFLP primer combinations. This map consists of 31 linkage groups with the number of markers ranging from 2 to 21, indicating that this was a partial map as gaps remained on the 22 basic chromosomes this map represented. The total length of the map was 1,802.8 cM with an average distance of 10.2 cM between adjacent markers. This genetic map was used for initial mapping of QTLs controlling source-sink traits in the same population. The dramatic differences in leaf and bean

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characteristics and coffee cupping quality between these two parental cultivars make this segregating population an attractive resource for mapping QTLs controlling source-sink traits. A true F2 population was developed and a new genetic map using the true F2 population is currently under construction (Nagai and Ming, unpublished data). B. Mapping Genes and QTLs Controlling Biotic Stress and Economic Traits Mapping QTLs in coffee is more challenging than in annual diploid crops due to its longer generation time and the difficulty in producing and maintaining large mapping populations with multiple replications in the field. Mapping of major genes controlling disease resistance and self-incompatibility has been successful and has provided linked DNA markers for introgression and marker-assisted selection. Three RAPD markers were tagged to the T gene conferring resistance to coffee berry disease caused by Colletotrichum kahawae J.M. Waller & Bridge (Agwanda et al. 1997). These three markers were identified by screening 120 RAPD markers with resistant and susceptible cultivars and segregating progenies of the first and second backcrosses between the donor and the recurrent parent. The self-incompatible (S) locus in C. canephora was mapped on a short linkage group using AFLP markers and backcross populations derived from (C. heterocalyx C. canephora) C. canephora (Coulibaly et al. 2002). The S locus was 10 cM from a closely linked AFLP marker. Fructification time in coffee is an additive trait, and a major gene, Ft1, controlling this trait was located on a linkage group flanked by two tightly linked DNA markers with 1.7 and 3.3 cM, respectively (Akaffou et al. 2003). This gene also showed pleiotropic effect to lower caffeine content and seed weight. A major gene (Mex-1) from C. canephora conferring resistance to the root-knot nematode, Meloidogyne exigua, was identified using a modified bulk segregant analysis (Noir et al. 2003). Mex-1 was mapped with 14 linked AFLP markers within an 8.2 cM interval. There are nine major dominant resistant genes (SH1 – SH9) for coffee leaf rust (Hemileia vastatrix Berk. & Br.) conferring resistance either individually or in combination. A major leaf rust resistance gene (SH3) from C. liberica was located at the end of a short linkage group co-segregating with an AFLP marker (Prakash et al. 2004). The successful tagging of these major genes with AFLP markers confirmed their pronounced effects on specific traits and generated markers for coffee improvement programs. Important economic traits, such as yield and cupping quality, are typical quantitative traits affected by accumulated small effects of


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multiple genes that require large mapping populations tested at multiple sites and in multiple years. It is technically challenging as well as costly to carry out large-scale experiments fitting these requirements. Nevertheless, attempts have been made to map QTLs in smaller populations that would detect those with relatively large effect. Two QTLs were detected for pollen viability in backcross populations derived from (C. heterocalyx C. canephora) C. canephora (Coulibaly et al. 2003b). One QTL contributed to trigonelline accumulation in green coffee beans in reciprocal backcross populations derived from an interspecific cross between C. pseudozanguebariae and C. liberica var. dewevrei (Ky et al. 2000). As sequencing costs continue to decline and more sequencespecific DNA markers become available and affordable, large-scale QTL mapping experiments will be possible in order to target the complex traits involved in the production of high-quality coffee. C. Construction of Large Insert Genomic Libraries Large insert genomic libraries are essential genomic resources for positional cloning, physical mapping, integration of genetic and physical maps, and eventual complete sequencing of the genomes. Two bacterial artificial chromosome (BAC) libraries have been constructed for C. arabica and C. canephora. The C. arabica cultivar ‘IAPAR 59’ was used for BAC library construction for its resistance to leaf rust and rootknot nematode (Sera 2001). This commercial cultivar, widely cultivated in Latin America, was derived from a spontaneous interspecific hybrid between C. arabica and C. canephora known as the ‘Timor hybrid’, and selfed for five generations. The BAC library consists of 88,813 clones with an average insert size of 130 kilobases (kb), representing about eight C. arabica genome equivalents (Noir et al. 2004). Fifteen low-copy RFLP probes on 11 chromosomes of the C. canephora genome were used to characterize the BAC library. Twelve of the 15 probes hybridized to two loci or two alleles of the same locus from the two subgenomes of the allotetraploid C. arabica genome, while the other three probes hybridized to only one allele. This BAC library was obtained from four ligation reactions with different average insert sizes, and 55% of all BAC clones contained insert sizes above 200 kb, which is particularly useful for physical mapping to identify a minimum tiling path for BAC by BAC sequencing of the genome. The BAC library of C. canephora was constructed from genotype 126 and contains 55,296 clones with an average insert size of 135 kb, representing about nine genome equivalents (Leroy et al. 2005). This BAC library was used to study the genomic organization of genes on the

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sucrose biosynthesis pathway and to clone one of the two sucrose synthase genes (CcSUS1). Additional genes with favorable agronomic and organoleptic characteristics in the genotype 126 can be discovered as coffee genomic research moves forward. D. Coffee Expressed Sequence Tags Large-scale genome projects on a variety of plants, animals, and microbes have yielded vast amounts of genomic sequence and expressed sequence tags (ESTs) that have revolutionized genetic and genomic research. In the coffee research community, several EST sequencing projects have been conducted in Brazil, Italy, Colombia, and the United States. The Brazilian EST sequencing project yielded 33,000 unigenes from 214,916 single-pass EST sequences (Vieira et al. 2006). This large collection of ESTs was generated from 37 cDNA libraries of C. arabica, C. canephora, and C. racemosa, and resulted in 130,792, 12,382, and 10,566 clean sequences for each species, respectively. Additional EST resources include 13,175 unigenes from 46,914 ESTs of C. canephora from S. D. Tanksley’s team at Cornell University in the United States (Lin et al. 2005) and 1,512 unigenes from 2,696 ESTs of C. arabica from G. Graziosi’s team at the University of Trieste in Italy (www. coffeedna.net). Detailed comparative genomic analysis revealed a greater degree of similarity on DNA sequences and gene inventory between coffee and tomato than between coffee and Arabidopsis genomes (Lin et al. 2005). About 21% of the coffee unigenes had no match in the Arabidopsis genome, but 90% of these unmatched unigenes have a match in the Solanaceae unigene collection that represent about 75% of the genes in the Solanaceae genome. Moreover, the DNA sequences of coffee unigenes shared greater similarity with Solanaceae homologs than withArabidopsis homologs. These results are consistent with the close phylogenetic relationship between coffee and tomato that shared a common ancestor 89 million years ago, while coffee and Arabidopsis diverged 125 million years ago (Wikstro¨m et al. 2001).

IV. BREEDING A. Historical and New Trends The history of C. arabica introduction into Latin America explains the narrow genetic base of most commercial C. arabica cultivars to this


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continent. These varieties are characterized by a number of desirable homogenous traits, such as high and stable yield and good quality, but at the same time they also present disadvantages, such as high susceptibility to a wide range of diseases and insect pests and low adaptability to specific agro-ecological conditions. The constant threat caused by the emergence of new strains of existing diseases makes it essential to broaden the genetic base of modern varieties. For more than a century, coffee has been an export crop of many Latin American countries, accounting for up to one-third of export earnings (Rice and Ward 1996). In the past 20 years, farmers in Latin America were advised to convert to more intensive production systems, based on high-yielding coffee varieties, growing without shade, but requiring high inputs of fertilizers and pesticides. The increase in yield obtained with new varieties, such as ‘Caturra’ and ‘Catuai’, was generally accompanied by a slight decrease in coffee quality compared with the traditional varieties ‘Typica’ and ‘Bourbon’ (Bertrand et al. 1999). With a clear rise in world coffee production in recent years and concomitant stagnation of consumption, excessively low coffee prices prevailed. These low prices meant that many producers have been barely able to recover their production costs, leading in consequence to a reduction of labor-intensive management practices up to the total abandonment of coffee plantations. However, the gourmet and specialty coffee sectors have consistently been the fastest-growing international coffee markets and are expected to continue to expand by 5% to 10% per year (Giovannucci 2001). Only a few countries have endeavored to make use of these niche markets, which address consumer preferences for single-origin specialty products, high-grade gourmet blends, flavored coffees, and organically grown coffees. Based on consumer demands, these markets are placing special emphasis on sustainable and environmentally friendly production systems. Organic, shade, and Fair Trade coffees are collectively known as sustainable coffees (Giovannucci 2001); these coffees are rewarded with a substantial premium price for being produced in an environmentally friendly and socially responsible manner. A survey conducted among retailers in the United States, representing the largest specialty coffee market in the world, revealed that purchasing decisions were mainly based on coffee quality rather than price and customer demand. Consistency in supply ranked second in importance (Giovannucci 2001). Given the high and further increasing labor costs in Latin American countries, such as Costa Rica, a major shift from high-yielding, averagequality coffee varieties to gourmet or specialty coffee varieties, which

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are suited for sustainable coffee production systems, is required. To address these market needs, coffee breeding programs have to reevaluate their priorities, moving away from yield increase as numberone objective toward the selection and creation of varieties with high cup quality and a broader genetic base. The latter is required to enable the integration of resistance genes for the genetic control of prevalent diseases and pests, thus paving the way for more environmentally friendly or organic production systems. According to van der Vossen (2001), coffee breeders are faced with the challenge of making substantial contributions to:

High bean and cup quality in C. arabica, including caffeine-free coffee. Lower production costs through easier harvesting (compact growth), higher yields per hectare, and reduced disease and pest control by host resistance. Ecologically grown coffee through zero or minimal pesticide use in disease- and pest-resistant varieties.

B. Exploiting Genetic Diversity The wild and semi-domesticated genotypes collected in Ethiopia and conserved in CATIE’s collection present high variability in traits that are of special interest to breeders, such as fertility, bean size, and yield of green coffee (Anthony et al. 1999). Incomplete resistance to coffee leaf rust was also observed in genotypes from Ethiopia (Gil et al. 1990), as well as resistance to Meloidogyne incognita (Anzueto et al. 2001). Thus, the wild genotypes and diploid species of CATIE’s collection are extremely valuable for the breeding of new varieties: They constitute a reservoir of valuable genes for disease resistance and special flavors and are characterized by a high-combining ability in crosses with traditional commercial varieties. An average heterosis effect of 30% has been reported (Bertrand 2002). Speciation in the coffee gene pool was not accompanied by the development of strong crossing barriers. This offers good prospects for the introgression of desirable characteristics from wild into cultivated species for the development of new cultivars with host resistance against major diseases and pests or tolerance against drought, low temperatures, or flooding. Markers are already being developed for the identification of genes controlling the resistance to coffee berry disease (Agwanda et al. 1997) and nematodes (Noir et al. 2003).


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1. Disease Resistance. As early as 1868, leaf rust disease had devastated coffee plantations in Sri Lanka and led to their replacement with tea (Medina-Filho et al. 1984). The disease reached the northeastern coast of Brazil in 1970 and from there spread rapidly to other South and Central American countries, given the high genetic uniformity of cultivated coffee in the Americas. Leaf rust resistance is frequently observed in C. canephora and C. pseudozanguebariae, but can also be found in C. liberica, C. eugenioides, and C. salvatrix (Rodrı´gues Jr. et al. 1975). As mentioned earlier, nine major dominant resistant genes (SH1 – SH9) have been identified conferring leaf rust resistance either individually or in combination. The alleles SH1, SH2, SH4, and SH5 have been traced in C. arabica germplasm of Ethiopian origin (MedinaFilho et al. 1984), while the SH3 gene seems to have introgressed from C. liberica into C. arabica, as shown by Prakash et al. (2004) in a F2 progeny derived from a cross between ‘Matari’ (C. arabica) and the C. liberica-introgressed line ‘S.288’ with a segregation ratio of 3:1, indicating single dominance. Linkage analysis revealed that 23 markers were strongly associated with the SH3 resistance gene and grouped together in a short single linkage group on chromosome fragments introgressed from C. liberica. The resistance gene SH6 is linked to the ‘Timor Hybrid’, the product of a spontaneous natural hybridization between C. arabica and C. canephora, having the phenotype and genotype from C. arabica but showing resistance to all types of leaf rust (Illy and Viani 2005). Cultivars with horizontal, polygenic coffee leaf rust resistance are progressively replacing traditional cultivars in some countries. For example, cultivars selected from ‘Catimor’ and ‘Sarchimor’ populations have resulted in the use of the ‘Colombia’ cultivar in Colombia, ‘IAPAR 59’ in Brazil, ‘IHCAFE 90’ and ‘CR 95’ in Central America, and ‘Cauvery’ in India (Illy and Viani 2005). Similarly, ‘Icatu’, developed in Brazil, originated from an artificial hybrid between C. arabica ‘Bourbon’ and a tetraploid C. canephora. This hybrid was backcrossed with the C. arabica ‘Mundo Novo’ and/or ‘Catuai amarillo’. ‘Icatu’ is also considered as a source of resistance for coffee berry disease and several nematodes (Bertrand et al. 1999). ‘Ruiru 11’ in Kenya and ‘Ababuna’ in Ethiopia are hybrids carrying resistance genes against coffee berry disease (Illy and Viani 2005; van der Vossen 2001). Coffee berry disease (CBD) first infected C. arabica coffee plantations in Kenya and seems to have originated from C. eugenioides, which is indigenous to the mountain forests in western Kenya and Uganda (Illy and Viani 2005). Latin American countries are still free from the disease. The control of CBD by fungicide sprays may account for one-third

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of the total production costs, while crop losses due to CBD may still occur at higher elevations during periods of excessive rains (van der Vossen 1987). Thus, only cultivars with durable CBD resistance can provide sustainable cash income to coffee farmers in Africa. Resistance can be found in C. canephora, the Kenyan cultivar ‘Ruiru 11’, ‘Rume Sudan’, and ‘Timor hybrid’. The resistance is controlled by two major genes in ‘Rume Sudan’ (a dominant R and recessive K gene) and by one gene (the T gene with incomplete dominance) in the ‘Timor hybrid’ (van der Vossen and Walyaro 1980). Other cultivars, such as ‘K7’ and ‘Blue Mountain’, show moderate resistance, which is controlled by the same recessive gene (K gene) of ‘Rume Sudan’. Recently, three RAPD markers could be linked to the T gene in the ‘Timor hybrid’, one of the three genes responsible for CBD resistance (Agwanda et al. 1997). Studies on populations of C. arabica C. canephora hybrids confirmed the presence of CBD resistance in C. canephora coffee (Carvalho et al. 1976). Thus, the resistance gene found in the ‘Timor hybrid’ might have introgressed from C. canephora (van der Vossen and Walyaro 1980). 2. Nematode Resistance. Root-knot nematodes of the genus Meloidogyne constitute a major threat in most countries where C. arabica is grown. Among the most damaging species is M. exigua, which is widespread in Latin America (Campos et al. 1990; Noir et al. 2003). All commercial cultivars grown in Central America (‘Typica’, ‘Bourbon’, ‘Caturra’, ‘Catuai’, ‘Costa Rica 95’, and ‘IHCAFE90’) are susceptible to M. exigua (Bertrand et al. 2001). The long-term economic feasibility of nematicide applications is questionable and not a viable option for sustainable and environmentally friendly production systems. The most promising option would be the selection and breeding of resistant genotypes. While C. arabica is highly susceptible to root-knot nematodes, resistance has been reported in C. canephora accessions (Curi et al. 1970; Bertrand et al. 2001). Studies undertaken by Bertrand et al. (2001) revealed immunity to M. exigua in more than 78% of the C. canephora plants tested and resistance in 100% of the plants. Similarly, the ‘Timor hybrid’ was found to be resistant to M. exigua (Bertrand et al. 2001). In a multi-institutional effort among IRD, CATIE, and other partners, it was shown that the resistance to M. exigua is controlled by a simply inherited major gene (Mex-1) with incomplete dominant expression (Noir et al. 2003). This conclusion was based on an extensive segregation analysis carried out in a large F2 population at CATIE. Inoculation studies conducted in the same institution revealed that nematode multiplication is possible in resistant tissues, but at a very low rate compared with susceptible tissues (Anthony et al. 2005). Fourteen AFLP markers were


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found to be associated with the resistance to M. exigua (Noir et al. 2003). Resistance to root-knot nematodes can also be introgressed into C. arabica from C. bengalis,C. congensis, C. dewvrei, C. eugenioides, C. kapakata, C. liberica, C. racemosa, C. salvatrix, and C. stenophylla (Medina-Filho et al. 1984; Illy and Viani 2005). For many years, C. canephora rootstocks have been successfully employed in C. arabica cultivars in Guatemala to control Pratylenchus and Meloidogyne nematodes (Bertrand et al. 1999). C. arabica cultivars grafted on C. canephora rootstocks presented four times higher productivity than ungrafted C. arabica cultivars (Villain et al. 1999). C. canephora rootstocks have been shown to be highly tolerant against Pratylenchus sp. (Villain et al. 1996), but seem to be much less efficient against M. incognita, which is prevalent in Guatemala (Bertrand et al. 1999). In recent years, progress has been made in Central America with the selection of specific C. canephora rootstocks for C. arabica cultivars that are highly resistant against several Meloidogyne species (Bertrand et al. 2000). Crosses made between two C. canephora accessions of CATIE’s collection (T3561 and T3751) resulted in the cultivar ‘Nemaya’, which is highly resistant against Meloidogyne sp. of El Salvador, M. incognita of Guatemala, as well as M. exigua and M. arabicida of Costa Rica (Bertrand et al. 1999). While among unselected C. canephora rootstocks only 35% of the plants are resistant against the prevalent nematodes in El Salvador and Guatemala, the ‘Nemaya’ cultivar raises the level of resistant plants to 80%. Concerning M. exigua and M. arabicida, ‘Nemaya’ reaches resistance levels of 90% and 80%, respectively. The ‘Nemaya’ cultivar is currently being propagated and in high demand in PROMECAFE (Programa Cooperativo para la Proteccioń y Modernizacioń del Cultivo de Cafe´ en Me´xico, Ame´rica Central, Panama´ y la Repu´blica Dominicana) member countries. At medium (800–1,000 meters [m]) to high altitudes (>1000 m), the lower temperature range negatively affects C. canephora, resulting in yield losses of 10 to 20% in areas with low nematode infestation, and the ‘Nemaya’ cultivar is therefore not recommended (Bertrand et al. 1999). Where Pratylenchus sp. is not a problem, nematode-resistant ‘Catimors’ could be used as rootstocks at higher elevation instead, without the negative effects on vigor and yield as observed with ‘Nemaya’ rootstocks. Selections of the interspecific hybrid ‘Arabusta’ (developed in the Ivory Coast by crossing a tetraploid C. canephora with C. arabica) with the aim of improving the quality of C. canephora (van der Vossen 2001), could offer another alternative. If ‘Arabusta’, having one parent from the cultivar ‘Nemaya’, is crossed with a M. incognita– and M. arabicida–resistant Ethiopian C. arabica tree, the resulting hybrid

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should be nematode resistant and could serve at high altitudes as rootstock for commercial C. arabica cultivars. Chromosome duplication of the ‘Nemaya’ parents has already started at CATIE as a basis for crosses with nematode-resistant C. arabica genotypes (Bertrand et al. 1999). 3. Other Types of Resistance or Tolerance. Most modern distributed C. arabica cultivars, as well as C. canephora and C. congensis, are susceptible to leaf miner in the genus Leucoptera (Medina-Filho et al. 1984). Only the C. arabica cultivar ‘Mokka’ shows limited resistance. Immunity to the leaf miner is found in C. stenophylla, while resistance has been reported for C. racemosa, C.eugenioides, C. kapakata, and C. dewevrei (Medina-Filho et al. 1984; Illy and Viani 2005). As C. arabica cannot be crossed with C. stenophylla, C. canephora could serve as a bridge to transfer leaf miner immunity from C. stenophylla to the C. arabica genome. Global warming has added a new objective to coffee breeding programs: the need to focus on drought tolerance. In Brazil, the largest coffee producer worldwide, drought is considered as a principal environmental stress factor having great impact on coffee production (DaMatta 2004). Drought and high temperature tolerance has been reported for C. racemosa (Guerreiro Filho 1992). The species C. stenophylla is also well adapted to dry conditions (Illy and Viani 2005). C. arabica is characterized by a deeper root system and, therefore, is more drought resistant than C. canephora (Illy and Viani 2005). Within C. arabica, good candidates for drought tolerance seem to be some East African cultivars like ‘SL28’ due to a well-developed root system, outstanding plant vigor, and an ability to retain their leaves under waterstress conditions (van der Vossen and Browning 1978). Indian lines of C. arabica and crosses of these lines with the modern cultivars ‘Mundo Novo’ and ‘Catuai’ are also reported to be drought-tolerant (MedinaFilho et al. 1984). Progenies of C. arabica C. racemosa hybrids are highly resistant to drought, highlighting the value of C. racemosa germplasm in breeding programs. Low-temperature tolerance can be found in C. liberica (Ahmad and Vishveshwara 1980, cited by Anthony et al. 1999), and flooding adaptation can be found in C. congensis. This has been exploited in Madagascar with the ‘Congusta’ hybrids, resulting from spontaneous hybridization between C. canephora and C. congensis (Charrier 1972). C. humilis is also known to grow well in wet environments (Charrier and Berthaud 1985). Uniform flowering and fruit ripening can be imparted from C. racemosa (Illy and Viani 2005). Under severe drought, this species drops all


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its leaves. Regrowth starts with the beginning of the rains, leading to a uniform flowering and berry ripening, which together with compact growth and ease to induce berry abscission, are important selection criteria for mechanical harvesting as practiced in Hawaii. 4. Low Caffeine Content. There are significant differences in the caffeine content among C. arabica cultivars. While the leaf caffeine content of C. laurina is approximately 23% less than in the commercial cultivar ‘Catuai’, the difference in the bean caffeine content is approximately 54% less than in ‘Catuai’ (Illy and Viani 2005). Coffea salvatrix also shows low caffeine content (0.71%). Recently, an Ethiopian C. arabica cultivar has been reported to have close to zero caffeine content in Brazil (Silvarolla et al. 2004); this plant had been obtained from the CATIE coffee collection, and had been deposited at CATIE after the FAO 1964–1965 coffee mission to Ethiopia. In the study by Silvarolla et al. (2004), three plants out of one accession composed of 12 plants showed an average caffeine content of 0.076 % dry weight compared to 1.2 % dry weight of the widely grown ‘Mundo Novo’ cultivar. No caffeine synthase activity could be detected in the leaves of mutated plants leading to an accumulation of theobromine, the precursor of caffeine. Genes involved in the caffeine biosynthesis pathway have been identified (Moisyadi et al. 1998; Kato et al. 2000; Ogawa et al. 2001; Uefuji et al. 2003). Mazzafera and Carvalho (1992) found very low to low seed caffeine content in diploid F1 hybrids of C. eugenioides C. salvatrix and in tetraploid hybrids of C. arabica C. salvatrix and C. arabica C. eugenioides. The development of low-caffeine or caffeine-free cultivars would be of enormous economic potential. Seven to 10 million bags of green coffee are used annually for the costly process of decaffeination. Water and organic solvents are required to remove caffeine from the coffee beans before they are roasted. Decaffeinated coffee beans are sensitive to molds (Illy and Viani 2005). Uneven bean drying may lead to earthy flavors from microorganism spoilage and to the formation of mycotoxins. Moreover, overdrying renders the beans brittle, leading to high losses during the roasting process. C. Propagation Systems 1. Seed Propagation. Propagation by seed is common practice in most coffee breeding programs worldwide (van der Vossen 2001). Seed multiplication of C. arabica F1 hybrids requiring hand pollination of previously emasculated and bagged flowers has been successfully applied on a large scale in Kenya for the propagation of the leaf rust– and coffee

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berry disease–resistant hybrid ‘Ruiru 11’. The production cost of 1 kilogram hybrid seed through emasculation and hand pollination has been estimated at US $55 (Bertrand et al. 1999). Another interesting alternative is the use of male sterility, which has been detected in five coffee trees of CATIE’s C. arabica germplasm collection. This would significantly lower seed production costs of hybrids. Male sterility is controlled by one recessive gene (Dufour et al. 1997). Introgressive breeding efforts have started to produce pure lines of ‘Costa Rica 95’ and ‘IAPAR 59’ based on male sterility (Bertrand et al. 1999). Genetic transformation could be an option to shorten the time required to produce male-sterile female parents for seed production (van der Vossen 2001). 2. Clonal Propagation. A clonal mass propagation system for coffee based on high-frequency somatic embryogenesis and cultivation in liquid medium using temporary immersion has been developed at CATIE (Etienne-Barry et al. 1999; Barry-Etienne et al. 2002a, b). A culture density of 1,600 somatic embryos per 1-L bioreactor and the addition of high levels of sucrose two weeks before direct sowing of the embryos into soil resulted in a 78% plant conversion rate, followed by vigorous plant growth (Etienne-Barry et al. 1999). Barry-Etienne et al. (2002b) reported differences in morphological development from bioreactorproduced embryos that were still evident 40 weeks after planting in the greenhouse. The use of this bioreactor-based clonal mass propagation method allows for large-scale rapid multiplication of high-quality genetic material, as evidenced by the recently completed mass propagation of 100,000 clonal coffee plantlets in CATIE’s tissue culture laboratory. Although differences in genotype response were noted (unpublished data), all 19 hybrids of the PROMECAFE-CIRAD-CATIE regional breeding program were successfully multiplied and transferred to the Instituto de Cafe´ de Costa Rica (ICAFE´) for trials on multiple sites. Coffee micropropagation in bioreactors has been recently reviewed by Etienne et al. (2006). Using a different liquid culture propagation method, Ducos et al. (2003) have reported no differences in C. canephora plant growth when comparing 5,067 trees derived from liquid culture somatic embryos to 548 trees derived from microcuttings. These were planted in the field in the Philippines and in Thailand. 3. F1 Hybrids. Hybrid vigor, or heterosis, has been observed in many intra- and interspecific crosses, resulting in increased yield, better yield stability, and improved adaptation of the F1 hybrids to suboptimal growing conditions (van der Vossen 1987, 2001; Bertrand et al. 1999).


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Better performance under suboptimal climate and soil conditions is seen as a major advantage of hybrids over pure lines by Bertrand et al. (1999), confirming earlier reports of Mońaco and Carvalho (1964). The yield superiority over ‘Caturra’ observed in the ‘Catimor’ cultivars ‘CR 95’ and ‘IHCAFE 90’, resulting from a cross between ‘Caturra’ (CIFC 19/1) and ‘Timor hybrid’ (CIFC 832/1), can be explained by a higher number of berries per node in ‘CR 95’ and increased vigor in ‘IHCAFE 90’ (Bertrand et al. 1999). Ameha and Belachew (1985) observed a heterosis effect of 36% to 60% in Ethiopia when indigenous wild genotypes were hybridized. In Costa Rica an average heterosis effect of 30% was observed in ‘Sarchimor’ and ‘Catimor’ hybrids when compared with the best parent (Bertrand et al. 1997). However, hybrid vigor often expresses itself also in an increase in total biomass, requiring more space per plant, thus reducing the number of plants per hectare in high-density plantations. Nevertheless, increased plant vigor is a considerable advantage under suboptimal growing conditions. Variations in bean size and cup quality parameters of F1 hybrids are mainly due to additive genetic effects (van der Vossen 1987). F1 hybrids tend to produce a higher percentage of defective beans (peaberries and empty berries) than cultivars derived from breeding lines (van der Vossen 1987; Bertrand et al. 1997). Withinfamily selection of the best plants for clonal multiplication is recommended to minimize this problem. Intraspecific hybridization was used as strategy to broaden the genetic base of new C. arabica cultivars and to accumulate in one genotype resistance genes against various diseases and pests in the context of a regional collaborative breeding program between PROMECAFE, CIRAD, and CATIE, initiated in 1991. This regional breeding program selected 19 F1 hybrids resulting from crosses made between wild genotypes from CATIE’s collection, which originated from Ethiopia, Sudan, Kenya, and Yemen, and commercial varieties (‘Caturra’, ‘Catuai’), as well as ‘Catimors’ and ‘Sarchimors’. After four years of evaluation in Costa Rica, Honduras, El Salvador, and Guatemala, 3 out of the 19 F1 hybrids are about to be released to farmers for commercial production. Two breeding lines (LI_L13A44 and LI_L12A28) were derived from the same cross T05296 ‘Rume Sudan’; the third breeding line resulted from a cross between ‘Caturra’ and the Ethiopian genotype T-16725 or ET-41 (FONTAGRO-PROMECAFE 2006). The coffee trees of these three clones are of the dwarf type, but clearly more vigorous than the traditional commercial varieties and show an average yield increase of up to 150%. Bean size of the selected clones is comparable or slightly superior to the traditional varieties, but the percentage of peaberries and empty

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grains is higher. At medium to high altitudes, the organoleptic quality of the coffee is equal to the commercial varieties. At lower elevations, there are indications of improved cupping quality of the selected clones, but this needs to be validated in further trials. D. Future Based on Biotechnology The progress in coffee genomics has provided a wealth of data and has revealed important characteristics for both C. arabica and C. canephora genomes. A collection of genomic resources and molecular tools is available to coffee breeders and geneticists to assist and advance their research and development programs. Future advancements and additional EST and genomic sequencing will provide increasingly practical tools for coffee breeders to develop coffee cultivars with improved quality and enhanced disease and pest resistance. Research efforts in these areas will have immediate and lasting impact on coffee genomics programs and in crop improvement:

Generate low-coverage whole-genome shotgun sequence of the C. arabica genome in order to: (1) provide a large collection of sequence-based DNA markers, such as SSRs and SNPs; (2) overcome the low rate of polymorphism among the majority of the C. arabica coffee varieties for genetic and QTL mapping and cultivar identification; (3) increase the gene inventory complementing to the EST unigenes; and (4) provide genomic sequences of regulatory elements of expressed genes. Identify specific C. arabica DNA markers through genome-wide scanning of thousands of markers and apply these markers for cultivar verification using DNA samples extracted from green beans or even roasted beans (Martellossi et al. 2005). Construct high-density linkage maps of the C. arabica and C. canephora genomes using sequence-based anchor markers as frame scaffolds and high-throughput random DNA markers such as AFLPs to fill the gaps. Develop the capacity to scan whole coffee genomes for QTLs controlling economic traits through the use of saturated genetic maps and microarrays of unigene collections. Integrate coffee genetic and physical maps with most of the abundant EST on the map to generate the ultimate tools for QTL mapping and gene isolation. Uncover the principle of genome evolution in allotetraploid coffee and the mechanism causing the DNA content discrepancy between


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C. arabica (1,176 Mbp) and its progenitors C. canephora (809 Mbp) and C. eugenioides (662 Mbp) (Kiehn 1986; Marie and Brown 1993). Various interesting questions could be answered with the help of molecular biology tools, including: (1) How does C. arabica ‘Maragogipe’, discovered in the state of Bahia (Brazil), in 1870 (Jones 1956) and believed to be a mutant of C. arabica ‘Typica’, compare to other C. arabica varieties? ‘Maragogipe’ coffee beans are also known as elephant beans due to their enormous size. Could the molecular basis for this attribute be identified?; (2) Do truly ‘‘wild’’ Coffea species still occur in Ethiopia (Meyer et al. 1968)? This question might be answered by comparing genetic material from plants found in the forests to that of plants cultivated in nearby areas; (3) How does ‘‘wild’’ C. canephora germplasm (e.g., that found at the Kibale National Park in Uganda; Oryem-Origa et al., 2004) differ from cultivated varieties closeby and elsewhere?; and (4) What genes do C. arabica share with C. canephora? In terms of coffee germplasm, it is imperative for the coffee industry to realize the vital importance of these collections and to actively search for funding mechanisms aimed at ensuring their long-term survival. A combination of sustained germplasm conservation and genomics research is essential for the future of the coffee industry.

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Plant Breeding Reviews: Subject Index Volume 30 index A Allelopathy, 231–258 B Biography, Francesco Salamini, 1–47 Blueberry (highbush) 353–414 Breeding allelopathy, 231–258 blueberry (highbush), 353–414 citrus, 323–352 coffee, 415–447 epigenetics, 49–177 peanut, 295–322 soybean, fatty acid manipulation, 259–294 wild relatives, 149–230 C Citrus breeding (seedlessness), 323–352 Coffee, breeding genomics, germplasm, 415–437 E Epigenetics, 49–177 F Fatty acid genetics and breeding, 259–294

Fruit breeding: blueberry (highbush), 353–414 citrus, seedlessness, 323–352 coffee, 415–437 G Genetics: epigenetics, 49–177 fatty acids in soybean, 259–294 Genetic resources: coffee, 415–437 wild relatives, 149–230 Genomics, coffee, 415–437 Germplasm coffee, 415–437 wild relatives 149–230 L Legume breeding, peanut, 295–322 soybean fatty acid manipulation, 259–294 O Oilseed breeding peanut, 295–322 soybean, 259–294


449

450

PLANT BREEDING REVIEWS: SUBJECT INDEX

P Peanut breeding, 295–322 Plant Breeding and epigenetics, 49–177 Polyploidy, citrus, 323–352 S Salamini, Francisco (biography), 1–47

Seed, citrus, 322–352 Soybean, fatty acid manipulation, 259–294 T Transformation and transgenesis, allelopathy, 231–258

Cumulative Subject Index (Volumes 1–30)

A Adaptation: blueberry, rabbiteye, 5:351–352 durum wheat, 5:29–31 genetics, 3:21–167 testing, 12:271–297 Aglaonema breeding, 23:267–269 Allelopathy, 30:231–258 Alexander, Denton, E. (biography), 22:1–7 Alfalfa: honeycomb breeding, 18:230–232 inbreeding, 13:209–233 in vitro culture, 2:229–234 somaclonal variation, 4:123–152 unreduced gametes, 3:277 Allard, Robert W. (biography), 12:1– 17 Allium cepa, see Onion Almond: breeding self–compatible, 8:313– 338 domestication, 25:290–291 transformation, 16:103 Alocasia breeding, 23:269 Alstroemaria, mutation breeding, 6:75 Amaranth: breeding, 19:227–285

cytoplasm, 23:191 genetic resources, 19:227–285 Animals, long term selection 24(2):169–210, 211–234– Aneuploidy: alfalfa, 10:175–176 alfalfa tissue culture, 4:128–130 petunia, 1:19–21 wheat, 10:5–9 Anther culture: cereals, 15:141–186 maize, 11:199–224 Anthocyanin maize aleurone, 8:91–137 pigmentation, 25:89–114 Anthurium breeding, 23:269–271 Antifungal proteins, 14:39–88 Antimetabolite resistance, cell selection, 4:139–141, 159–160 Apomixis: breeding, 18:13–86 genetics, 18:13–86 reproductive barriers, 11:92–96 rice, 17:114–116 Apple: domestication, 25:286–289 fire blight resistance, 29:315–358 genetics, 9:333–366 rootstocks, 1:294–394


451

452

Apple: (cont.) transformation, 16:101–102 Apricot: domestication, 25:291–292 transformation, 16:102 Arachis, see Peanut in vitro culture, 2:218–224 Artichoke breeding, 12:253–269 Avena sativa, see Oat Avocado domestication, 25:307 Azalea, mutation breeding, 6: 75–76 B Bacillus thuringensis, 12:19–45 Bacteria, long-term selection, 24(2):225–265 Bacterial diseases: apple rootstocks, 1:362–365 cell selection, 4:163–164 cowpea, 15:238–239 fire blight, 29:315–358 maize, 27:156–159 potato, 19:113–122 raspberry, 6:281–282 soybean, 1:209–212 sweet potato, 4:333–336 transformation fruit crops, 16:110 Banana: breeding, 2:135–155 domestication, 25:298–299 transformation, 16:105–106 Barley: anther culture, 15:141–186 breeding methods, 5:95–138 diversity, 21:234–235 doubled haploid breeding, 15:141– 186 gametoclonal variation, 5:368–370 haploids in breeding, 3:219–252 molelcular markers, 21:181–220 photoperiodic response, 3:74, 89– 92, 99 vernalization, 3:109 Bean (Phaseolus):

CUMULATIVE SUBJECT INDEX

breeding, 1:59–102; 10:199–269; 23:21–72 breeding mixtures, 4:245–272 breeding (tropics), 10:199–269 heat tolerance, 10:149 in vitro culture, 2:234–237 long-term selection, 24(2):69–74 photoperiodic response, 3:71–73, 86–92; 16:102–109 protein, 1:59–102 rhizobia interaction, 23:21–72 seed color genetics, 28:239–315 Beet (table) breeding, 22:357–388 Beta, see Beet Biochemical markers, 9:37–61 Biography: Alexander, Denton E., 22:1–7 Allard, Robert W., 12:1–17 Bliss, Frederick A., 27:1–14 Borlaug, Norman E., 28:1–37 Bringhurst, Royce S., 9:1–8 Burton, Glenn W., 3:1–19 Coyne, Dermot E., 23:1–19 Downey, Richard K., 18:1–12 Dudley, J.W., 24(1):1–10 Draper, Arlen D., 13:1–10 Duvick, Donald N., 14:1–11 Gabelman, Warren H., 6:1–9 Hallauer, Arnel R., 15:1–17 Hymowitz, Theodore, 29:1–18 Harlan, Jack R., 8:1–17 Jones, Henry A., 1:1–10 Laughnan, John R. 19:1–14 Munger, Henry M., 4:1–8, Re´dei, George, P., 26:1–33 Peloquin, Stanley J., 25:1–19 Ryder, Edward J., 16:1–14 Sears, Ernest Robert, 10:1–2 Salamini, Francesco, 30:1–47 Simmonds, Norman W., 20:1–13 Sprague, George F., 2:1–11 Vogel, Orville A., 5:1–10 Vuylsteke, Dirk R., 21:1–25 Weinberger, John H., 11:1–10 Yuan, Longping, 17:1–13


Biotechnology: Cucurbitaceae, 27:213–244 Douglas-fir, 27:331–336 politics, 25:21–55 Rosaceae, 27:175–211 Birdsfoot trefoil, tissue culture, 2:228– 229 Blackberry, 8:249–312, 29:19–144 mutation breeding, 6:79 Black walnut, 1:236–266 Bliss, Frederick A. (biography), 27:1– 14 Blueberry: breeding, 5:353–414;13:1–10; 30:353–414 domestication, 25:304 highbush, 30:353–414 rabbiteye, 5:307–357 Borlaug, Norman, E.(biography), 28:1– 37 Brachiaria, apomixis, 18:36–39, 49–51 Bramble: domestication, 25:303 transformation, 16:105 Brassica, see Cole crops napus, see Canola, Rutabaga rapa, see Canola Brassicaceae: incompatibility, 15:23–27 molecular mapping, 14:19–23 Breeding: Aglaonema, 23:267–269 alfalfa via tissue culture, 4:123–152 allelopathy, 30:231–258 almond, 8:313–338 Alocasia, 23:269 amaranth, 19:227–285 apomixis, 18:13–86 apple, 9:333–366 apple fire blight resistance, 29:315– 358 apple rootstocks, 1:294–394 banana, 2:135–155 barley, 3:219–252; 5:95–138; 26:125–169

453

bean, 1:59–102; 4:245–272; 23:21– 72 beet (table), 22:357–388 biochemical markers, 9:37–61 blackberry, 8:249–312; 29:19–144 black walnut, 1:236–266 blueberry, 5:307–357; 30:353–414; bromeliad, 23:275–276 cactus, 20:135–166 Calathea, 23:276 carbon isotope discrimination, 12:81–113 carrot, 19:157–190 cassava, 2:73–134 cell selection, 4:153–173 chestnut, 4:347–397 chimeras, 15:43–84 chrysanthemum, 14:321–361 citrus, 8:339–374; 30:323–352 coffee, 2:157–193; 30:415–447 coleus, 3:343–360 competitive ability, 14:89–138 cowpea, 15:215–274 cucumber, 6:323–359 Cucurbitaceae 27:213–244 cucurbits, 27:213–244 currant, 145–175 cytoplasmic DNA, 12:175–210 diallel analysis, 9:9–36 Dieffenbachia, 271–272 doubled haploids, 15:141–186; 25:57–88 Dougas-fir, 27:245–253 Dracaena, 23:277 drought tolerance, maize, 25:173– 253 durum wheat, 5:11–40 Epepremnum, 23:272–273 epigenetics, 30:49–177 epistasis, 21:27–92 exotic maize, 14:165–187 fern, 23:276 fescue, 3:313–342 Ficus, 23:276 fire blight resistance, 29:315–358

454

Breeding: (cont.) flower color, 25:89–114 foliage plant, 23:245–290 forest tree, 8:139–188 fruit crops, 25:255–320 gene action 15:315–374 genotype x environment interaction, 16:135–178 gooseberry, 29:145–175 grapefruit, 13:345–363 grasses, 11:251–274 guayule, 6:93–165 heat tolerance, 10:124–168 Hedera, 23:279–280 herbicide-resistant crops, 11:155– 198 heritability, 22:9–111 heterosis, 12:227–251 homeotic floral mutants, 9:63–99 honeycomb, 13:87–139; 18:177–249 hybrid, 17:225–257 hybrid wheat, 2:303–319; 3:169–191 induced mutations, 2:13–72 insect and mite resistance in cucurbits, 10:199–269 isozymes, 6:11–54 legumes, 26:171–357 lettuce, 16:1–14; 20:105–133 maize, 1:103–138, 139–161; 4:81– 122; 9:181–216; 11:199–224; 14:139–163, 165–187, 189–236; 25:173–253; 27:119– 173; 28:59–100 meiotic mutants, 28:163–214 mitochondrial genetics, 25:115–238 molecular markers, 9:37–61, 10:184–190; 12:195–226; 13:11– 86; 14:13–37, 17:113–114, 179, 212–215; 18:20–42; 19:31–68, 21:181–220, 23:73–174 mosaics, 15:43–84 mushroom, 8:189–215 negatively associated traits, 13:141– 177 oat, 6:167–207


oil palm, 4:175–201; 22:165–219 onion, 20:67–103 ornamental transgenesis, 28:125– 216 papaya, 26:35–78 palms, 23:280–281 pasture legumes, 5:237–305 pea, snap, 212:93–138 peanut, 22:297–356; 30:295–322 pear fire blight resistance, 29:315– 358 pearl millet, 1:162–182 perennial rye, 13:265–292 persimmon, 19:191–225 Philodendron, 23:2 phosphate efficiency, 29:394–398 plantain, 2:150–151; 14:267–320; 21:211–25 potato, 3:274–277; 9:217–332; 16:15–86; 19:59–155, 25:1–19 proteins in maize, 9:181–216 quality protein maize (QPM), 9:181– 216 raspberry, 6:245–321 recurrent restricted phenotypic selection, 9:101–113 recurrent selection in maize, 9:115– 179; 14:139–163 rice, 17:15–156; 23:73–174 rol genes, 26:79–103 Rosaceae, 27:175–211 rose, 17:159–189 rubber (Hevea), 29:177–283 rutabaga, 8:217–248 sesame, 16:179–228 snap pea, 21:93–138 somatic hybridization, 20:167–225 sorghum male sterility, 25:139– 172 soybean, 1:183–235; 3:289–311; 4:203–243; 21:212–307; 30:250– 294 soybean fatty acids, 30:259–294 soybean hybrids, 21:212–307 soybean nodulation, 11:275–318


soybean recurrent selection, 15:275–313 spelt, 15:187–213 statistics, 17:296–300 strawberry, 2:195–214 sugarcane, 16:272–273; 27:15–158 supersweet sweet corn, 14:189–236 sweet cherry, 9:367–388 sweet corn, 1:139–161; 14:189–236 sweet potato, 4:313–345 Syngonium, 23:274 tomato, 4:273–311 transgene technology, 25:105–108 triticale, 5:41–93; 8:43–90 Vigna, 8:19–42 virus resistance, 12:47–79 wheat, 2:303–319; 3:169–191; 5:11– 40; 11:225–234; 13:293–343, 28:1–37, 39–78 wheat for rust resistance, 13:293– 343 white clover, 17:191–223 wild relatives, 30:149–230 wild rice, 14:237–265 Bringhurst, Royce S. (biography), 9:1–8 Broadbean, in vitro culture, 2:244–245 Bromeliad breeding, 23:275–276 Burton, Glenn W. (biography), 3:1–19 C Cactus: breeding, 20:135–166 domestication, 20:135–166 Cajanus, in vitro culture, 2:224 Calathea breeding, 23:276 Canola, R.K. Downey, designer, 18:1–12 Carbohydrates, 1:144–148 Carbon isotope discrimination, 12:81–113 Carica papaya, see Papaya Carnation, mutation breeding, 6:73–74

455

Carrot breeding, 19: 157–190 Cassava: breeding, 2:73–134 long-term selection, 24(2):74–79 Castanea, see Chestnut Cell selection, 4:139–145, 153–173 Cereal breeding, see Grain breeding Cereal diversity, 21:221–261 Cherry, see Sweet cherry domestication, 25:202–293 Chestnut breeding, 4:347–397 Chickpea, in vitro culture, 2:224–225 Chimeras and mosaics, 15:43–84 Chinese cabbage, heat tolerance, 10:152 Chromosome, petunia, 1:13–21, 31–33 Chrysanthemum: breeding, 14:321–361 mutation breeding, 6:74 Cicer, see Chickpea Citrus: Breeding (seedlessness), 30:323– 352 domestication, 25:296–298 protoplast fusion, 8:339–374 Clonal repositories, see National Clonal Germplasm Repository Clover: in vitro culture, 2:240–244 molecular genetics, 17:191–223 Coffea arabica, see Coffee Coffee, 2:157–193; 30:415–437 Cold hardiness: breeding nectarines and peaches, 10:271–308 wheat adaptation, 12:124–135 Cole crops: Chinese cabbage, heat tolerance, 10:152 gametoclonal variation, 5:371–372 rutabaga, 8:217–248 Coleus, 3:343–360 Competition, 13:158–165 Competitive ability breeding, 14:89– 138

456

Controlling elements, see Transposable elements Corn, see Maize; Sweet corn Cotton, heat tolerance 10:151 Cowpea: breeding, 15:215–274 heat tolerance, 10:147–149 in vitro culture, 2:245–246 photoperiodic response, 3:99 Coyne, Dermot E. (biography), 23:1–19 Cranberry domestication, 25:304–305 Crop domestication and selection, 24(2):1–44 Cryopreservation, 7:125–126,148– 151,167 buds, 7:168–169 genetic stability, 7:125–126 meristems, 7:168–169 pollen, 7:171–172 seed, 7:148–151,168 Cucumber, breeding, 6:323–359 Cucumis sativa, see Cucumber Cucurbitaceae: insect and mite resistance, 10:309– 360 mapping, 213–244 Cucurbits mapping, 213–244 Currant breeding, 29:145–175 Cybrids. 3:205–210; 20: 206–209 Cytogenetics: alfalfa, 10:171–184 blueberry, 5:325–326 cassava, 2:94 citrus, 8:366–370 coleus, 3:347–348 durum wheat, 5:12–14 fescue, 3:316–319 Glycine, 16:288–317 guayule, 6:99–103 maize mobile elements, 4:81–122 maize-tripsacum hybrids, 20:15–66 meiotic mutants, 28:163–214 oat, 6:173–174 polyploidy terminology, 26:105– 124


pearl millet, 1:167 perennial rye, 13:265–292 petunia, 1:13–21, 31–32 potato, 25:1–19 rose, 17:169–171 rye, 13:265–292 Saccharum complex, 16:273–275 sesame, 16:185–189 sugarcane, 27:74–78 triticale, 5:41–93; 8:54 wheat, 5:12–14; 10:5–15; 11:225– 234 Cytoplasm: breeding, 23: 175–210; 25:115–138 cybrids, 3:205–210; 20:206–209 incompatibility, 25:115–138 male sterility, 25:115–138,139–172 molecular biology of male sterility, 10:23–51 organelles, 2:283–302; 6:361–393 pearl millet, 1:166 petunia, 1:43–45 sorghum male sterility, 25:139–172 wheat, 2:308–319 D Dahlia, mutation breeding, 6:75 Date palm domestication, 25:272–277 Daucus, see Carrot Diallel cross, 9:9–36 Dieffenbachia breeding, 23:271–272 Diospyros, see Persimmon Disease and pest resistance: antifungal proteins, 14:39–88 apple rootstocks, 1:358–373 banana, 2:143–147 barley, 26:135–169 blackberry, 8:291–295 black walnut, 1:251 blueberry, rabbiteye, 5:348–350 cassava, 2:105–114 cell selection, 4:143–145, 163–165 citrus, 8:347–349 coffee, 2:176–181 coleus, 3:353


cowpea, 15:237–247 durum wheat, 5:23–28 fescue, 3:334–336 herbicide-resistance, 11:155–198 host-parasite genetics, 5:393–433 induced mutants, 2:25–30 lettuce, 1:286–287 maize, 27:119–173 ornamental transgenesis, 28:145– 147 papaya, 26:161–357 potato, 9:264–285, 19:69–155 raspberry, 6:245–321 rutabaga, 8:236–240 soybean, 1:183–235 spelt, 15:195–198 strawberry, 2:195–214 virus resistance, 12:47–79 wheat rust, 13:293–343 Diversity: landraces, 21:221–261 legumes, 26:171–357 DNA methylation, 18:87–176; 49–177 Doubled haploid breeding, 15:141– 186; 25:57–88 Douglas-fir breeding, 27:245–353 Downey, Richard K. (biography), 18:1–12 Dracaena breeding, 23:277 Draper, Arlen D. (biography), 13:1–10 Drought resistance: durum wheat, 5:30–31 maize, 25:173–253 soybean breeding, 4:203–243 wheat adaptation, 12:135–146 Dudley, J.W. (biography), 24(1):1–10 Durum wheat, 5:11–40 Duvick, Donald N. (biography), 14:1–11 E Elaeis, see Oil palm Embryo culture: in crop improvement, 5:181–236 oil palm, 4:186–187

457

pasture legume hybrids, 5:249–275 Endosperm: balance number, 25:6–7 maize, 1:139–161 sweet corn, 1:139–161 Endothia parasitica, 4:355–357 Epepremnum breeding, 23:272–273 Epistasis, 21:27–92. Escherichia coli, long-term selection, 24(2):225–224 Evolution: coffee, 2:157–193 fruit, 25: 255–320 grapefruit, 13:345–363 maize, 20:15–66 sesame, 16:189 Exploration, 7:9–11, 26–28, 67–94 F Fabaceae, molecular mapping, 14:24– 25 Fatty acid genetics and breeding, 30:259–294 Fern breeding, 23:276 Fescue, 3:313–342 Festuca, see Fescue Fig domestication, 25:281–285 Fire blight resistance, 29:315–358 Flavonoid chemistry, 25:91–94 Floral biology: almond, 8:314–320 blackberry, 8:267–269 black walnut, 1:238–244 cassava, 2:78–82 chestnut, 4:352–353 coffee, 2:163–164 coleus, 3:348–349 color, 25:89–114 fescue, 3:315–316 garlic: 23:211–244 guayule, 6:103–105 homeotic mutants, 9:63–99 induced mutants, 2:46–50 pearl millet, 1:165–166 pistil in reproduction, 4:9–79

458

Floral biology: (cont.) pollen in reproduction, 4:9–79 reproductive barriers, 11:11–154 rutabaga, 8:222–226 sesame, 16:184–185 sweet potato, 4:323–325 Flower: color genetics, 25:89–114 color transgenesis, 28:1128–142 Forage breeding: alfalfa inbreeding, 13:209–233 diversity, 21:221–261 fescue, 3:313–342 perennials, 11:251–274 white clover, 17:191–223 Foliage plant breeding, 23:245–290 Forest crop breeding: black walnut, 1:236–266 chestnut, 4:347–397 Douglas-fir, 27:245–353 ideotype concept, 12:177–187 molecular markers, 19:31–68 quantitative genetics, 8:139–188 rubber (Hevea), 29:177–283 Fragaria, see Strawberry Fruit, nut, and beverage crop breeding: almond, 8:313–338 apple, 9:333–366 apple fire blight resistance, 29:315– 358 apple rootstocks, 1:294–394 banana, 2:135–155 blackberry, 8:249–312; 29:19–144 blueberry, 13:1–10 blueberry, 5:307–357; 30:323–414 breeding, 25:255–320 cactus, 20:135–166 cherry, 9:367–388 citrus, 8:339–374; 30:323–352 coffee, 2:157–193; 30:415–437 currant, 29:145–175 domestication, 25:255–320 fire blight resistance, 29:315–358 genetic transformation, 16:87–134


gooseberry, 29:145–175 grapefruit, 13:345–363 ideotype concept, 12:175–177 incompatability, 28:215–237 mutation breeding, 6:78–79 nectarine (cold hardy), 10: 271–308 origins, 25:255–320 papaya, 26:35–78 peach (cold hardy), 10:271–308 pear fireblight resistance, 29:315– 358 persimmon, 19:191–225 plantain, 2:135–155 raspberry, 6:245–321 strawberry, 2:195–214 sweet cherry, 9:367–388 Fungal diseases: apple rootstocks, 1:365–368 banana and plantain, 2:143– 145, 147 barley, Fusarium head blight, 26:125–169 cassava, 2:110–114 cell selection, 4:163–165 chestnut, 4:355–397 coffee, 2:176–179 cowpea, 15:237–238 durum wheat, 5:23–27 Fusarium head blight (barley), 26:125–169 host-parasite genetics, 5:393–433 lettuce, 1:286–287 maize foliar, 125–142 potato, 19:69–155 raspberry, 6:245–281 soybean, 1:188–209 spelt, 15:196–198 strawberry, 2:195–214 sweet potato, 4:333–336 transformation, fruit crops, 16:111– 112 wheat rust, 13:293–343 Fusarium head blight (barley), 26:125–169


G Gabelman, Warren H. (biography), 6:1–9 Gametes: almond, self compatibility, 7:322– 330 blackberry, 7:249–312 competition, 11:42–46 epigenetics, 30:49–177 forest trees, 7:139–188 maize aleurone, 7:91–137 maize anthocynanin, 7:91–137 mushroom, 7:189–216 polyploid, 3:253–288 rutabaga, 7:217–248 transposable elements, 7:91–137 unreduced, 3:253–288 Gametoclonal variation, 5:359–391 barley, 5:368–370 brassica, 5:371–372 potato, 5:376–377 rice, 5:362–364 rye, 5:370–371 tobacco, 5:372–376 wheat, 5:364–368 Garlic breeding, 6:81, 23:211–244 Genes: action, 15:315–374 apple, 9:337–356 Bacillus thuringensis, 12:19–45 incompatibility, 15:19–42 incompatibility in sweet cherry, 9:367–388 induced mutants, 2:13–71 lettuce, 1:267–293 maize endosperm, 1:142–144 maize protein, 1:110–120, 148–149 petunia, 1:21–30 quality protein in maize, 9:183–184 Rhizobium, 23:39–47 rol in breeding, 26:79–103 rye perenniality, 13:261–288 soybean, 1:183–235 soybean nodulation, 11:275–318 sweet corn, 1:142–144

459

wheat rust resistance, 13:293–343 Genetic engineering: bean, 1:89–91 DNA methylation, 18:87–176 fire blight resistance, 29:315–358 fruit crops, 16:87–134 host-parasite genetics, 5:415–428 legumes, 26:171–357 maize mobile elements, 4:81–122 ornamentals, 125–162 papaya, 26:35–78. rol genes, 26:79–103 salt resistance, 22:389–425 sugarcane, 27:86–97 transformation by particle bombardment, 13:231–260 transgene technology, 25:105–108 virus resistance, 12:47–79 Genetic load and lethal equivalents, 10:93–127 Genetics: adaptation, 3:21–167 almond, self compatibility, 8:322– 330 amaranth, 19:243–248 Amaranthus, see Amaranth apomixis, 18:13–86 apple, 9:333–366 Bacillus thuringensis, 12:19–45 bean seed color: 28:219–315 bean seed protein, 1:59–102 beet, 22:357–376 blackberry, 8:249–312; 29:19–144 black walnut, 1:247–251 blueberry, 13:1–10 blueberry, rabbiteye, 5:323–325 carrot, 19:164–171 chestnut blight, 4:357–389 chimeras, 15:43–84 chrysanthemums, 14:321 clover, white, 17:191–223 coffee, 2:165–170 coleus, 3:3–53 cowpea, 15:215–274 Cucurbitaceae, 27:213–344

460

Genetics: (cont.) cytoplasm, 23:175–210 DNA methylation, 18:87–176 domestication, 25:255–320 durum wheat, 5:11–40 epigenetics, 30:49–177 fatty acids in soybean, 30:259–294 fire blight resistance, 29:315–358 forest trees, 8:139–188 flower color, 25:89–114 fruit crop transformation, 16:87– 134 gene action, 15:315–374 history, 24(1):11–40 host-parasite, 5:393–433 incompatibility: circumvention, 11:11–154 molecular biology, 11:19–42; 28:215–237 sweet cherry, 9:367–388 induced mutants, 2:51–54 insect and mite resistance in Cucurbitaceae, 10:309–360 isozymes, 6:11–54 lettuce, 1:267–293 maize aleurone, 8:91–137 maize anther culture, 11:199–224 maize adaptedness, 28 :101–123 maize anthocynanin, 8:91–137 maize foliar diseases, 27:118–173 maize endosperm, 1:142–144 maize male sterility, 10:23–51 maize mobile elements, 4:81–122 maize mutation, 5:139–180 maize seed protein, 1:110–120, 148– 149 maize soil acidity tolerance, 28:59– 123 male sterility, maize, 10:23–51 mapping, 14:13–37 markers to manage germplasm, 13:11–86 maturity, 3:21–167 meiotic mutants, 163–214 metabolism and heterosis, 10:53–59


mitochondrial, 25:115–138. molecular mapping, 14:13–37 mosaics, 15:43–84 mushroom, 8:189–216 oat, 6:168–174 organelle transfer, 6:361–393 overdominance, 17:225–257 pea, 21:110–120 pearl millet, 1:166, 172–180 perennial rye, 13:261–288 petunia, 1:1–58 phosphate mechanisms, 29: 359– 419 photoperiod, 3:21–167 plantain, 14:264–320 polyploidy terminology, 26:105– 124 potato disease resistance, 19:69–165 potato ploidy manipulation, 3:274– 277; 16:15–86 quality protein in maize, 9:183–184 quantitative trait loci, 15:85–139 quantitative trait loci in animals selection, 24(2):169–210, 211– 224 reproductive barriers, 11:11–154 rhizobia, 21–72 rice, hybrid, 17:15–156, 23:73–174 Rosaceae, 27:175–211 rose, 17:171–172 rubber (Hevea), 29:177–283 rutabaga, 8:217–248 salt resistance, 22:389–425 selection, 24(1):111–131, 143–151, 269–290 snap pea, 21:110–120 sesame, 16:189–195 soybean, 1:183–235 soybean nodulation, 11:275–318 spelt, 15:187–213 supersweet sweet corn, 14:189–236 sweet corn, 1:139–161; 14:189–236 sweet potato, 4:327–330 temperature, 3:21–167 tomato fruit quality, 4:273–311


transposable elements, 8:91–137 triticale, 5:41–93 virus resistance, 12:47–79 wheat gene manipulation, 11:225– 234 green revolution, 28:1–37, 39–78 wheat male sterility, 2:307–308 wheat molecular biology, 11:235–250 wheat rust, 13:293–343 white clover, 17:191–223 yield, 3:21–167 Genome: Glycine, 16:289–317 Poaceae, 16:276–281 Genomics: coffee, 30:415–437 grain legumes, 26:171–357 Genotype æ environment, interaction, 16:135–178 Germplasm, see also National Clonal Germplasm Repositories; National Plant Germplasm System acquisition and collection, 7:160–161 apple rootstocks, 1:296–299 banana, 2:140–141 blackberry, 8:265–267 black walnut, 1:244–247 cactus, 20:141–145 cassava, 2:83–94, 117–119 chestnut, 4:351–352 coffee, 2:165–172 distribution, 7:161–164 enhancement, 7:98–202 evaluation, 7:183–198 exploration and introduction, 7:9– 18,64–94 genetic markers, 13:11–86 guayule, 6:112–125 isozyme, 6:18–21 grain legumes, 26:171–357 legumes, 26:171–357 maintenance and storage, 7:95–110, 111–128, 129–158, 159–182; 13:11–86

461

maize, 14:165–187 management, 13:11–86 oat, 6:174–176 peanut, 22:297–356 pearl millet, 1:167–170 plantain, 14:267–320 potato, 9:219–223 preservation, 2:265–282; 23:291– 344 rights, 25:21–55 rutabaga, 8:226–227 sampling, 29:285–314 sesame, 16:201–204 spelt, 15:204–205 sweet potato, 4:320–323 triticale, 8:55–61 wheat, 2:307–313 wild relatives, 30:149–230 Gesneriaceae, mutation breeding, 6:73 Gladiolus, mutation breeding, 6:77 Glycine, genomes, 16:289–317 Glycine max, see Soybean Gooseberry breeding, 29:145–175 Grain breeding: amaranth, 19:227–285 barley, 3:219–252, 5:95–138; 26:125–169 diversity, 21:221–261 doubled haploid breeding, 15:141– 186 ideotype concept, 12:173–175 maize, 1:103–138, 139–161; 5:139– 180; 9:115–179, 181–216; 11:199– 224; 14:165–187; 22:3–4; 24(1): 11–40, 41–59, 61–78; 24(2): 53– 64, 109–151; 25:173–253: 27:119– 173; 28:59–100, 101–123 maize history, 24(2):31–59, 41–59, 61–78 oat, 6:167–207 pearl millet, 1:162–182 rice, 17:15–156; 24(2):64– 67 sorghum, 25:139–172 spelt, 15:187–213

462

Grain breeding: (cont.) transformation, 13:231–260 triticale, 5:41–93; 8:43–90 wheat, 2:303–319; 5:11–40; 11:225– 234, 235–250; 13:293–343; 22:221–297; 24(2):67–69; 28:1–37, 39–78 wild rice, 14:237–265 Grape: domestication, 25:279–281 transformation, 16:103–104 Grapefruit: breeding, 13:345–363 evolution, 13:345–363 Grass breeding: breeding, 11:251–274 mutation breeding, 6:82 recurrent selection, 9:101–113 transformation, 13:231–260 Growth habit, induced mutants, 2:14– 25 Guayule, 6:93–165 H Hallauer, Arnel R. (biography), 15:1– 17 Haploidy, see also unreduced and polyploid gametes apple, 1:376 barley, 3:219–252 cereals, 15:141–186 doubled, 15:141–186; 25:57–88 maize, 11:199–224 petunia, 1:16–18, 44–45 potato, 3:274–277; 16:15–86 Harlan, Jack R. (biography), 8:1–17 Heat tolerance breeding, 10:129– 168 Herbicide resistance: breeding needs, 11:155–198 cell selection, 4:160–161 decision trees, 18:251–303 risk assessment, 18:251–303 transforming fruit crops, 16:114 Heritability estimation, 22:9–111


Heterosis: gene action, 15:315–374 overdominance, 17:225–257 plant breeding, 12:227–251 plant metabolism, 10:53–90 rice, 17:24–33 soybean, 21:263–320 Hevea, see Rubber Honeycomb: breeding, 18:177–249 selection, 13:87–139, 18:177–249 Hordeum, see Barley Host-parasite genetics, 5:393–433 Hyacinth, mutation breeding, 6:76–77 Hybrid and hybridization, see also Heterosis barley, 5:127–129 blueberry, 5:329–341 chemical, 3:169–191 interspecific, 5:237–305 maize high oil selection, 24(1):153– 175 maize long-term selection, 24(2):43– 64, 109–151 maize history, 24(1): 31–59, 41–59, 61–78 overdominance, 17:225–257 rice, 17:15–156 soybean, 21:263;–320 wheat, 2:303–319 Hymowitz, Theodore (biography), 29:1–18 I Ideotype concept, 12:163–193 In vitro culture: alfalfa, 2:229–234; 4:123–152 barley, 3:225–226 bean, 2:234–237 birdsfoot trefoil, 2:228–229 blackberry, 8:274–275 broadbean, 2:244–245 cassava, 2:121–122 cell selection, 4:153–173 chickpea, 2:224–225


citrus, 8:339–374 clover, 2:240–244 coffee, 2:185–187 cowpea, 2:245–246 embryo culture, 5:181–236, 249– 275 germplasm preservation, 7:125,162–167 introduction, quarantines, 3:411– 414 legumes, 2:215–264 mungbean, 2:245–246 oil palm, 4:175–201 pea, 2:236–237 peanut, 2:218–224 petunia, 1:44–48 pigeon pea, 2:224 pollen, 4:59–61 potato, 9:286–288 sesame, 16:218 soybean, 2:225–228 Stylosanthes, 2:238–240 wheat, 12:115–162 wingbean, 2:237–238 zein, 1:110–111 Inbreeding depression, 11:84–92 alfalfa, 13:209–233 cross pollinated crops, 13:209–233 Incompatibility: almond, 8:313–338 molecular biology, 15:19–42, 28:215–237 pollen, 4:39–48 reproductive barrier, 11:47–70 sweet cherry, 9:367–388 Incongruity, 11:71–83 Industrial crop breeding: guayule, 6:93–165 rubber (Hevea), 29:177–283 sugarcane, 27:5–118 Insect and mite resistance: apple rootstock, 1:370–372 black walnut, 1:251 cassava, 2:107–110 clover, white, 17:209–210

463

coffee, 2:179–180 cowpea, 15:240–244 Cucurbitaceae, 10:309–360 durum wheat, 5:28 maize, 6:209–243 raspberry, 6:282–300 rutabaga, 8:240–241 sweet potato, 4:336–337 transformation fruit crops, 16:113 wheat, 22:221–297 white clover, 17:209–210 Intergeneric hybridization, papaya, 26:35–78 Interspecific hybridization: blackberry, 8:284–289 blueberry, 5:333–341 citrus, 8:266–270 pasture legume, 5:237–305 rose, 17:176–177 rutabaga, 8:228–229 Vigna, 8:24–30 Intersubspecific hybridization, rice, 17:88–98 Introduction, 3:361–434; 7:9–11, 21– 25 Ipomoea, see Sweet potato Isozymes, in plant breeding, 6:11–54 J Jones, Henry A. (biography), 1:1–10 Juglans nigra, see Black walnut K Karyogram, petunia, 1:13 Kiwifruit: domestication, 25:300–301 transformation, 16:104 L Lactuca sativa, see Lettuce Landraces, diversity, 21:221–263 Laughnan, Jack R. (bibliography), 19:1–14 Legume breeding, see also Oilseed, Peanut, Soybean:

464

Legume breeding (cont.) cowpea, 15:215–274 genomics, 26:171–357 pasture legumes, 5:237–305 peanut, 22:297–356; 30:295–322 soybean fatty acid manipulation, 259–294 Vigna, 8:19–42 Legume tissue culture, 2:215–264 Lethal equivalents and genetic load, 10:93–127 Lettuce: genes, 1:267–293 breeding, 16:1–14; 20:105–133 Lingonberry domestication, 25:300–301 Linkage: bean, 1:76–77 isozymes, 6:37–38 lettuce, 1:288–290 maps, molecular markers, 9:37–61 petunia, 1:31–34 Lotus: hybrids, 5:284–285 in vitro culture, 2:228–229 Lycopersicon, see Tomato M Maize: anther culture, 11:199–224; 15:141– 186 anthocyanin, 8:91–137 apomixis, 18:56–64 breeding, 1:103–138, 139–161; 27:119–173 carbohydrates, 1:144–148 cytoplasm, 23:189 doubled haploid breeding, 15:141– 186 drought tolerance, 25:173–253 exotic germplasm utilization, 14:165–187 foliar diseases, 27:119–173 high oil, 22:3–4; 24(1):153–175 history of hybrids, 23(1): 11–40, 41– 59, 61–78


honeycomb breeding, 18:226–227 hybrid breeding, 17:249–251 insect resistance, 6:209–243 long-term selection 24(2):53–64, 109–151 male sterility, 10:23–51 marker-assisted selection. 24(1):293–309 mobile elements, 4:81–122 mutations, 5:139–180 origins, 20:15–66 origins of hybrids, 24(1):31–50, 41– 59, 61–78 overdominance, 17:225–257 physiological changes with selection, 24(1):143–151 protein, 1:103–138 quality protein, 9:181–216 recurrent selection, 9:115–179; 14:139–163 RFLF changes with selection, 24(1):111–131 selection for oil and protein, 24(1):79–110, 153–175 soil acidity tolerance, 28:59–100 supersweet sweet corn, 14:189– 236 transformation, 13:235–264 transposable elements, 8:91–137 unreduced gametes, 3:277 Male sterility: chemical induction, 3:169–191 coleus, 3:352–353 genetics, 25:115–138, 139–172 lettuce, 1:284–285 molecular biology, 10:23–51 pearl millet, 1:166 petunia, 1:43–44 rice, 17:33–72 sesame, 16:191–192 sorghum, 139–172 soybean, 21:277–291 wheat, 2:303–319 Malus spp, see Apple Malus C ¸ domestica, see Apple


Malvaceae, molecular mapping, 14:25–27 Mango: domestication, 25:277–279 transformation, 16:107 Manihot esculenta, see Cassava Mapping: Cucurbitaceae, 27:213–244 Rosaceae, 27:175–211 Medicago, see also Alfalfa in vitro culture, 2:229–234 Meiosis: mutants, 28:239–115 petunia, 1:14–16 Metabolism and heterosis, 10:53–90 Microprojectile bombardment, transformation, 13:231–260 Mitochondrial genetics, 6:377–380; 25:115–138 Mixed plantings, bean breeding, 4:245–272 Mobile elements, see also transposable elements: maize, 4:81–122; 5:146–147 Molecular biology: apomixis, 18:65–73 comparative mapping, 14:13–37 cytoplasmic male sterility, 10:23–51 DNA methylation, 18:87–176 herbicide-resistant crops, 11:155– 198 incompatibility, 15:19–42 legumes, 26:171–357 molecular mapping, 14:13–37; 19:31–68 molecular markers, 9:37–61, 10:184– 190; 12:195–226; 13:11–86; 14:13–37; 17:113–114, 179, 212–215; 18:20–42; 19:31–68, 21:181–220, 23:73–174, 26:292–299 papaya, 26 :35–78 quantitative trait loci, 15:85–139 rol genes, 26 :790103 salt resistance, 22:389–425

465

somaclonal variation, 16:229–268 somatic hybridization, 20:167–225 soybean nodulation, 11:275–318 strawberry, 21:139–180 transposable (mobile) elements, 4:81–122; 8:91–137 virus resistance, 12:47–79 wheat improvement, 11:235–250 Molecular markers, 9:37–61, 10:184– 190; 12:195–226; 13:11–86; 14:13–37; 17:113–114, 179, 212–215; 18:20–42; 19:31–68, 21:181–220, 23:73–174 alfalfa, 10:184–190 apomixis, 18:40–42 barley, 21:181–220 clover, white, 17:212–215 forest crops, 19:31–68 fruit crops, 12:195–226 maize selection, 24(1):293–309 mapping, 14:13–37 plant genetic resource mangement, 13:11–86 rice, 17:113–114, 23:73–124 rose, 17:179 somaclonal variation, 16:238–243 wheat, 21:181–220 white clover, 17:212–215 Monosomy, petunia, 1:19 Mosaics and chimeras, 15:43–84 Mungbean, 8:32–35 in vitro culture, 2:245–246 photoperiodic response, 3:74, 89– 92 Munger, Henry M. (biography), 4:1–8 Musa, see Banana, Plantain Mushroom, breeding and genetics, 8:189–215 Mutants and mutation: alfalfa tissue culture, 4:130–139 apple rootstocks, 1:374–375 banana, 2:148–149 barley, 5:124–126 blackberry, 8:283–284 cassava, 2:120–121

466

Mutants and mutation: (cont.) cell selection, 4:154–157 chimeras, 15:43–84 coleus, 3:355 cytoplasmic, 2:293–295 gametoclonal variation, 5:359–391 homeotic floral, 9:63–99 induced, 2:13–72 long term selection variation, 24(1):227–247 maize, 1:139–161, 4:81–122; 5:139– 180 mobile elements, see Transposable elements mosaics, 15:43–84 petunia, 1:34–40 sesame, 16:213–217 somaclonal variation, 4:123–152; 5:147–149 sweet corn, 1:139–161 sweet potato, 4:371 transposable elements, 4:181–122; 8:91–137 tree fruits, 6:78–79 vegetatively-propagated crops, 6:55–91 zein synthesis, 1:111–118 Mycoplasma diseases, raspberry, 6:253–254 N National Clonal Germplasm Repository (NCGR), 7:40–43 cryopreservation, 7:125–126 genetic considerations, 7:126–127 germplasm maintenance and storage, 7:111–128 identification and label verification, 7:122–123 in vitro culture and storage, 7:125 operations guidelines, 7:113–125 preservation techniques, 7:120– 121 virus indexing and plant health, 7:123–125


National Plant Germplasm System (NPGS), see also Germplasm history, 7:5–18 information systems, 7:57–65 operations, 7:19–56 preservation of genetic resources, 23:291–34 National Seed Storage Laboratory (NSSL), 7:13–14, 37–38, 152– 153 Nectarines, cold hardiness breeding, 10:271–308 Nematode resistance: apple rootstocks, 1:368 banana and plantain, 2:145–146 coffee, 2:180–181 cowpea, 15:245–247 soybean, 1:217–221 sweet potato, 4:336 transformation fruit crops, 16:112– 113 Nicotiana, see Tobacco Nodulation, soybean, 11:275–318 O Oat, breeding, 6:167–207 Oil palm: breeding, 4:175–201, 22:165–219 in vitro culture, 4:175–201 Oilseed breeding: canola, 18:1–20 oil palm, 4:175–201; 22:165–219 peanut, 22:295–356; 30:295–322 sesame, 16:179–228 soybean, 1:183–235; 3:289–311; 4:203–245; 11:275–318; 15:275– 313 Olive domestication, 25:277–279 Onion, breeding history, 20:57–103 Opuntia, see Cactus Organelle transfer, 2:283–302; 3:205– 210; 6:361–393 Ornamentals breeding: chrysanthemum, 14:321–361 coleus, 3:343–360


petunia, 1:1–58 rose, 17:159–189 transgenesis, 28:125–162 Ornithopus, hybrids, 5:285–287 Orzya, see Rice Overdominance, 17:225–257 Ovule culture, 5:181–236 P Palm (Arecaceae): foliage breeding, 23:280–281 oil palm breeding, 4:175–201; 22:165–219. Panicum maximum, apomixis, 18:34– 36, 47–49 Papaya: Breeding, 26:35–78 domestication, 25:307–308 transformation, 16:105–106 Parthenium argentatum, see Guayule Paspalum, apomixis, 18:51–52 Paspalum notatum, see Pensacola bahiagrass Passionfruit transformation, 16:105 Pasture legumes, interspecific hybridization, 5:237–305 Pea: breeding, 21:93–138 flowering, 3:81–86, 89–92 in vitro culture, 2:236–237 Peach: cold hardiness breeding, 10:271– 308 domestication, 25:294–296 transformation, 16:102 Peanut: breeding, 22:297–356 in vitro culture, 2:218–224 Pear: domestication, 25:289–290 transformation, 16:102 Pearl millet: apomixis, 18:55–56 breeding, 1:162–182

467

fire blight resistance, 315–358 Pecan transformation, 16:103 Peloquin, Stanley, J. (biography), 25:1–19 Pennisetum americanum, see Pearl millet Pensacola bahiagrass, 9:101–113 apomixis, 18:51–52 selection, 9:101–113 Pepino transformation, 16:107 Peppermint, mutation breeding, 6:81– 82 Perennial grasses, breeding, 11:251– 274 Perennial rye breeding, 13:261–288 Persimmon: breeding, 19:191–225 domestication, 25:299–300 Petunia spp., genetics, 1:1–58 Phaseolin, 1:59–102 Phaseolus vulgaris, see Bean Philodendrum breeding, 23:273 Phytophthora fragariae, 2:195–214 Phosphate molecular mechanisms, 29:359–419 Pigeon pea, in vitro culture, 2:224 Pineapple domestication, 25:305– 307 Pistil, reproductive function, 4:9–79 Pisum, see Pea Plant breeders; rights, 25:21–55 Plant breeding: epigenetics, 30:49–177 politics, 25:21–55 prediction, 15–40 Plant introduction, 3:361–434; 7:9–11, 21–25 Plant exploration, 7:9–11, 26–28, 67– 94 Plantain: breeding, 2:135–155; 14:267–320; 21:1–25 domestication, 25: 298 Plastid genetics, 6:364–376, see also Organelle

468

Plum: domestication, 25:293–294 transformation, 16:103–140 Poaceae: molecular mapping, 14:23–24 Saccharum complex, 16:269–288 Pollen: reproductive function, 4:9–79 storage, 13:179–207 Polyploidy, see also Haploidy alfalfa, 10:171–184 alfalfa tissue culture, 4:125–128 apple rootstocks, 1:375–376 banana, 2:147–148 barley, 5:126–127 blueberry, 13:1–10 citrus, 30:322–352 gametes, 3:253–288 isozymes, 6:33–34 petunia, 1:18–19 potato, 16:15–86; 25:1–19 reproductive barriers, 11:98–105 sweet potato, 4:371 terminology, 26:105–124 triticale, 5:11–40 Pomegranate domestication, 25:285– 286 Population genetics, see Quantitative Genetics Potato: breeding, 9:217–332, 19:69–165 cytoplasm, 23:187–189 disease resistance breeding, 19:69– 165 gametoclonal variation, 5:376–377 heat tolerance, 10:152 honeycomb breeding, 18:227–230 mutation breeding, 6:79–80 photoperiodic response, 3:75–76, 89–92 ploidy manipulation, 16:15–86 unreduced gametes, 3:274–277 Protein: antifungal, 14:39–88 bean, 1:59–102


induced mutants, 2:38–46 maize, 1:103–138, 148–149; 9:181– 216 Protoplast fusion, 3:193–218; 20: 167– 225 citrus, 8:339–374 mushroom, 8:206–208 Prunus: amygdalus, see Almond avium, see Sweet cherry Pseudograin breeding, amaranth, 19:227–285 Psophocarpus, in vitro culture, 2:237– 238 Q Quantitative genetics: epistasis, 21:27–92 forest trees, 8:139–188 gene interaction, 24(1):269–290 genotype x environment interaction, 16:135–178 heritability, 22:9–111 maize RFLP changes with selection, 24(1):111–131 mutation variation, 24(1): 227–247 overdominance, 17:225–257 population size & selection, 24(1):249–268 selection limits, 24(1):177–225 statistics, 17:296–300 trait loci (QTL), 15:85–139; 19:31–68 variance, 22:113–163 Quantitative trait loci (QTL), 15:85– 138; 19:31–68 animal selection, 24(2):169–210, 211–224 selection limits: 24(1):177–225 Quarantines, 3:361–434; 7:12, 35 R Rabbiteye blueberry, 5:307–357 Raspberry, breeding, 6:245–321 Recurrent restricted phenotypic selection, 9:101–113


Recurrent selection, 9:101–113, 115– 179; 14:139–163 soybean, 15:275–313 Red stele disease, 2:195–214 Re´dei, George P. (bibliography), 26: 1–33. Regional trial testing, 12:271–297 Reproduction: barriers and circumvention, 11:11– 154 foliage plants, 23:255–259 garlic, 23:211–244 Rhizobia, 23:21–72 Rhododendron, mutation breeding, 6:75–76 Ribes, see Currant, Gooseberry Rice, see also Wild rice: anther culture, 15:141–186 apomixis, 18:65 cytoplasm, 23:189 doubled haploid breeding, 15:141– 186 gametoclonal variation, 5:362–364 heat tolerance, 10:151–152 honeycomb breeding, 18:224–226 hybrid breeding, 17:1–15, 15–156; 23:73–174 long-term selection 24(2): 64–67 molecular markers, 73–174 photoperiodic response, 3:74, 89–92 Rosa, see Rose Rosaceae, synteny, 27:175–211 Rose breeding, 17:159–189 Rubber (Hevea) breeding, 29:177–283 Rubus, see Blackberry, Raspberry Rust, wheat, 13:293–343 Rutabaga, 8:217–248 Ryder, Edward J. (biography), 16:1–14 Rye: gametoclonal variation, 5:370–371 perennial breeding, 13:261–288 triticale, 5:41–93 S Saccharum complex, 16:269–288

469

Salamini, Francisco (biography), 30:1–47 Salt resistance: cell selection, 4:141–143 durum wheat, 5:31 yeast systems, 22:389–425 Sears, Ernest R. (biography), 10:1–22 Secale, see Rye Seed: apple rootstocks, 1:373–374 banks, 7:13–14, 37–40, 152–153 bean, 1:59–102; 239–315 citrus, 30:322–350 garlic, 23:211–244 lettuce, 1:285–286 maintenance and storage, 7:95–110, 129–158, 159–182 maize, 1:103–138, 139–161, 4:81–86 pearl millet, 1:162–182 protein, 1:59–138, 148–149 rice production, 17:98–111, 118– 119, 23:73–174 soybean, 1:183–235, 3:289–311 synthetic, 7:173–174 variegation, 4:81–86 wheat (hybrid), 2:313–317 Selection, see also Breeding bacteria, 24(2): 225–265 bean, 24(2): 69–74 cell, 4:139–145, 153–173 crops of the developing world, 24(2):45–88 divergent selection for maize ear length, 24(2):153–168 domestication, 24(2):1–44 Escherichia coli, 24(2): 225–265 gene interaction, 24(1):269–290 genetic models, 24(1):177–225 honeycomb design, 13:87–139; 18:177–249 limits, 24(1):177–225 maize high oil, 24(1):153–175 maize history, 24(1):11–40, 41–59, 61– 78 maize inbreds, 28:101–123

470

maize long term, 24(1):79–110, 111– 131, 133–151; 24(2):53- 64, 109–151 maize oil & protein, 24(1):79–110, 153–175 maize physiological changes, 24(1):133–151 maize RFLP changes, 24(1):111–131 marker assisted, 9:37–61, 10:184–190; 12:195–226; 13:11–86; 14:13– 37; 17:113–114, 179, 212–215; 18:20–42; 19:31–68, 21:181– 220, 23:73–174, 24(1):293–309; 26:292–299 mutation variation, 24(1):227–268 population size, 24(1):249–268 prediction, 19: 15–40 productivity gains in US crops, 24(2):89–106 quantitative trait loci, 24(1):311–335 recurrent restricted phenotypic, 9:101–113 recurrent selection in maize, 9:115– 179; 14:139–163 rice, 24(2): 64–67 wheat, 24(2): 67–69 Sesame breeding, 16:179–228 Sesamum indicum, see Sesame Simmonds, N.W. (biography), 21:1–13 Snap pea breeding, 21:93–138 Solanaceae: incompatibility, 15:27–34 molecular mapping, 14:27–28 Solanum tuberosum, see Potato Somaclonal variation, see also Gametoclonal variation alfalfa, 4:123–152 isozymes, 6:30–31 maize, 5:147–149 molecular analysis, 16:229–268 mutation breeding, 6:68–70 rose, 17:178–179 transformation interaction, 16:229– 268 utilization, 16:229–268


Somatic embryogenesis, 5:205–212; 7:173–174 oil palm, 4:189–190 Somatic genetics, see also Gametoclonal variation; Somaclonal variation: alfalfa, 4:123–152 legumes, 2:246–248 maize, 5:147–149 organelle transfer, 2:283–302 pearl millet, 1:166 petunia, 1:43–46 protoplast fusion, 3:193–218 wheat, 2:303–319 Somatic hybridization, see also Protoplast fusion 20:167–225 Sorghum: male sterility, 25:139–172 photoperiodic response, 3:69–71, 97–99 transformation, 13:235–264 Southern pea, see Cowpea Soybean: cytogenetics, 16:289–317 disease resistance, 1:183–235 drought resistance, 4:203–243 fatty acid manipulation, 259–294 genetics and evolution, 29:1–18 hybrid breeding, 21:263–307 in vitro culture, 2:225–228 nodulation, 11:275–318 photoperiodic response, 3:73–74 recurrent selection, 15:275–313 semidwarf breeding, 3:289–311 Spelt, agronomy, genetics, breeding, 15:187–213 Sprague, George F. (biography), 2:1– 11 Sterility, see also Male sterility, 11:30–41 Starch, maize, 1:114–118 Statistics: advanced methods, 22:113–163 history, 17:259–316


Strawberry: biotechnology, 21: 139–180 domestication, 25:302–303 red stele resistance breeding, 2:195–214 transformation, 16:104 Stress resistance: cell selection, 4:141–143, 161–163 transformation fruit crops, 16:115 Stylosanthes, in vitro culture, 2:238– 240 Sugarcane: Breeding, 27:15–118 mutation breeding, 6:82–84 Saccharum complex, 16:269–288 Synteny, Rosaceae, 27:175–211 Sweet cherry: Domestication, 25:202–293 pollen-incompatibility and selffertility, 9:367–388 transformation, 16:102 Sweet corn, see also Maize: endosperm, 1:139–161 supersweet (shrunken2), 14:189– 236 Sweet potato breeding, 4:313–345; 6:80–81 T Tamarillo transformation, 16:107 Taxonomy: amaranth, 19:233–237 apple, 1:296–299 banana, 2:136–138 blackberry, 8:249–253 cassava, 2:83–89 chestnut, 4:351–352 chrysanthemum, 14:321–361 clover, white, 17:193–211 coffee, 2:161–163 coleus, 3:345–347 fescue, 3:314 garlic, 23:211–244 Glycine, 16:289–317 guayule, 6:112–115

471

oat, 6:171–173 pearl millet, 1:163–164 petunia, 1:13 plantain, 2:136; 14:271–272 rose, 17:162–169 rutabaga, 8:221–222 Saccharum complex, 16:270–272 sweet potato, 4:320–323 triticale, 8:49–54 Vigna, 8:19–42 white clover, 17:193–211 wild rice, 14:240–241 Testing: adaptation, 12:271–297 honeycomb design, 13:87–139 Tissue culture, see In vitro culture Tobacco, gametoclonal variation, 5:372–376 Tomato: breeding for quality, 4:273–311 heat tolerance, 10:150–151 Toxin resistance, cell selection, 4:163–165 Transformation and transgenesis alfalfa, 10:190–192 allelopathy, 30:231–258barley, 26:155–157 cereals, 13:231–260 fire blight resistance, 29:315–358 fruit crops, 16:87–134 maize breeding, 142–156 mushroom, 8:206 ornamentals, 28:125–162 papaya, 26:35–78 rice, 17:179–180 somaclonal variation, 16:229–268 sugarcane, 27:86–97 white clover, 17:193–211 Transpiration efficiency, 12:81–113 Trilobium, long-term selection, 24(2):211–224 Transposable elements, 4:81–122; 5:146–147; 8:91–137 Tree crops, ideotype concept, 12:163– 193

472

Tree fruits, see Fruit, nut, and beverage crop breeding Trifolium, see Clover, White Clover Trifolium hybrids, 5:275–284 in vitro culture, 2:240–244 Tripsacum: apomixis, 18:51 maize ancestry, 20:15–66 Trisomy, petunia, 1:19–20 Triticale, 5:41–93; 8:43–90 Triticosecale, see Triticale Triticum: Aestivum, see Wheat Turgidum, see Durum wheat Tulip, mutation breeding, 6:76 U United States National Plant Germplasm System, see National Plant Germplasm System Unreduced and polyploid gametes, 3:253–288; 16:15–86 Urd bean, 8:32–35 V Vaccinium, see Blueberry Variance estimation, 22:113–163 Vegetable breeding: artichoke, 12:253–269 bean, 1:59–102; 4:245–272, 24(2):69–74; 28:239–315 bean (tropics), 10:199–269 beet (table), 22:257–388 carrot 19: 157–190 cassava, 2:73–134; 24(2):74–79 cucumber, 6:323–359 cucurbit insect and mite resistance, 10:309–360 lettuce, 1:267–293; 16:1–14; 20:105:–133 mushroom, 8:189–215 onion, 20:67–103 pea, 21:93–138 peanut, 22:297–356


potato, 9:217–232; 16:15–861; 19:69–165 rutabaga, 8:217–248 snap pea, 21: 93–138 tomato, 4:273–311 sweet corn, 1:139–161; 14:189–236 sweet potato, 4:313–345 Vicia, in vitro culture, 2:244–245 Vigna, see Cowpea, Mungbean in vitro culture, 2:245–246; 8:19–42 Virus diseases: apple rootstocks, 1:358–359 clover, white, 17:201–209 coleus, 3:353 cowpea, 15:239–240 indexing, 3:386–408, 410–411, 423–425 in vitro elimination, 2:265–282 lettuce, 1:286 maize, 142–156 papaya, 26:35–78 potato, 19:122–134 raspberry, 6:247–254 resistance, 12:47–79 soybean, 1:212–217 sweet potato, 4:336 transformation fruit crops, 16:108– 110 white clover, 17:201–209 Vogel, Orville A. (biography), 5:1–10 Vuylsteke, Dirk R. (biography), 21:1– 25 W Walnut (black), 1:236–266 Walnut transformation, 16:103 Weinberger, John A. (biography), 11:1–10 Wheat: anther culture, 15:141–186 apomixis, 18:64–65 chemical hybridization, 3:169–191 cold hardiness adaptation, 12:124– 135 cytogenetics, 10:5–15


cytoplasm, 23:189–190 diversity, 21:236–237 doubled haploid breeding, 15:141– 186 drought tolerance, 12:135–146 durum, 5:11–40 gametoclonal variation, 5:364–368 gene manipulation, 11:225–234 green revolution, 2*; 1–37, 39–58 heat tolerance, 10:152 hybrid, 2:303–319; 3:185–186 insect resistance, 22:221–297 in vitro adaptation, 12:115–162 long-term selection, 24(2):67–69 molecular biology, 11:235–250 molecular markers, 21:191–220 photoperiodic response, 3:74

473

rust interaction, 13:293–343 triticale, 5:41–93 vernalization, 3:109 White clover, molecular genetics, 17:191–223 Wild rice, breeding, 14:237–265 Winged bean, in vitro culture, 2:237– 238 Y Yeast, salt resistance, 22:389–425 Yuan, Longping (biography), 17:1–13. Z Zea mays, see Maize, Sweet corn Zein, 1:103–138 Zizania palustris, see Wild rice

Cumulative Contributor Index (Volumes 1–30)

Abbott, A.G., 27:175 Abdalla, O.S., 8:43 Acquaah, G., 9:63 Aldwinckle, H.S., 1:294; 29:315 Alexander, D.E., 24(1):53 Anderson, N.O., 10:93; 11:11 Aronson, A.I., 12:19 Aruna, R., 30:295 Aru´s, P., 27:175 Ascher, P.D., 10:93 Ashri, A., 16:179 Baggett, J.R. 21:93 Balaji, J., 26:171 Baltensperger, D.D., 19:227 Barker, T., 25:173 Bartels, D., 30:1 Basnizki, J., 12:253 Bassett, M.J., 28:239 Beck, D.L., 17:191 Beebe, S., .23:21-72 Beineke, W.F., 1:236 Bell, A.E., 24(2):211 Below, F.E., 24(1):133 Bertin, C. 30:231

Bertioli, D.J., 30:179 Berzonsky, W.A., 22:221 Bingham, E.T., 4:123; 13:209 Binns, M.R., 12:271 Bird, R. McK., 5:139 Bjarnason, M., 9:181 Blair, M.W., 26; 30:179 Bliss, F.A., 1:59; 6:1 Boase, M.R., 14:321 Borlaug, N.E., 5:1 Boyer, C.D., 1:139 Bravo, J.E., 3:193 Brenner, D.M., 19:227 Bressan , R.A., 13:235; 14:39; 22:389 Bretting, P.K., 13:11 Broertjes, C., 6:55 Brown, A.H.D., 221 Brown, J.W.S., 1:59 Brown, S.K., 9:333, 367 Buhariwalla, H.K., 26:171 Buńger, L., 24(2):169 Burnham, C.R., 4:347 Burton, G.W., 1:162; 9:101 Burton, J.W., 21:263 Byrne, D., 2:73

Camadro, E.L., 26:105 Campbell, K.G., 15:187 Campos, H., 25: 173 Cantrell, R.G., 5:11 Cardinal, A.J., 30:259 Carputo, D., 25:1; 26:105; 28:163 Carvalho, A., 2:157 Casas, A.M., 13:235 Cervantes-Martinez, C.T., 22:9 Chen, J., 23: 245 Cherry, M., 27:245. Chew, P.S., 22:165 Choo, T.M., 3:219; 26:125 Christenson, G.M., 7:67 Christie, B.R., 9:9 Clark, J.R., 29:19 Clark, R.L., 7:95 Clarke, A.E., 15:19 Clegg, M.T., 12:1 Cle´ment-Demange, A, 29:177 Comstock, J.G., 27:15 Consiglio, F., 28:163 Condon, A.G., 12:81 Conicella, C., 28:163


475

476

Cooper, M, 24(2):109; 25:173 Cooper, R.L., 3:289 Cornu, A., 1:11 Costa, W.M., 2:157 Cregan, P., 12:195 Crouch, J.H., 14:267; 26:171 Crow, J.F., 17:225 Cummins, J.N., 1:294 Dambier, D. 30:323 Dana, S., 8:19 Dean, R.A., 27:213 De Jong, H., 9:217 D’Hont, A., 27:15 Dekkers, J.C.M., 24(1):311 Deroles, S.C., 14:321 Dhillon, B.S., 14:139 Dickmann, D.I., 12:163 Ding, H., 22:221 Dirlewanger, E., 27:175 Dodds, P.N., 15:19 Dolan, D., 25:175 Donini, P., 21:181 Dowswell, C., 28:1 Draper, A.D., 2:195 Drew, R., 26:35 Dudley, J.W. 24(1):79 Dumas, C., 4:9 Duncan, D.R., 4:153 Duvick, D.N., 24(2):109 Dwivedi, S.L., 26:171; 30:179 Ebert, A.W., 30:415 Echt, C.S., 10:169 Edmeades, G., 25:173 Ehlers, J.D., 15:215 England, F., 20:1 Eubanks, M.W., 20:15 Evans, D.A., 3:193; 5:359 Everett, L.A., 14:237 Ewart, L.C., 9:63

CUMULATIVE CONTRIBUTOR INDEX

Farquhar, G.D., 12:81 Fasoula, D.A., 14:89; 15:315; 18:177 Fasoula, V.A., 13:87; 14:89; 15:315; 18:177 Fasoulas, A.C., 13:87 Fazuoli, L.C., 2:157 Fear, C.D., 11:1 Ferris, R.S.B., 14:267 Finn, C.E., 29:19 Flore, J.A., 12:163 Forsberg, R.A., 6:167 Forster, B.P., 25:57 Forster, R.L.S., 17:191 Fowler, C., 25:21 Frei, U., 23:175 French, D.W., 4:347 Friesen, D.K., 28:59 Froelicher, Y. 30:323 Frusciante, L., 25:1; 28:163 Gai, J., 21:263 Galiba, G., 12:115 Galletta, G.J., 2:195 Gepts, P., 24(2):1 Glaszmann, J.G., 27:15 Gmitter, F.G., Jr., 8:339; 13:345 Gold, M.A., 12:163 Goldman, I.L. 19:15; 20:67; 22:357; 24(1):61; 24(2):89 Goldway, M., 28:215 Gonsalves, D., 26:35 Goodnight, C.J, 24(1):269 Gordon, S.G., 27:119 Gradziel, T.M., 15:43 Gressel, J., 11:155; 18:251 Gresshof, P.M., 11:275 Griesbach, R.J., 25:89 Grombacher, A.W., 14:237

Grosser, J.W., 8:339 Grumet, R., 12:47 Gudin, S., 17:159 Guimara˜es, C.T., 16:269 Gustafson, J.P., 5:41; 11:225 Guthrie, W.D., 6:209 Habben, J., 25:173 Haley, S.D., 22:221 Hall, A.E., 10:129; 12:81; 15:215 Hall, H.K., 8:249; 29:19 Hallauer, A.R., 9:115; 14:1,165; 24(2):153 Hamblin, J., 4:245 Hancock, J.F., 13:1 Hancock, J.R., 9:1 Hanna, W.W., 13:179 Harlan, J.R., 3:1 Harris, M.O., 22: 221 Hasegawa, P.M. 13:235; 14:39: 22:389 Havey, M.J., 20:67 Henny, R.J., 23:245 Hill, W.G., 24(2):169 Hillel, J., 12:195 Hjalmarsson, I., 29:145 Hoa, T.T.T., 29:177 Hodgkin, T., 21:221 Hokanson, S.C., 21: 139 Holbrook, C.C., 22: 297 Holland, J.B: 21: 27; 22: 9 Hor, T.Y., 22:165 Howe, G.T., 27:245 Hunt, L.A., 16:135 Hutchinson, J.R., 5:181 Hymowitz, T., 8:1; 16:289 Ivañ Ortiz-Monasterio, J., 28:39


Jain, A., 29:359 Janick, J., 1:xi; 23:1; 25:255 Jansky, S., 19:77 Jayaram, Ch., 8:91 Jayawickrama, K., 27:245 Jenderek, M.M., 23:211 Jifon, J., 27:15 Johnson, A.A.T., 16:229; 20:167 Johnson, G.R., 27:245 Johnson, R., 24(1):293 Jones, A., 4:313 Jones, J.S., 13:209 Joobeur, T., 27:213 Ju, G.C., 10:53 Kang, H., 8:139 Kann, R.P., 4:175 Kapazoglou, A. 30:49 Karmakar, P.G., 8:19 Kartha, K.K., 2:215, 265 Kasha, K.J., 3:219 Kaur, H. 30:231 Keep, E., 6:245 Kleinhofs, A., 2:13 Keightley, P.D., 24(1):227 Knox, R.B., 4:9 Koebner, R.M.D., 21:181 Kollipara, K.P., 16:289 Koncz, C., 26:1 Kononowicz, A.K., 13:235 Konzak, C.F., 2:13 Kovacˇevie´l, N.M., 30:49 Krikorian, A.D., 4:175 Krishnamani, M.R.S., 4:203 Kronstad, W.E., 5:1 Kuehnle, A.R., 28:125 Kulakow, P.A., 19:227 Lamb, R.J., 22:221

Lambert, R.J., 22: 1; 24(1):79, 153 Lamborn, C., 21:93 Lamkey, K.R., 15:1; 24(1):xi; 24(2):xi Lavi, U., 12:195 Layne, R.E.C., 10:271 Lebowitz, R.J., 3:343 Lee, M., 24(2):153 Lehmann, J.W., 19:227 Lenski, R.E., 24(2):225 Levings, III, C.S., 10:23 Lewers, K.R., 15:275 Li, J., 17:1,15 Liedl, B.E., 11:11 Lin, C.S., 12:271 Lockwood, D.R., 29:285 Lovell, G.R., 7:5 Lower, R.L., 25:21 Lukaszewski, A.J., 5:41 Luro, F., 30:323 Lyrene, P.M., 5:307; 30:353 Maas, J. L., 21: 139 Mackenzie, S.A., 25:115 Maheswaran, G., 5:181 Maizonnier, D., 1:11 Malnoy, M., 29:285 Marcotrigiano, M., 15:43 Martin, F.W., 4:313 Matsumoto, T.K. 22:389 McCoy, T.J., 4:123; 10:169 McCreight, J.D., 1:267; 16:1 McDaniel, R.G., 2:283 McKeand, S.E., 19:41 McKenzie, R.I.H., 22:221 McRae, D.H., 3:169 Medina-Filho, H.P., 2:157 Mejaya, I.J., 24(1): 53 Mikkilineni, V., 24(1):111

477

Miles, D., 24(2):211 Miles, J.W., 24(2):45 Miller, R., 14:321 Ming, R., 27:15; 30:415 Mirkov, T.E., 27:15 Mobray, D., 28:1 Mondragon Jacobo, C., 20:135 Monti, L.M., 28 :163 Moose, S.P., 24(1):133 Moore, P.H., 27:15 Morrison, R.A., 5:359 Mowder, J.D., 7:57 Mroginski, L.A., 2:215 MudaligeJayawickrama, 28:125 Muir, W.M., 24(2):211 Mumm, R.H., 24(1):1 Murphy, A.M., 9:217 Mutschler, M.A., 4:1 Myers, J.R., 21:93 Myers, O., Jr., 4:203 Myers, R.L., 19:227. Namkoong, G., 8:1 Narro Leoń, L.A., 28:59 Navazio, J., 22:357 Neuffer, M.G., 5:139 Newbigin, E., 15:19 Nigam, S.N. 30:295 Nielen, S., 30:179 Nyquist, W.E., 22:9 Ohm, H.W., 22:221 Ollitrault, P., 30:323 O’Malley, D.M., 19:41 Ortiz, R., 14:267; 16:15; 21:1; 25:1, 139; 26:171; 28:1, 39; 30:179 Osborn, T.C., 27:1 Palmer, R.G., 15:275, 21:263; 29:1

478

Pandy, S., 14:139; 24(2):45; 28:59 Pardo, J. M., 22:389 Parliman, B.J., 3:361 Paterson, A.H., 14:13; 26:15 Patterson, F.L., 22:221 Peairs, F.B., 22:221 Pedersen, J.F., 11:251 Perdue, R.E., Jr., 7:67 Peiretti, E.G., 23:175 Peloquin, S.J., 26 :105 Peterson, P.A., 4:81; 8:91 Polidoros, A.N., 18:87; 30:49 Porter, D.A., 22:221 Porter, R.A., 14:237 Powell, W., 21:181 Prasartsee, V., 26:35 Pratt, R.C., 27:119 Priyadarshan, P.M., 29:177 Proudfoot, K.G., 8:217 Rackow, G., 18:1 Raghothama, K.G., 29:359 Rai, M., 27:15 Raina, S.K., 15:141 Rajaram, S. 28:1 Ramage, R.T., 5:95 Ramesh, S., 25:139 Ramming, D.W., 11:1 Ratcliffe, R.H., 22:221 Ray, D.T., 6:93 Reddy, B.V.S., 25:139 Redei, G.P., 10:1; 24(1):11 Reimann-Phillipp, R., 13:265 Reinbergs, E., 3:219 Reynolds, M.P., 28:39 Rhodes, D., 10:53 Richards, C.M., 29:285


Richards, R.A., 12:81 Roath, W.W., 7:183 Robinson, R.W., 1:267; 10:309 Rochefored.T.R., 24(1):111 Ron Parra, J., 14:165 Roos, E.E., 7:129 Ross, A.J., 24(2):153 Rotteveel, T., 18:251 Rowe, P., 2:135 Russell, W.A., 2:1 Rutter, P.A., 4:347 Ryder, E.J., 1:267; 20:105 Sahi, S.V., 2:359. Samaras, Y., 10:53 Sansavini, S., 16:87 Sapir, G., 28:215 Saunders, J.W., 9:63 Savidan, Y., 18:13 Sawhney, R.N., 13:293 Schaap, T., 12:195 Schaber, M.A. 24(2):89 Schneerman, M.C. 24(1):133 Schnell, R.J., 27:15 Schroeck, G., 20:67 Schussler, J., 25:173 Scott, D.H., 2:195 Seabrook, J.E.A., 9:217 Sears, E.R., 11:225 Seebauer, J.R., 24(1):133 Serraj, R., 26:171 Shands, Hazel L., 6:167 Shands, Henry L., 7:1, 5 Shannon, J.C., 1:139 Shanower, T.G., 22:221 Shattuck, V.I., 8:217; 9:9 Shaun, R., 14:267 Sidhu, G.S., 5:393 Silva, da, J., 27:15 Simmonds, N.W., 17:259

Simon, P.W., 19:157; 23:211 Singh, B.B., 15:215 Singh, R.J., 16:289 Singh, S.P., 10:199 Singh, Z., 16:87 Slabbert, M.M., 19:227 Sleper, D.A., 3:313 Sleugh, B.B., 19 Smith, J.S.C., 24(2):109 Smith, S.E., 6:361 Snoeck, C., 23:21 Socias i Company, R., 8:313 Sobral, B.W.S., 16:269 Soh, A.C., 22:165 Sondahl, M.R., 2:157 Spoor, W., 20: 1 Stafne, E.T., 29:19 Stalker, H.T., 22:297; 30:179 Steadman, J.R., 23:1 Steffensen, D. M., 19:1 Stern, R.A., 28:215 Stevens, M.A., 4:273 Stoner, A.K., 7:57 Stuber, C.W., 9:37; 12:227 Sugiura, A., 19:191 Sun, H. 21:263 Suzaki, J.Y., 26 :35 Tai, G.C.C., 9:217 Talbert, L.E., 11:235 Tan, C.C., 22:165 Tani, E., 30:49 Tarn, T.R., 9:217 Tehrani, G., 9:367 Teshome, A., 21:221 Tew, T.L., 27:15 Thomas, W.T.B., 25:57 Thompson, A.E., 6:93 Tiefenthaler, A.E. 24(2):89


Towill, L.E., 7:159, 13:179 Tracy, W.F., 14:189; 24(2):89 Trethowan, R.M., 28:39 Tripathi, S., 26:35 Troyer, A.F., 24(1):41; 28:101 Tsaftaris, A.S., 18:87; 30:49 Tsai, C.Y., 1:103 Ullrich, S.E., 2:13 Upadhyaya, H.D., 26:171; 39:179 Uribelarrea, M., 24(1):133 Vanderleyden, J., .23:21 Van Harten, A.M., 6:55 Varughese, G., 8:43 Vasal, S.K., 9:181; 14:139 Vasconcelos, M.J., 29:359 Vega, F.E., 30:415 Vegas, A., 26:35 Veilleux, R., 3:253; 16:229; 20:167 Venkatachalam, P., 29 :177

Villareal, R.L., 8:43 Vogel, K.P., 11:251 Volk, G.M., 23:291; 29:285 Vuylsteke, D., 14:267 Wallace, B., 29:145 Wallace, D.H., 3:21; 13:141 Walsh, B. 24(1):177 Wan, Y., 11:199 Wang, Y.-H., 27:213 Waters, C., 23:291 Weber, K., 24(1):249 Weeden, N.F., 6:11 Wehner, T.C., 6:323 Welander, M., 26:79 Wenzel, G. 23:175 Weston, L.A. 30:231 Westwood, M.N., 7:111 Wheeler, N.C., 27:245 Whitaker, T.W., 1:1 White, D.W.R., 17:191 White, G.A., 3:361; 7:5 Widholm, J.M., 4:153; 11:199 Widmer, R.E., 10:93 Widrlechner, M.P., 13:11 Wilcox, J.R., 1:183

479

Williams, E.G., 4:9; 5:181, 237 Williams, M.E., 10:23 Wilson, J.A., 2:303 Wong, G., 22: 165 Woodfield, D.R., 17:191 Wright, D., 25:173 Wright, G.C., 12:81 Wu, K.-K., 27:15 Wu, L., 8:189 Wu, R., 19:41 Xin, Y., 17:1,15 Xu, S., 22:113 Xu, Y., 15:85; 23:73 Yamada, M., 19:191 Yamamoto, T., 27:175 Yan, W., 13:141 Yang, W.-J., 10:53 Yonemori, K., 19:191 Yopp, J.H., 4:203 Yun, D.-J., 14:39 Zeng, Z.-B., 19:41 Zhu, L.-H., 26:79 Zimmerman, M.J.O., 4:245 Zinselmeier, C., 25:173 Zohary, D., 12:253

Plate 7.3. Comparison of diploid (left) and autoPlate 7.2. Fruits of triploid tetraploid (right) Citrus ‘Tahiti lime’ (left) and dipl- deliciosa seedlings. oid ‘Mexican lime’ (right). Plate 7.1. Polyembryonic seeds of Citrus volkameriana.

Plate 7.4. Achievements at CIRAD/INRA of triploid breeding of citrus with the 2x 2x strategy.


Plate 7.5. Achievements at CIRAD of triploid breeding of citrus with the 2x 4x strategy.

Plate 7.6. Achievements at CIRAD of triploid breeding of citrus with the 2x x strategy.

Plant Breeding Reviews, Volume 30

Plant Breeding Reviews (Volume 23)

Plant Breeding Reviews (Volume 26)

Plant Breeding Reviews (Volume 25)

Plant Breeding Reviews (Volume 35)

Plant Breeding Reviews (Volume 31)

Plant Breeding Reviews, Volume 17

Plant Breeding Reviews, Volume 20

Plant Breeding Reviews, Volume 22

Plant Breeding Reviews (Volume 27)

Plant Breeding Reviews - Raspberry Breeding and Genetics (Volume 32)

Light and Plant Development (Annual Plant Reviews, Volume 30)

Horticultural Reviews (Volume 30)

Plant Epigenetics (Annual Plant Reviews, Volume 19)

Annual Plant Reviews, Plant Proteomics (Volume 28)

Plant Reproduction (Annual Plant Reviews, Volume 6)

Cereals (Handbook of Plant Breeding, Volume 3)

Plant Molecular Breeding

Fruit Breeding (Handbook of Plant Breeding, volume 8)

Annual Plant Reviews, Plant Mitochondria

Plant Breeding and Biotechnology

Plant-Pathogen Interactions (Annual Plant Reviews S.) (Annual Plant Reviews)

Molecular Plant Breeding (Cabi)

Plant Hormone Signaling (Annual Plant Reviews, Volume 24)

Plastids (Annual Plant Reviews, Volume 13)

Root Development (Annual Plant Reviews, Volume 37)

Biology of the Plant Cuticle (Annual Plant Reviews, Volume 23)

Annual Plant Reviews, Plant Polysaccharides: Biosynthesis and Bioengineering (Volume 41)

Plant Cell Separation and Adhesion (Annual Plant Reviews, Volume 25)

Annual Plant Reviews, Plant Hormone Signaling (Volume 24)

Plant Breeding Reviews, Volume 30

Plant Breeding Reviews (Volume 23)

Plant Breeding Reviews (Volume 26)

Plant Breeding Reviews (Volume 25)

Plant Breeding Reviews (Volume 35)

Plant Breeding Reviews (Volume 31)

Plant Breeding Reviews, Volume 17

Plant Breeding Reviews, Volume 20

Plant Breeding Reviews, Volume 22

Plant Breeding Reviews (Volume 27)

Plant Breeding Reviews - Raspberry Breeding and Genetics (Volume 32)

Light and Plant Development (Annual Plant Reviews, Volume 30)

Horticultural Reviews (Volume 30)

Plant Epigenetics (Annual Plant Reviews, Volume 19)

Annual Plant Reviews, Plant Proteomics (Volume 28)

Plant Reproduction (Annual Plant Reviews, Volume 6)

Cereals (Handbook of Plant Breeding, Volume 3)

Plant Molecular Breeding

Fruit Breeding (Handbook of Plant Breeding, volume 8)

Annual Plant Reviews, Plant Mitochondria

Plant Breeding and Biotechnology

Plant-Pathogen Interactions (Annual Plant Reviews S.) (Annual Plant Reviews)

Molecular Plant Breeding (Cabi)

Plant Hormone Signaling (Annual Plant Reviews, Volume 24)

Plastids (Annual Plant Reviews, Volume 13)

Root Development (Annual Plant Reviews, Volume 37)

Biology of the Plant Cuticle (Annual Plant Reviews, Volume 23)

Annual Plant Reviews, Plant Polysaccharides: Biosynthesis and Bioengineering (Volume 41)

Plant Cell Separation and Adhesion (Annual Plant Reviews, Volume 25)

Annual Plant Reviews, Plant Hormone Signaling (Volume 24)

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