Transgenic Crops VI (Biotechnology in Agriculture and Forestry)

Biotechnology in Agriculture and Forestry Edited by T. Nagata (Managing Editor) H. Lörz J. M. Widholm

Biotechnology in Agriculture and Forestry Volumes already published and in preparation are listed at the end of this book.

Biotechnology in Agriculture and Forestry 61 Transgenic Crops VI Edited by E.C. Pua and M.R. Davey

With 51 Figures, 3 in Color and 26 Tables

123

Series Editors Professor Dr. Toshiyuki Nagata University of Tokyo Graduate School of Science Department of Biological Sciences 7-3-1 Hongo, Bunkyo-ku Tokyo 113-0033, Japan Professor Dr. Horst Lörz Universität Hamburg Institut für Allgemeine Botanik Angewandte Molekularbiologie der Pflanzen II Ohnhorststraße 18 22609 Hamburg, Germany

Professor Dr. Jack M. Widholm University of Illinois 285A E.R. Madigan Laboratory Department of Crop Sciences 1201 W. Gregory Urbana, IL 61801, USA

Volume Editors Professor Dr. Eng-Chong Pua School of Arts and Sciences Monash University Malaysia 2 Jalan Kolej, Bandar Sunway 46150 Petaling Jaya, Selangor, Malaysia

Professor Dr. Michael R. Davey Plant Sciences Division School of Biosciences University of Nottingham Sutton Bonington Campus Loughborough LE12 5RD, UK

Library of Congress Control Number: 2007931238

ISSN 0934-943X ISBN 978-3-540-71710-2 Springer Berlin Heidelberg New York This work is subject to copyright. All rights reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable for prosecution under the German Copyright Law. Springer is a part of Springer Science + Business Media springer.com © Springer-Verlag Berlin Heidelberg 2007 The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Editor: Dr. Dieter Czeschlik, Heidelberg, Germany Desk Editor: Dr. Andrea Schlitzberger, Heidelberg, Germany Cover design: WMXdesign GmbH, Heidelberg, Germany Typesetting and production: LE-TEX Jelonek, Schmidt & Vöckler GbR, Leipzig, Germany Printed on acid-free paper SPIN 11376859 31/3180 5 4 3 2 1 0

Preface

Exciting developments in crop biotechnology in recent years have prompted the necessity to update the first series of Transgenic Crops I, II and III, published in 1999 and 2001. In this current endeavor, 69 chapters have been compiled, contributed by a panel of experts in crop biotechnology from 26 countries. These chapters are grouped into three volumes, namely Transgenic Crops IV, V and VI. This new series not only reviews recent advances in cell and tissue culture and genetic transformation methodologies, but also presents aspects of the molecular genetics of target crops and the practical applications of transgenic plants. In addition, more than 30% of crop species that were not discussed previously are included in the present series. This new series commences with the volume Transgenic Crops IV, consisting of 23 chapters that focus on cereals, vegetables, root crops, herbs and spices. Section I is an introductory chapter that places into perspective the impact of plant biotechnology in agriculture. Section II focuses on cereals (rice, wheat, maize, rye, pearl millet, barley, oats), while Section III is directed to vegetable crops (tomato, cucumber, eggplant, lettuce, chickpea, common beans and cowpeas, carrot, radish). Root crops (potato, cassava, sweet potato, sugar beet) are included in Section IV, with herbs and spices (sweet and hot peppers, onion, garlic and related species, mint) in Section V. Transgenic Crops V also consists of 23 chapters in three sections devoted to fruit (Section I), trees (Section II) and beverage crops (Section III). Fruit crops target banana, citrus, mango, papaya, pineapple, watermelon, avocado, grape, melon, apple, Prunus spp, strawberry and kiwifruit, while trees include rubber, eucalyptus, legumes and conifers. Section III, on beverage crops, reports studies on coffee, cacao, tea and sugarcane. As in volumes IV and V, Transgenic Crops VI has 23 chapters organized in five Sections. Section I targets oil and fiber crops (soybean, rapeseed, sunflower, oil palm, peanut, cotton, flax), followed by medicinally important plants (including ginseng, opium poppy, herbane, bellandonna, Datura, Duboisia, Taxus) in Section II. Ornamentals (roses, carnation, chrysanthemum, orchids, gladiolus, forsythia) are discussed in Section III, while Section IV involves forages and grains (alfalfa, clovers, tall fescue, ryegrasses, lupin). Section V has one chapter that discusses aspects of the freedom to commercialize transgenic plants, together with regulatory and intellectual property issues. The editors express their sincere thanks to Maggie Yap Lan from Monash University, Malaysia, for her excellent secretarial and editorial assistance. She

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Preface

forwarded to contributors timely reminders of deadlines, where appropriate, and assisted in editing the manuscripts for typographical errors and formatting. This series will serve as a key reference for advanced students and researchers in crop sciences, genetics, horticulture, agronomy, cell and molecular biology, biotechnology and other disciplines in life sciences. E.C. Pua and M.R. Davey

Contents

Section I Oils and Fibers I.1 1 2 3 4 5 I.2 1 2 3 4 5 6 7 8 I.3 1 2 3 4 5 6 7

Soybean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P.M. Olhoft and D.A. Somers Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Somatic Embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Organogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Regeneration and Transformation Methods . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Canola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Cardoza and C.N. Stewart Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Economic Importance of Canola . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Genetics of Rapeseed . . . . . . . . . . . . . . . . . . . . . . . . . . . Tissue Culture of Canola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transformation of Canola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transgenic Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Impact of Transgenic Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sunflower . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Lu, X. Hu, and D.L. Bidney Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tissue Culture and Transformation . . . . . . . . . . . . . . . . . . . . . . . . . Genomics and Molecular Biology . . . . . . . . . . . . . . . . . . . . . . . . . . Transgenic Improved Input Agronomic Traits . . . . . . . . . . . . . . . . . Transgenic Output Quality Traits . . . . . . . . . . . . . . . . . . . . . . . . . . Gene Flow and Biosafety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 3 4 14 20 23 24 29 29 29 30 30 31 32 34 34 34 39 39 40 42 45 48 50 50 51

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I.4 1 2 3 4 5 6 7 I.5 1 2 3 4 I.6 1 2 3 4 5 6 7 8 9 10 I.7 1 2 3 4 5 6

Contents

Oil Palm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Rival Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Economic Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Impact of Biotechnology in Breeding Strategies . . . . . . . . . . . . Advances in Tissue Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic Transformation Technologies . . . . . . . . . . . . . . . . . . . . . . . Transgenic Plants for Oil Palm Improvement . . . . . . . . . . . . . . . . . Conclusion and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peanut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. Ozias-Akins Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of Molecular Markers . . . . . . . . . . . . . . . . . . . . . . . . . Peanut Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cotton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K.S. Rathore Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Importance of Genetic Engineering in Cotton . . . . . . . . . . . . . . . . . Modification of Cotton via Genetic Transformation . . . . . . . . . . . . Transformation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternative Methods Used to Transform Cotton . . . . . . . . . . . . . . . Selectable Marker Genes Used for Generating Transgenic Cotton . . . . . . . . . . . . . . . . . . . . . . . . . . Reporter Genes Used in Cotton . . . . . . . . . . . . . . . . . . . . . . . . . . . . Traits Introduced into Cotton Through Genetic Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . New Technological Advances and Their Role in Cotton Improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Flax . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Preˇtová, B. Obert, and Z. Bartoˇsová Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tissue and Organ Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Somatic Embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protoplast and Cell Suspension Cultures . . . . . . . . . . . . . . . . . . . . . Anther, Microspore and Ovary Cultures . . . . . . . . . . . . . . . . . . . . . Gene Transfer in Flax . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

59 59 59 61 66 70 71 74 75 81 81 81 90 99 99 107 107 107 108 108 118 119 119 120 122 123 124 129 129 129 131 134 135 136

Contents

7 8 9

Potential Applications of Transgenic Flax . . . . . . . . . . . . . . . . . . . . Molecular Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks and Further Prospects . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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137 138 138 139

Section II Medicinal Crops II.1 1 2 3 4 5 6 7 8 II.2 1 2 3 4 5 6 7 II.3 1 2 3 4 5 6 7

Ginseng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y.E. Choi Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Culture of P. ginseng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hairy Root Culture of P. ginseng . . . . . . . . . . . . . . . . . . . . . . . . . . . Adventitious Root Culture in P. ginseng . . . . . . . . . . . . . . . . . . . . . Plant Regeneration of P. ginseng via Organogenesis and Somatic Embryogenesis . . . . . . . . . . . . . . . Genetic Transformation and Metabolic Engineering . . . . . . . . . . . . Genomics in P. ginseng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Opium Poppy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J.M. Hagel, B.P. Macleod, and P.J. Facchini Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Origins and History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modern Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classic Breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biochemistry and Molecular Biology . . . . . . . . . . . . . . . . . . . . . . . Biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Henbane, Belladonna, Datura and Duboisia . . . . . . . . . . . . . . . . . . R. Arroo, J. Woolley, and K.-M. Oksman-Caldentey Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tropane Alkaloids, Uses and Outlook . . . . . . . . . . . . . . . . . . . . . . . Economic Importance of Tropane Alkaloid-Containing Crops . . . . Tropane Alkaloid Biosynthetic Pathway . . . . . . . . . . . . . . . . . . . . . Current Research and Development in Transgenic Technology . . . . Use of Hairy Root Cultures for Tropane Alkaloid Production . . . . . Novel Developments and Future Challenges . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

149 149 150 152 153 155 158 161 163 164 169 169 170 171 177 178 181 184 185 189 189 189 192 193 195 198 200 201

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Taxus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M.T. Piñol, R.M. Cusidó, J. Palazón, and M. Bonfill Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biosynthesis of Taxol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Vitro Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

205 205 207 209 220 221

Section III Ornamental Crops III.1 Roses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S.S. Korban 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Advances in Molecular Markers for Genetic Studies and Breeding . 3 Cloning and Characterization of Genes of Economic Value . . . . . . . 4 Advances in Genetic Transformation and Recovery of Transgenic Plants . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III.2 Carnation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. Moyal-Ben Zvi and A. Vainstein 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Recent Developments in Carnation Biotechnology . . . . . . . . . . . . . 3 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III.3 Chrysanthemum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P.B. Visser, R.A. de Maagd, and M.A. Jongsma 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Chrysanthemum Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Future Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III.4 Orchids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Yu and Y. Xu 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Genetic Transformation of Orchids . . . . . . . . . . . . . . . . . . . . . . . . . 3 Potential Genes for Genetic Engineering of Orchids . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

227 227 227 230 232 235 236 241 241 242 250 250 253 253 255 268 269 273 273 274 284 286

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III.5 Gladiolus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. Kamo and Y.H. Joung 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Tissue Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Genetic Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Promoters and Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Resistance to Bean Yellow Mosaic Virus . . . . . . . . . . . . . . . . . . . . . 6 Resistance to Cucumber Mosaic Virus . . . . . . . . . . . . . . . . . . . . . . 7 Future Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III.6 Forsythia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Rosati, A. Cadic, M. Duron, and P. Simoneau 1 Botanical Origin and Genetic Information . . . . . . . . . . . . . . . . . . . 2 Genetic Resources and Breeding Programs . . . . . . . . . . . . . . . . . . . 3 In Vitro Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Forsythia Biotechnology Research . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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289 289 289 291 292 294 295 295 296 299 299 305 307 310 316 316

Section IV Forages and Grains IV.1 Alfalfa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Sengupta-Gopalan 1 Introduction and Economic Importance . . . . . . . . . . . . . . . . . . . . . 2 Breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Genetic Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

321

IV.2 Clover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Mouradov, S. Panter, M. Emmerling, M. Labandera, E. Ludlow, J. Simmonds, and G. Spangenberg 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Improvement of Forage Quality by Modification of Secondary Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Improvement of Tolerance to Abiotic and Biotic Stresses . . . . . . . . 4 Functional Genomics and Metabolomics as Key Technologies for Characterisation and Modification of Natural Product Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusions, Challenges and Future Developments . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

337

321 322 323 323 331 331

337 338 342 345 347 349

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IV.3 Tall Fescue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Z-Y. Wang and G. Spangenberg 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Economic Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Current Research and Development . . . . . . . . . . . . . . . . . . . . . . . . 4 Practical Applications of Transgenic Plants . . . . . . . . . . . . . . . . . . . 5 Conclusions and Future Challenges . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

357 357 358 359 364 367 368

IV.4 Ryegrasses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 Y. Ran, C. Ramage, S. Felitti, M. Emmerling, J. Chalmers, N. Cummings , N. Petrovska, A. Mouradov, and G. Spangenberg 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 2 Economic Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 3 Current Research and Development . . . . . . . . . . . . . . . . . . . . . . . . 374 4 Practical Applications of Transgenic Plants . . . . . . . . . . . . . . . . . . . 380 5 Conclusions and Future Challenges . . . . . . . . . . . . . . . . . . . . . . . . 386 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387 IV.5 Lupins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L.M. Tabe and L. Molvig 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Genetic Transformation of Lupins . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Lupin Improvement Through Biotechnology . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

397 397 399 402 407

Section V Regulatory and Intellectual Property of GM Plants V.1

1 2 3 4

Freedom to Commercialize Transgenic Plant Products: Regulatory and Intellectual Property Issues . . . . . . . . . . . . . . . . . . S. Chandler and J. Rosenthal Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intellectual Property . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory Approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

411 411 412 419 428 428

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431

List of Contributors

R. Arroo Leicester School of Pharmacy, Natural Products Research, De Montfort University, The Gateway, Leicester LE1 9BH, United Kingdom S. Bagga Department of Plant and Environmental Sciences, New Mexico State University, Las Cruces, NM 88003, USA Z. Bartoˇsová Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, P.O. Box 39A, 95007 Nitra, Slovak Republic, e-mail: [email protected] D.L. Bidney Pioneer Hi-Bred International, Inc., 7300 NW 62nd Avenue, P.O. Box 1004, Johnston, Iowa 50131 M. Bonfill Seccion de Fisiología Vegetal, Facultad de Farmacia, Universidad de Barcelona, Avd. Diagonal 643, E-08028 Barcelona, Spain A. Cadic INRA, C.R. Angers, UMR Génétique et Horticulture (GenHort) – INRA/INH/UA, BP 60057, 49071 Beaucouzé Cedex, France V. Cardoza Department of Plant Sciences, University of Tennessee, 2431 Joe Johnson Drive, Room 252 Ellington Plant Sciences, Knoxville, TN 37996-4561, USA J. Chalmers Primary Industries Research Victoria, Victorian Agribiosciences Centre, La Trobe University, Bundoora, Victoria 3086, Australia

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List of Contributors

S. Chandler Florigene Ltd, 1 Park Drive, Bundoora, VIC 3083, Australia, e-mail: schandler@florigene.com.au Y.E. Choi Division of Forest Resources, College of Forest and Environmental Sciences, Kangwon National University, Chunchon 200-701, Korea, e-mail: [email protected] N. Cummings Primary Industries Research Victoria, Victorian Agribiosciences Centre, La Trobe University, Bundoora, Victoria 3086, Australia R.M. Cusidó Seccion de Fisiología Vegetal, Facultad de Farmacia, Universidad de Barcelona, Avd. Diagonal 643, E-08028 Barcelona, Spain R.A. de Maagd Business Unit Bioscience, Plant Research International, Wageningen University and Research Centre, P.O. Box 16, NL-6700 AA Wageningen, The Netherlands M. Duron INRA, C.R. Angers, UMR Génétique et Horticulture (GenHort) – INRA/INH/UA, BP 60057, 49071 Beaucouzé Cedex, France M. Emmerling Primary Industries Research Victoria, Victorian Agribiosciences Centre, La Trobe University, Bundoora, Victoria 3086, Australia P.J. Facchini Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada, e-mail: [email protected] S. Felitti Primary Industries Research Victoria, Victorian Agribiosciences Centre, La Trobe University, Bundoora, Victoria 3086, Australia J.M. Hagel Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada X. Hu Pioneer Hi-Bred International, Inc., 7300 NW 62nd Avenue, P.O. Box 1004, Johnston, Iowa 50131, USA

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XV

M.A. Jongsma Business Unit Bioscience, Plant Research International, Wageningen University and Research Centre, P.O. Box 16, NL-6700 AA Wageningen, The Netherlands, e-mail: [email protected] Y.H. Joung School of Biological Science and Technology, Chonnam National University, Gwangju 500-757, Korea K. Kamo Floral and Nursery Plants Research Unit, US National Arboretum, USDA, Beltsville, MD 20705, USA, e-mail: [email protected] S.S. Korban Department of Natural Resources & Environmental Sciences, 310 ERML, University of Illinois, 1201 W. Gregory, Urbana, IL 61801, USA, e-mail: [email protected] M. Labandera Primary Industries Research Victoria, Victorian Agribiosciences Centre, La Trobe University, Bundoora, Victoria 3086, Australia G. Lu Pioneer Hi-Bred International, Inc., 7300 NW 62nd Avenue, P.O. Box 1004, Johnston, Iowa 50131, USA, e-mail: [email protected] E. Ludlow Primary Industries Research Victoria, Victorian Agribiosciences Centre, La Trobe University, Bundoora, Victoria 3086, Australia B.P. Macleod Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada L. Molvig CSIRO Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia A. Mouradov Primary Industries Research Victoria, Victorian Agribiosciences Centre, La Trobe University, Bundoora, Victoria 3086, Australia M. Moyal-Ben Zvi The Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel

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List of Contributors

B. Obert Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, P.O. Box 39A, 95007 Nitra, Slovak Republic K.-M. Oksman-Caldentey VTT Technical Research Centre of Finland, Plant Biotechnology, P.O. Box 1000, FIN-02044 Espoo, Finland, e-mail: Kirsi-Marja.Oksman@vtt.fi P.M. Olhoft BASF Plant Science, 26 Davis Drive, P.O. Box 13528, Research Triangle Park, NC 27709, USA, e-mail: [email protected] J.L. Ortega Department of Plant and Environmental Sciences, New Mexico State University, Las Cruces, NM 88003, USA P. Ozias-Akins Department of Horticulture, The University of Georgia Tifton Campus, Tifton, GA 31793, USA, e-mail: [email protected] J. Palazón Seccion de Fisiología Vegetal, Facultad de Farmacia, Universidad de Barcelona, Avd. Diagonal 643, E-08028 Barcelona, Spain S. Panter Primary Industries Research Victoria, Victorian Agribiosciences Centre, La Trobe University, Bundoora, Victoria 3086, Australia N. Petrovska Primary Industries Research Victoria, Victorian Agribiosciences Centre, La Trobe University, Bundoora, Victoria 3086, Australia M.T. Piñol Seccion de Fisiología Vegetal, Facultad de Farmacia, Universidad de Barcelona, Avd. Diagonal 643, E-08028 Barcelona, Spain, e-mail: [email protected] C. Potenza Department of Plant and Environmental Sciences, New Mexico State University, Las Cruces, NM 88003, USA A. Preˇtová Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, P.O. Box 39A, 95007 Nitra, Slovak Republic, e-mail: [email protected]

List of Contributors

XVII

C. Ramage Primary Industries Research Victoria, Victorian Agribiosciences Centre, La Trobe University, Bundoora, Victoria 3086, Australia Y. Ran Primary Industries Research Victoria, Victorian Agribiosciences Centre, La Trobe University, Bundoora, Victoria 3086, Australia K.S. Rathore Institute for Plant Genomics & Biotechnology, and Department of Soil and Crop Science, Texas A & M University, College Station, TX 77843-2123, USA, e-mail: [email protected] A. Rival CIRAD, UMR DAP, BP 64501, F-34394 Montpellier Cedex 5, France, e-mail: [email protected] C. Rosati ENEA, Trisaia Research Center,BAS-BIOTECGEN, S.S.106, km 419+500, 75026 Rotondella (MT), Italy, e-mail: [email protected] J. Rosenthal Alchemia Oncology Ltd, Pacific Towers, 737 Burwood Rd, Hawthorn, VIC 3122, Australia C. Sengupta-Gopalan Department of Plant and Environmental Sciences, New Mexico State University, Las Cruces, NM 88003, USA, e-mail: [email protected] J. Simmonds Primary Industries Research Victoria, Victorian Agribiosciences Centre, La Trobe University, Bundoora, Victoria 3086, Australia P. Simoneau UMR PaVé 77, Faculté des Sciences, Boulevard Lavoisier 2, 49045 Angers, France D.A. Somers Monsanto Company, Agracetus Campus, 8520 University Green, P.O. Box 620999, Middleton, Wisconsin, USA

XVIII

List of Contributors

G. Spangenberg Primary Industries Research Victoria, Victorian Agribiosciences Centre, La Trobe University, Bundoora, Victoria 3086, Australia, e-mail: [email protected] C.N. Stewart, Jr Department of Plant Sciences, University of Tennessee, 2431 Joe Johnson Drive, Room 252 Ellington Plant Sciences, Knoxville, TN 37996-4561, USA, e-mail: [email protected] L.M. Tabe CSIRO Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia, e-mail: [email protected] A. Vainstein The Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel, e-mail: [email protected] P.B. Visser Business Unit Bioscience, Plant Research International, Wageningen University and Research Centre, P.O. Box 16, NL-6700 AA Wageningen, The Netherlands J. Woolley Leicester School of Pharmacy, Natural Products Research, De Montfort University, The Gateway, Leicester LE1 9BH, United Kingdom Y. Xu Plant Functional Genomics Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Science Drive 4, Singapore 117543 H. Yu Plant Functional Genomics Group, Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, and Plant Functional Genomics Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Science Drive 4, Singapore 117543, e-mail: [email protected] Z-Y. Wang Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA, e-mail: [email protected]

Section I Oils and Fibers

I.1 Soybean P.M. Olhoft1 and D.A. Somers2

1 Introduction Soybean [Glycine max (L.) Merr.] is grown in more than 50 countries and is the leading oilseed crop produced and consumed worldwide (Wilcox 2004). In addition to oil, soybeans are a rich and efficiently produced source of protein. They are consumed directly and, following fermentation or processing, are consumed as food, food ingredients, food additives and dietary supplements, and they are also used in animal feeds and for the production of a range of industrial compounds and materials. World soybean production has nearly doubled since 1985, by increasing yields on a per area basis and by increasing the overall area planted with the crop (Wilcox 2004). Substantial quantities of soybeans are traded on world markets and, therefore, the crop contributes significantly to the global economy (Wilcox 2004). The worldwide scale of soybean production and the economic importance of the crop dictate that substantial private industry and public research efforts are directed towards crop improvement, management and production. Conventional plant breeding has been and will continue to be the mainstay of soybean improvement (Sleper and Shannon 2003). Over the past 30 years, major advances in molecular genetics and, more recently, genomics have been applied to crop improvement, resulting in molecular approaches that augment conventional breeding efforts. In soybean, a number of molecular marker maps have been constructed and applied to marker-assisted breeding. For example, mapping the rhg-1 locus conferring resistance to soybean cyst nematode is used routinely for introgressing that region into new soybean varieties. The next step on this path of genome characterization is the completion of the alignment of genome sequences to genetic and physical maps and the overwhelming task of providing this information to the scientific community (Jackson et al. 2006). The majority of expressed genes in soybean have been at least partially sequenced, providing an extensive library of expressed sequenced tags (ESTs) for gene identification. Taken together, the molecular and genomic resources now available in soybean provide and will continue to provide a range of tools for investigating gene function, the genetics and regulation 1 BASF

Plant Science, 26 Davis Drive, P.O. Box 13528, Research Triangle Park, NC 27709, USA, e-mail: [email protected] 2 Monsanto Company, Agracetus Campus, 8520 University Green, P.O. Box 620999, Middleton, Wisconsin, USA Biotechnology in Agriculture and Forestry, Vol. 61 Transgenic Crops VI (ed. by E.C. Pua and M.R. Davey) © Springer-Verlag Berlin Heidelberg 2007

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P.M. Olhoft and D.A. Somers

of important traits, and for crop improvement through both conventional and marker-assisted breeding, and genetic engineering approaches (Stacey et al. 2004). Introgression of desired traits into crops using transformation approaches augments traditional breeding and add value. Genetic transformation of soybean has been and will likely continue to be an important component in crop improvement. For example, Roundup Ready soybeans are the most extensively planted transgenic crop worldwide occupying 109 million acres (ca. 44 × 106 ha) in 2004. Roundup Ready soybean was produced from some of the first successful soybean transformation experiments conducted in 1988, as described by Padgette et al. (1995). Since that time, other approaches have emerged as the predominant transformation methods (Trick et al. 1997; Parrot and Clemente 2004). Transformation of soybean remains to be highly challenging with relatively few laboratories routinely able to transform soybean. The methods commonly used for soybean transformation require personnel skilled in the art to produce healthy, fertile transgenic plants that transmit the transgene to the next generation. Currently, many soybean transformation systems are dependent on the production of tissue cultures as sources of regenerable target cells for DNA delivery. Regeneration of plants from cell cultures in soybeans occurs through either somatic embryogenesis or shoot organogenesis from apical and axillary meristematic cells. For somatic embryogenesis, embryo development is mainly induced from explants derived from immature zygotic embryos that were initially exposed to high concentrations of auxin in the medium, especially 2,4-dichlorophenoxyacetic acid (2,4-D). In contrast, shoots that regenerate via organogenesis are derived from explants exposed to low levels of cytokinins like 6-benzylaminopurine (BAP). In this review, various methods are discussed that successfully and repeatedly regenerate whole fertile plants from tissue culture, with a focus on those methods predominately used for transformation.

2 Somatic Embryogenesis 2.1 Background Somatic embryo development progresses through the distinct phases of histodifferentiation, maturation, desiccation, germination and conversion, as reviewed by Parrott et al. (1995). Histodifferentiation begins with the induction of the embryogenic stage and lasts until auxin concentration is lowered and somatic embryo development commences. Embryos during this stage are actively undergoing cell division and differentiate through the globular, heart and torpedo stages, ending with the cotyledonary stage. At somatic embryo maturation, mitosis ceases and cell expansion occurs, with the accumulation of storage reserves and acquisition of desiccation tolerance. Physiological matu-

Soybean

5

rity is followed by a period of desiccation that is associated with the synthesis of proteins that enables germination of the cotyledon-stage embryo. Conversion is completed with whole plant regeneration of the germinated embryo. The primary method for producing somatic embryogenic cultures is through direct induction from immature zygotic cotyledons, meaning that embryos develop with few mitotic cell cycles before becoming embryogenetically determined. Thus, there is little intervening callus phase before induction (Parrott et al. 1995). Somatic embryo development is usually stimulated by culturing the immature cotyledons with high concentrations of auxin such as 2,4-D. However, α-naphthaleneacetic acid (NAA) is also used to induce embryogenesis. Exposure of the soybean cotyledon explants with 2,4-D results in the development of somatic embryos from most of the epidermal surface (Hartweck et al. 1988); and cotyledon explants exposed to NAA develop fewer normal embryos that are limited to the crescent-shaped distal periphery of explants (Lazzeri et al. 1987; Hartweck et al. 1988). Indirect somatic embryogenesis from callus cultures derived from immature zygotic embryos has been reported (Christianson 1983; Barwale et al. 1986; Ghazi et al. 1986). A smooth, green, shiny embryogenic callus was induced from cotyledon tissue that was capable of regeneration when cultured in the presence of 10 mg l−1 2,4-D (45 μM; Ghazi et al. 1986). By changing the hormones in the callus induction medium, Barwale et al. (1986) reported plant regeneration from callus cultures derived from immature embryos from 54 genotypes through either embryogenesis or organogenesis. Embryogenic cultures capable of plant regeneration were best obtained by adding 43 μM NAA to an MS-based medium (Murashige and Skoog 1962) after 4 weeks growth. When the MS media was amended with 13.3 μM BAP, 0.2 μM NAA and four times the normal concentration of MS salts, regenerable organogenic callus cultures from the immature embryos resulted (see Section 3: Organogenesis). Although these studies indicate callus cultures capable of regeneration were established, it is much more common to directly induce somatic embryogenesis without going through the callus phase. Genotype is a critical factor in determining the somatic embryo quality and quantity and in regeneration competency and, therefore, affects all stages of somatic embryogenesis from induction, histodifferentiation, maturation and conversion (Delzer et al. 1990; Simmonds and Donaldson 2000; Meurer et al. 2001; Tomlin et al. 2002). Soybean genotypes are grouped according to maturity zones in North America from 000 to 10, with lower maturity groups belonging to Northern genotypes and higher maturity groups to the Southern genotypes. No relationship between maturity group and induction of embryogenesis was apparent in studies by Bailey et al. (1993). However, a few studies indicate that some of the most responsive genotypes are from lower maturity groups (Meurer et al. 2001; Tomlin et al. 2002). In particular, one of the most responsive cultivars used across many laboratories because of its consistent embryogenic response and reliable plant regeneration is the maturity group II cultivar ‘Jack’. Because soybeans are photoperiod-sensitive, it is important to

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P.M. Olhoft and D.A. Somers

factor in regeneration conditions with the maturity group of the cultivar being used for somatic embryogenesis. Problems associated with photoperiod sensitivity are partially overcome in somatic embryogenesis protocols by supplying the in vitro tissue cultures with a continuous light regime, thereby ensuring that plant development remains in the vegetative phase (Ghazi et al. 1986). After comparing both embryogenic induction and proliferation from immature cotyledons of nine cultivars, Meurer et al. (2001) concluded that, to successfully regenerate soybean plants via somatic embryogenesis, an established protocol should be used in conjunction with one of several embryogenic cultivars already identified, such as Jack. 2.2 Regeneration Development of a successful regeneration protocol via somatic embryogenesis in soybean involved many years of optimization and experimentation in many laboratories. Some of the first successful efforts in establishing embryonic tissues were from suspension cultures of soybean initiated from hypocotyl or cotyledon callus tissue (Beversdorf and Bingham 1977). The hypocotyl or cotyledon callus was found to differentiate into embryos in liquid medium containing high sucrose and 2,4-D, but full plant regeneration was not achieved (Beversdorf and Bingham 1977). An early report of plant regeneration via somatic embryogenesis was by Christianson et al. (1983). In this study, a morphogenetically competent suspension culture was initiated from a single callus piece derived from the embryonic axes from which a single plant was regenerated. Ranch et al. (1985) reported plant regeneration using an intact zygotic embryo as an explant source and Lazzeri et al. (1985) reported regeneration using excised cotyledons. Currently, somatic embryogenesis methods in soybeans use the excised immature cotyledons to induce embryoids. A general outline for somatic embryogenesis is given below, from initiation of somatic embryos to plant regeneration. 2.2.1 Induction The following protocol for induction of embryogenic tissues from immature cotyledons was described by Finer and Nagasawa (1988). Immature seeds from pods 7−14 days after flowering were collected and surface-sterilized before explant preparation. The optimal size of the cotyledons for induction of embryoids was 3−5 mm in length (Lazzeri et al. 1985; Finer 1988). The cotyledons from the immature seeds were separated from the embryo axes (Lazzeri et al. 1985) and cultured with the abaxial surface in contact with induction medium (MSD40) containing MS (Murashige and Skoog 1962) salts, B5 vitamins (Gamborg et al. 1968), 6% sucrose, 40 mg l−1 2,4-D (0.18 mM), and 0.8% agar (pH 5.7) to induce embryogenic callus growth. In some working protocols, lower concentrations of sucrose are used. Lippmann and Lippman (1984),

Soybean

7

Komatsuda et al. (1991), and Hofmann et al. (2004) all reported that initiation of embryo structures was greater when 2–3% sucrose was used instead of 6%. After 1−2 months, globule-stage embryos and proliferative embryogenic tissue were formed on the immature cotyledon. These tissues grew from protrusions and swollen ridges on the entire surface of the immature cotyledon when 2,4-D was used as the auxin. In addition, the embryos were friable and translucent yellowish-green in colour. When NAA was used as the auxin, the embryos were compact, opaque, pale green and more advanced in stage, forming cotyledonlike structures and usually forming on the cut edges of cotyledons (Hofmann et al. 2004). Adventitious roots were also reported to form on embryos incubated using NAA (Lazzeri et al. 1987). Embryogenic tissue can remain embryogenic indefinitely (Ranch et al. 1985; Finer 1988). Secondary somatic embryos arise from the apical or terminal portions of the older primary embryos, which may be highly responsive cotyledon tissue. In semi-solid medium, this is achieved using 20−40 mg l−1 (0.090−0.18 mM) 2,4-D (Finer 1988; Wright et al. 1991) and in liquid medium 5 mg l−1 (22.5 μM) 2,4-D (Finer and Nagasawa 1988). Secondary embryogenesis is inhibited and embryo differentiation and maturation are promoted when auxin is removed from the medium. Embryogenic suspension cultures are initiated by placing 20−50 mg of early-staged, highly embryogenic callus into a flask with 35 ml suspension culture medium 10A40N (also known as FN) consisting of MS salts (nitrogen replaced with 10 mM NH4 NO3 , 30 mMKNO3 ), B5 vitamins, 6% sucrose, 5 mg l−1 2,4-D (22.5 μM) and 15 mM glutamine (eventually replaced with 5 mM asparagine to prevent tissue necrosis and reduce clump size; Finer and Nagasawa 1988). Amino acids are added to the medium to increase the frequency of somatic embryogenesis. An optimization of FN medium, called FN Lite, was reported to improve the proliferation of soybean suspension cultures by replacing the macro salts with 27.9 mM KNO3 (NH4 NO3 is removed), 3.5 mM (NH4 )2 SO4 , 1.4 mM KH2 PO4 , 2.0 mM CaCl2 and 3% sucrose (Samoylov et al. 1998). High quality embryogenic tissues have fast growing, smooth, dense, nodular spheres that are green in colour. Eventually, the suspension cultures proliferate as clumps of globular embryos attached at their bases that range over 0.5–8.0 mm in diameter, which apparently arise from the apical surface of older embryos. Although it is possible to regenerate plants directly from these embryos (Finer and Nagasawa 1988), it is more common to undergo desiccation, germination and conversion. 2.2.2 Histodifferentiation The induction of histodifferentiation given below was described by Bailey et al. (1993). Globular stage somatic embryos were transferred to semi-solid (MSM6AC) MS salts, B5 vitamins, 6% maltose, 0.5% activated charcoal, 0.2% Gelrite, pH 5.8, to differentiate the embryos into cotyledon-stage somatic embryos. Activated charcoal was added during maturation to aid in the bind-

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ing and removal of auxins, thereby inducing embryo development (Ebert and Taylor 1990). After 28 days, cotyledon-stage embryos with a distinct hypocotyl/root and shoot axes were transferred to MSM6 medium (MSM6AC without charcoal) for maturation. Maltose was used in place of sucrose for enhanced embryo development and conversion during maturation (Finer and McMullen 1991). At this point, it is possible to produce plants directly from cotyledon-stage embryos without undergoing desiccation and germination. In a protocol developed by Wright et al. (1991), whole plant regeneration from embryos that reached the cotyledon- and torpedo-stage was obtained through shoot-bud propagation by transferring embryos to medium containing BAP. Alternatively, it is possible to induce somatic embryos from the cotyledonstage somatic embryos by culturing the embryos on MS media containing either 40 mg l−1 (0.18 mM) 2,4-D or 10 mg l−1 (0.054 mM) NAA, termed somatic embryo cycling (Liu et al. 1992). By inducing somatic embryos from cotyledonstage embryos, the difficulties with genotype-specific induction frequencies when applied to transformation protocols are of little relevance, since only a low percentage of cotyledon explants need to respond to embryogenesis. 2.2.3 Desiccation, Germination and Conversion The induction of histodifferentiation given below was described by Bailey et al. (1993). After 28 days on MSM6, embryos were partially desiccated in dry Petri dishes for 5 days to enhance conversion (Hammatt and Davey 1987; Parrott et al. 1988). ABA may be necessary during embryogenesis to initiate the synthesis of storage proteins and proteins involved in desiccation tolerance (Parrott et al. 1995). Partially desiccated embryos were then transferred to MSO medium (MS salts, B5 vitamins, 3% sucrose, pH 5.8) for germination. Germinating embryos enlarged, became green and then produced pubescent structures at the shoot apex. Germinating embryos were then placed on MSO medium for another 3 weeks before transplanting into soil, thereby completing the conversion phase. 2.3 Transformation of Somatic Embryogenic Cultures Transformation efforts using somatic embryogenesis regeneration methods have used Agrobacterium tumefaciens and particle bombardment for DNA delivery into target cells. The major improvements to protocols using somatic embryos or immature cotyledons for transformation focus on increasing fertility of regenerant plants and increasing and optimizing DNA delivery and selection techniques to produce fertile, healthy plants. An early report detailing the recovery of primary transformants was by Parrott et al. (1989), using Agrobacterium and targeting the immature cotyledons for T-DNA delivery. This study also reported that the embryonic tissues of soybean were amenable

Soybean

9

to Agrobacterium-mediated transformation. Because progeny of three primary regenerants did not inherit the T-DNA, it was suggested that stimulating cell division before the initiation of embryo development, or inducing secondary embryogenesis from transformed primary embryos, should increase the recovery of non-chimeric primary regenerants. To increase fertility, most Agrobacterium-mediated protocols eliminated the liquid suspension culture step and incorporated more stringent selection regimes primarily using hygromycin. Particle bombardment methods are also focused on reducing the culture times and increasing selection regimes. However, the liquid embryo proliferation step is still commonly used. To give readers an idea of transformation procedures using somatic embryos, several transformation protocols are briefly presented that concentrate on incorporating transformation and selection steps into previously outlined regeneration methods. 2.3.1 Agrobacterium-Mediated Transformation Agrobacterium-mediated transformation protocols for soybean somatic embryogenesis most commonly use immature cotyledons as the target explant for Agrobacterium infection and do not go through a significant secondary embryo proliferation step in liquid medium (Parrott et al. 1989; Yan et al. 2000; Ko et al. 2003, 2004). By selecting for transgenic cells on semi-solid induction and maturation media, the transformation method from induction of somatic embryos to plants in soil can be shortened from 9 months using a liquid proliferating step, to 4–5 months (Ko et al. 2003). The ability to produce transgenic plants within 5 months was, in large part, due to using hygromycin B selection (Ko et al. 2003). Soybean tissues are highly sensitive to this antibiotic and, therefore, the selection of transgenic cells over non-transgenic cells is very stringent and occurs very early in embryo development. The reduction of culture time is very important not only for the development of an efficient transformation method but also for the fertility of regenerated plants, which is known to be compromised as cultures become older. This is reflected in the studies below, since the Agrobacterium-mediated transformation methods all produced fertile plants. Although a significant phase for secondary-embryo cycling was not incorporated into many of the methods, some period of secondary embryo formation may be useful in increasing transformation efficiency, as long as fertility is not affected. A major factor to take into consideration when developing efficient Agrobacterium-mediated T-DNA delivery protocols is the Agrobacterium–plant interaction. During co-cultivation, enhanced results may be achieved by identifying a balance between the survival of the explant and the optimization of conditions for induction and delivery of the T-DNA into plant cells. For example, an investigator may consider using hypervirulent Agrobacterium strains, adjusting co-cultivation time, Agrobacterium concentration, medium composition, orientation of explant during co-culture and wounding, to name a few

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(Hansen and Wright 1999). Wounding of the explant tissue appears to be essential in soybean for T-DNA delivery by providing access to the target cells, as well as for induction of the Agrobacterium T-DNA transfer process. Wounding of immature cotyledons can be accomplished by applying mechanical pressure, using a steel or nylon mesh (Parrott et al. 1989), forceps (Yan et al. 2000), or a multi-needle wounding prong (Ko et al. 2003). Micro-wounding on somatic embryos and immature cotyledons by using brief periods of ultrasound called sonication-assisted Agrobacterium-mediated transformation (SAAT; Trick and Finer 1997, 1998), or by particle bombardment (Droste et al. 2000) has shown to increase stable β-glucuronidase (GUS) expression. Colonization of Agrobacterium was high on the immature cotyledons that underwent sonication and could be found on both epidermal and subepidermal cells up to 7–8 cell layers deep (Trick and Finer 1997). Without sonication, agrobacteria were found only on the surface of the cotyledons and GUS expression was much less than on sonication-treated explants. These techniques may be useful in increasing T-DNA delivery in future Agrobacterium-mediated transformation protocols. 2.3.2 Examples of Agrobacterium-Mediated Transformation Three protocols that exemplify some of the techniques used for transformation of somatic embryos are outlined, starting with the early report of the production of transgenic primary plants (Parrott et al. 1989) and progressing to more recent methods (Yan et al. 2000; Ko et al. 2003). Although Agrobacteriummediated transformation of somatic embryos is still in its infancy, there is much promise for the development of improved and reproducible systems from laboratory to laboratory. 1. Immature cotyledons approximately 5 mm in length from 14 genotypes were used as the target for Agrobacterium infection (Parrott et al. 1989). Explants were wounded by placing a steel or nylon mesh on top of 20 immature cotyledons and pressing with a spatula. The macerated cotyledons were placed adaxial side up on semi-solid N10 medium [MS salts, B5 vitamins, 1.5% sucrose, 10 mg l−1 (0.054 mM) NAA, 0.2% Gelrite, pH 5.8] and the explants were co-cultivated overnight at 28 ◦ C with Agrobacterium. Transformed cells were selected using 10 mg l−1 geneticin (G418) for 30 days on N10 medium with antibiotics. Regeneration of somatic embryos was followed according to Lazzeri et al. (1985), using a semi-solidified MS salts medium amended with 0.017 mg l−1 of BAP, kinetin and zeatin, and 0.05 mg l−1 NAA. Three independent transformation events with simple T-DNA integrations were obtained from the cultivars Peking and PI 283332. These plants were chimeric for the transgene and did not transmit the T-DNA into T1 progenies. 2. Yan et al. (2000) used immature cotyledons from the soybean cv. Jack for transformation. Immature cotyledons, ranging in size from 4−10 mm in length, were chosen for transformation and embryo production, GUS expression and explant survival. Although 4−7 mm is the optimal size for

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embryoid production (Finer 1988), explant survival after Agrobacterium infection was found to be optimal when the cotyledons were larger than 8 mm (Yan et al. 2000; Ko and Korban 2004). Therefore, immature cotyledons ∼ 8 mm in size were best for Agrobacterium infection when the Agrobacterium concentration had an optical density (at 600 nm; OD600 ) of 0.2–1.0. Explants were wounded with forceps and incubated at room temperature for 30 min with the Agrobacterium strain EHA105 containing 35S-gusA and 35Shpt on a binary vector. Explants were placed with adaxial side up on solidified MSD40 medium containing 100 μM acetosyringone to induce T-DNA delivery and co-cultivated in the dark at 23−25 ◦ C for 3 days. Co-cultivation times longer than 3 days were found to be detrimental to explant survival (Yan et al. 2000). However, Ko and Korban (2004) found a 4-day co-cultivation to be optimal for transformation. After co-cultivation, explants were transferred to MSD40 medium containing antibiotics and 10 mg l−1 hygromycin for the first 2 weeks, then 25 mg l−1 hygromycin for an additional 2 weeks. Selection was continued during the maturation phase in MSM6 medium with 10 mg l−1 hygromycin plus antibiotics and transformed soybean plants were recovered after desiccation and germination as described above. One unique event was reported in this paper for a transformation frequency of only 0.03%. 3. Immature cotyledons 5−8 mm in length were collected from soybean cvs. Jack, Williams, Ina, Macon, Dwight and Rend and used for transformation experiments (Ko et al. 2003). The explants were wounded using a multineedle prong and then inoculated and co-cultivated with Agrobacterium as described by Yan et al. (2000). In these experiments, Agrobacterium strains KYRT1, GV3101 and EHA105 were used to deliver T-DNAs containing the cassettes 2x 35S-hpt and 35S-gusA. In addition, the orientation of the immature cotyledons was tested by placing the cotyledons with the abaxial side facing up or down during co-cultivation, initiation and selection. The explants were then placed on MSD40 medium with antibiotics without hygromycin for the first 2 weeks, then with 25 mg l−1 hygromycin for the second 2 weeks of somatic embryo initiation. In this study and in that of Ko and Korban (2004), induction of somatic embryos was found only on explants orientated with their abaxial side facing upwards. This is presumably due to the accessibility of 2,4-D, hygromycin and cefotaxime to the margins of the cotyledons where the embryos arise. Selection was also applied during maturation by adding 25 mg l−1 hygromycin and cefotaxime into D40 medium. Maturation and regeneration methods were as reported above. Fertile plants were recovered from primary embryos and the transformation frequency was reported at 1.1–1.7% using the cv. Jack, the Agrobacterium strain KYRT1 and co-cultivating/regenerating with abaxial side oriented upwards. Induction of somatic embryogenesis was possible using Agrobacterium strain pKYRT1, but induction was poor using both GV3101 and EHA105 (Ko et al. 2003). The helper plasmid pKYRT1 from the Chry5 strain is not com-

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pletely disarmed and has a portion of T-left (TL ) and the entire T-right (TR ) (Palanichelvam et al. 2000). Additional wild-type genes carried on the TR -DNA in conjunction with 2,4-D are postulated to promote somatic embryogenesis of transformed cells at a high frequency, since the fully disarmed T-DNA in Agrobacterium NTL/pKPSF2 and the bacterial chromosome background of Chry5c/pEHA105 were not conducive to somatic embryogenesis (Ko et al. 2004). Even though the TR -DNA was transmitted to the progeny, the TR -DNA can be segregated away in later generations, making this a useful technique to increase the transformation efficiency using somatic embryogenesis. 2.3.3 Particle Bombardment Most transformation protocols using particle bombardment include a step for secondary embryo formation in conjunction with increasing selection pressure and/or using more effective selection agents (e.g. hygromycin) to enrich for transgenic cells. The first report of obtaining transformed soybean plants with transmission of the transgene into T1 progenies using somatic embryogenesis was by establishing proliferating embryogenic cultures and subsequently bombarding somatic embryos for DNA delivery (Finer and McMullen 1991). Histological examination of soybean embryogenic cultures showed that the embryos arose from the surface or adjacent subsurface tissues establishing secondary embryogenesis and were homogeneous for the transgene, thereby resulting in the recovery of non-chimeric T0 plants. Transgenic plants recovered from bombarded embryogenic suspension cultures (Sato et al. 1993) were also found to be non-chimeric; and histological studies indicated that transformation, selection and development from single epidermal cells were responsible for initiating the secondary somatic embryos. Another technique to establish homogeneous plants used the somatic embryo cycling technique detailed by Liu et al. (1992, 1996). Instead of bombarding the zygotic immature cotyledons or globule-stage embryos, the hypocotyls of cotyledon-stage somatic embryos were targeted and secondary embryos arising from a single epidermal cell were selected and regenerated. More somatic embryos were found initiated from the hypocotyl somatic embryos than the zygotic immature cotyledons. The fertility of regenerated plants from embryogenic cultures over 6 months old decreases rapidly as mentioned earlier, which presents a problem with several of the transformation protocols that use liquid suspension cultures or somatic cycling (Liu et al. 1996; Hazel et al. 1998). In addition to the age of cultures used for transformation, both the stage of cells in the cell cycle and the stage of somatic embryos in development were also found to affect transformability using particle bombardment (Hazel et al. 1998). The highest transient GUS expression was found to peak at 4 days after transferring to new medium, which correlates with a burst in the mitotic activity. This may be due, in part, to the breakdown of the nuclear envelope or the incorporation of the transgene DNA during synthesis or repair (Hazel et al. 1998). In addition, the

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most transformable embryo structures were the tightly packed globule-stage embryos with cytoplast-rich cells. Therefore, the choice of materials to be used for particle bombardment is as important as the regeneration protocols that have been established for somatic embryos. 2.3.4 Examples of Particle Bombardment Methods 1. Embryogenic suspension cultures of the cultivar ‘Fayette’ were initiated and maintained as stated by Finer and McMullen (1991). After 3 weeks of subculture, 1 g of embryogenic suspension tissue was transferred to a sterile Petri dish and subjected to particle bombardment using a DuPont Biolistics particle delivery system with plasmid DNA precipitated onto 1.1 μm tungsten pellets. The plasmid DNA used for transformation included the 35S-HygCaMV and 35S-GUS-nos gene cassettes. Bombarded embryogenic tissues were first transferred to 10A40N maintenance medium without selection for 1–2 weeks, and then to 10A40N medium supplemented with 50 mg l−1 hygromycin for selection of transgenic cells for 3–4 months. Embryo maturation, desiccation and plant regeneration were as stated earlier. Using this method, plants were recovered 9 months after bombardment. On average, three stable transgenic events were obtained per bombardment. 2. Liu et al. (1996) selected somatic embryos of cultivar J103 that reached the cotyledon stage with both hypocotyl and radicals for bombardment using the Particle delivery system 1000 from DuPont. Thirty-two somatic embryos were arranged into a 2.5-cm circle and then bombarded with 1.1 μm tungsten particles with 5 μg plasmid DNA precipitated onto them. The plasmid DNA contained a double 35S-gusA coding sequence fused with phosphotransferase II (nptII). After bombardment, the somatic embryos were cultured for approximately 50 days on solid medium containing 40 mg l−1 (0.18 mM) 2,4-D until globular embryos initiated from the hypocotyls. The globular embryos were then proliferated in liquid medium containing 40 mg l−1 geneticin for selection of transgenic cells. Cotyledon-stage embryos were removed for desiccation and germination. All 52 lines recovered from 111 bombardments had abnormal floral development and, thus, seeds were not produced. 3. The cvs. Bragg and IAS5 were used for somatic embryogenesis (Droste et al. 2000). Pods 3−5 mm in length were chosen as explant material. Somatic embryos were induced by culture on MSD40 medium as described above; and 50 mg of proliferative embryos produced by growing on solid D40 medium (Wright et al. 1991) were bombarded with tungsten particles coated with plasmid DNA containing 35S-gusA and the hpt gene. Five transgenic clones per 0.5 g of bombarded IAS5 embryogenic tissues were obtained using the Particle inflow gun.

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3 Organogenesis 3.1 Background Organogenesis in soybean typically involves regeneration of shoots directly from meristematic cells or from tissues near meristematic cells. These shoots can arise from the explant tissues with or without an intervening callus phase. Reports of plant regeneration via organogenesis in soybean are predominately based on methods that induce shoot development at either the axillary meristem or apical meristem cells without undergoing a significant callus phase. Compared with somatic embryogenesis regeneration methods, organogenic methods are relatively cv.-independent, since every soybean plant has axillary meristems that are capable of plant regeneration. However, there are differences among the cvs., especially in the vigor of explant growth in vitro and their sensitivity to the media components, making some cvs. favourable to tissue culture techniques used in regeneration via organogenesis. Establishment of shoot-bud cultures in soybean is possible through the removal of apical dominance and stimulation of axillary meristem growth with or without the addition of low levels of hormones to plant regeneration media. Axillary meristematic cells are present at each node throughout the plant and plant regeneration can be induced with or without undergoing a significant callus phase. Plant regeneration has been reported from numerous different axillary meristems attached to a number of explant sources from germinated seeds and at different developmental stages. In a majority of the methods described below, stimulation of shoot-bud growth is possible using low concentrations of cytokinin in the regeneration medium (Cheng et al. 1980; Saka et al. 1980). Cytokinins promote cell division and induce lateral bud growth at the nodal regions, which is important for the following regeneration methods. To continue bud growth of an explant, young, elongating shoots are typically removed to stimulate the development of new buds at the base by reducing endogenous auxin concentrations that are produced at the apical tip in elongating shoots (Wright et al. 1986). In protocols that are used for genetic transformation purposes, the axillary buds on existing shoots are mechanically removed to induce adventitious shoot production in a de novo fashion from meristem cells lying adjacent to the previously initiated shoots to ensure genetic uniformity of regenerating transgenic plants. The regeneration time of plants from the axillary or apical meristem cells is much more rapid than undergoing somatic embryogenesis. Shoot primordia are first formed within 1 week on shoot induction media, and plant formation is common within 1 month of culture. Because of the short culture time, the fertility of the regenerated plant is usually not compromised. Below, regeneration protocols focusing on the establishment of multiple shoot-bud cultures from axillary meristem cells are described. As long as the cultures are maintained by regular transfer to new medium, these proliferating shoot cultures are capable of plant regeneration for extended periods of time (i.e., >1year ).

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3.2 Regeneration Numerous regeneration protocols have been developed for inducing shoot growth from axillary meristematic cells attached to various explant tissues in soybean. These protocols are divided in this section into three groups, in which the first describes direct plant regeneration methods at the primary node, the second describes direct regeneration methods from the cotyledon node and the third describes indirect plant regeneration from callus cultures derived from axillary meristem cells located at the cotyledon node. 3.2.1 Primary-Node Regeneration Methods 1. Epicotyl explants. Wright et al. (1987b) reported the recovery of fertile plants from shoot buds that were induced on epicotyl tissue segments derived from 14-day-old seedlings germinated in the presence of 5 μM BAP. The addition of BAP to the germination medium resulted in a seedling with thickened hypocotyls, epicotyls and primary radicals, with a reduction in secondary root growth and apical dominance (Cheng et al. 1980). On average, seven segments were excised from the epicotyl and callus/shoot pads were formed on SH medium (Schenk and Hildebrandt 1972) containing 5.2 mM monobasic ammonium phosphate, 74 μM 3-aminopyridine and 20 μM kinetin for 5 weeks. Most shoots were concentrated on the apicalmost sections where preformed meristems were located but 10% of epicotyls produced shoots on a majority of explants which contained no preformed meristems (i.e., not adjacent to either the cotyledon or primary node). 2. Seedling explants. Regeneration of fertile soybean plants from the primarynode of intact 7-day-old seedlings was reported by Kim et al. (1990). The seedling was cut at the top of the primary node and at the base of the cotyledon, leaving an explant that consisted of a primary node, a cotyledon node, the internode between the nodes and a single cotyledon. Addition of 2 mg l−1 (8.9 μM) BAP, 0.02 mg l−1 (0.1 μM) NAA, 2 g l−1 l-proline and 4-fold inorganic micronutrients to an MS basal salt medium with 3% sucrose enhanced shoot production from the primary nodes. Proline was found to increase the number of shoots initiated, but ultimately caused a decrease in the length of the elongating shoots (Kim et al. 1994). The decrease in length was overcome by the addition of 4-fold micronutrient salt solution to the culture, where six components, especially zinc, were found to be significantly deficient of the normal concentration. 3.2.2 Cotyledon-Node Regeneration Methods Perhaps the most prolific shoot-bud formation from axillary meristem tissue is from cells located at the cotyledon node (Cheng et al. 1980; Wright et al.

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1986). Several regeneration methods that use the axillary meristems attached to various explants are described below. 1. Hypocotyl explants. Regeneration via organogenesis from hypocotyl segments of germinated seedlings has been reported only from the acropetal end of the hypocotyl closest to the cotyledon node (Kimball and Bingham 1973; Kaneda et al. 1997; Dan and Reichert 1998). Thus far, other segments of the hypocotyl below the cotyledon node have not been shown to have regenerative potential unless they are dissected from very small hypocotyls from 4−6 h imbibed seeds (Yoshida 2002). Recovery of plants was first reported by stimulating shoot-bud growth at the base of the cotyledon node on the hypocotyl using a combination of 2.9 μM IAA and 2.3 μM kinetin in the regeneration medium (Kimball and Bingham 1973). In a soybean regeneration protocol described by Dan and Reichert (1998), the explant was prepared by cutting a 5-mm explant segment below the cotyledon nodes of 7-day-old seedlings germinated with 5 μM BAP in the dark. The explants were cut such that most axillary meristem cells were removed from the explant tissue. Shoots were initiated from the cut surface closest to the cotyledon node by culturing the explant in the dark with the basipetal end inserted into the medium for 4 weeks. Adventitious shoots arose from the central region of the acropetal cross-section as well as from the outer edge, where axillary meristem cells from the cotyledon node are located (Reichert et al. 2003). R0 plants and their R1 progenies from 18 soybean genotypes representative of nine maturity groups were found to be morphologically normal. In another method, shoot regeneration from the acropetal end of hypocotyls of 4−6 h imbibed mature seeds was reported using B5 medium containing TDZ as a hormone (Yoshida 2002). In addition, shoots were also shown to regenerate from basipetal ends of segments cut 1−2 mm below the cotyledon node when cultured vertically on the medium with the basipetal ends positioned downward. From 14-day-old seedlings, Kaneda et al. (1997) also reported optimal shoot regeneration from the acropetal end of hypocotyl segments closest to the cotyledon node when cultured in regeneration medium containing 1−2 mg l−1 (9 μM) TDZ with a low salt concentration (50% B5, 50% L2 salts). 2. Cotyledon node explants without cotyledons. Using 4-week-old seedlings germinated in the presence of BAP, Cheng et al. (1980) prepared explants by removing cotyledons adjacent to the stem axis and cutting away the stem and hypocotyl about 3 mm above and below the cotyledon node. In a similar study, Wright et al. (1986) used 14-day-old instead of 4-weekold seedlings. From the cotyledon-node segment, multiple shoot-buds were induced when cultured in B5 salts/B5 vitamin medium with 3% sucrose and 5−50 μM BAP. To allow further shoot development, it was found necessary to reduce the BAP concentration to 1 μM. Regenerated plants were found to be morphogenetically normal and fertile.

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3. Cotyledon node explants with cotyledons. Of the soybean regeneration protocols via organogenesis, probably the most recognized explant is the cotyledon-node explant that is used in transformation protocols, referred to as the cotyledon-node transformation method. The explants are isolated from 4- to 10-day-old seedlings and consist of a single cotyledon with the axillary meristems attached to the base (Hinchee et al. 1988). In the first transformation protocol by Hinchee et al. (1988), explants from germinated seedlings were placed adaxial side down on B5 BA medium [B5 salts, 20 mg l−1 sucrose, 1.15 mg l−1 (6.6 μM) BAP, 0.8% purified agar; pH 5.8] for 3–4 weeks. After this time, explants were transferred to B5 O medium (B5 BA without BAP) and subcultured onto new medium every 4 weeks. Elongating shoots were removed and placed onto 50% B5 O medium to induce root development. The regeneration potential of the shoot-bud that formed at the base of the cotyledon was shown to be reduced if the cotyledons were removed from the developing shoots, indicating that the attached cotyledons may be important for shoot formation by translocating nutrients and possibly hormones in this explant (Franklin et al. 2004). 4. Regeneration of plants from cotyledon nodes incorporated in transformation methods. These include various modifications of the original protocol by Hinchee et al. (1988); and a synopsis is presented of the resulting method that is used by numerous laboratories (Townsend and Thomas 1994; Di et al. 1996; Zhang et al. 1999; Olhoft and Somers 2001; Paz et al. 2004; Zeng et al. 2004). Although minor variations from the method described below exist, most protocols use similar media compositions at four developmental stages that are specific to germination, shoot induction, shoot elongation and rooting. The cotyledon-node explants are prepared from 5- to 7-day-old seedlings by removing the hypocotyl approximately 1−5 mm below the cotyledon, removing the epicotyl above the cotyledon and separating the cotyledons with a vertical cut through the remaining hypocotyl tissue, such that two cotyledon-node explants remain. The removal of preformed axillary shoots is important at this time if the explants are to be used for transformation. To induce shoots at the cotyledon node, the explants are placed with the cotyledons in contact with B5 media with 3% sucrose supplemented with 7.5 μM BAP for 4 weeks. Recently, a synergistic effect between BAP and TDZ in an MS medium was reported to enhance the regeneration efficiency from the cotyledon node of 6-day-old seedlings (Franklin et al. 2004). A shoot induction medium with a combination of BAP and IBA has also been reported (Townsend and Thomas 1994; Donaldson and Simmonds 2000). The size of the developing callus/shoot pad at the base of the cotyledons after 4 weeks can reach several centimetres in diameter, depending on the cv. used. The explants are then usually transferred to an MS-based medium with 3% sucrose containing 0.775 mM pyroglutamic acid, 0.378 mM asparagine, 1.44 μM gibberellic acid, 0.57 μM IAA and 2.85 μM trans-zeatin riboside to induce elongation of the shoot buds. Glutamine and

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asparagine was first reported to be critical components added to induce de novo organogenesis in soybean (Shetty et al. 1992). However, a subsequent study found that glutamine added to the medium after autoclaving was inhibitory to regeneration and that, in fact, a breakdown product of glutamine when heated, pyroglutamic acid, resulted in the stimulatory effect. Therefore, current protocols use the addition of pyroglutamic acid instead of glutamine in shoot regeneration protocols (Wright et al. 1987a; Townsend and Thomas 1994). Elongated shoots are then removed and placed in a rooting medium containing 50% B5 salts, 2% sucrose and 5 μM IBA or 0.5 mg l−1 (2.7 μM) NAA for plant regeneration. 3.2.3 Callus Regeneration Methods As mentioned earlier, it was found possible to induce organogenesis from immature cotyledon explants by exposing explant tissue to MS-based medium amended with 13.3 μM BAP, 0.2 μM NAA and 4-fold the normal concentration of MS minor salts (Barwale et al. 1986). This treatment resulted in the regeneration of healthy, fertile plants. Another tissue reported to undergo plant regeneration via organogenesis from callus cultures is the base of the primary leaf petiole (Wright et al. 1987a). After 4 weeks on a medium containing B5 salts, 40 mg l−1 adenine sulfate, 1 g l−1 l-glutamine, and 0.1 mg l−1 2,4,5-trichlorophenoxyacetic acid, the callus cultures developed small green leaf-like structures through apparent organogenesis that eventually regenerated into mature, fertile plants. Recently, Sairam et al. (2003) reported that callus induced from cotyledon nodes excised from 3- to 4-day-old seedlings and cultured on an MS-based basal medium containing 2.26 μM 2,4-D and sorbital was able to regenerate morphologically normal soybean plants on medium containing 8.8 μM BAP and maltose. Callus induction was shown to begin after 7 days on callus induction medium and shoot buds arose after 15 days on regeneration medium. 3.3 Transformation Of the organogenic regeneration methods presented above, successful and repeatable transformation protocols have been reported using cotyledon-node explants and Agrobacterium for T-DNA delivery (Hinchee et al. 1988; Townsend and Thomas 1994; Di et al. 1996; Zhang et al. 1999; Donaldson and Simmonds 2000; Olhoft and Somers 2001; Paz et al. 2004; Zeng et al. 2004). Particle bombardment of tissues amiable to regeneration is difficult due to the inaccessibility for particles to these meristematic cells that give rise to the transformed shoots. Selection of transformed cells using Agrobacterium is especially important using cotyledon-node explants to prevent development of chimeric plants and to reduce regeneration of non-transformed plants or ‘escapes’. This is due in part to the large callus/shoot pad that forms at the base of the cotyledon and

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to the short regeneration time to plants. Selection for transgenic cells has been reported using the nptII gene with kanamycin selection (Hinchee et al. 1988), the bar gene with glufosinate selection (Zhang et al. 1999), the epsps gene with glyphosate selection (Clemente et al. 2000) and the hpt gene with hygromycin B selection (Olhoft et al. 2003). Of the selectable marker genes, nptII selection was found the weakest for soybean resulting in the most non-transformed ‘escapes’ (Donovan C.M., personal communication). High transformation efficiencies (>10%) were reported using a stringent selection regime involving hygromycin B (Olhoft et al. 2003), with little to no production of escapes or chimeric plants (Olhoft et al. 2004). Addition of thiol compounds to the semi-solid co-cultivation medium results in a significant increase in transformation using soybean cotyledon-node explants (Olhoft et al. 2001). The thiol compounds are thought to reduce plant tissue necrosis from wounding and Agrobacterium infection by inhibiting the activity of plant pathogen- and wound-response enzymes, such as peroxidases and polyphenol oxidases. In studies using optimized glufosinate selection, coupled with the addition of thiols during co-cultivation, transformation efficiencies were reported to increase from, on average, 0.9% to 2.1% using l-cysteine at 3.3−8.8 mM (Olhoft and Somers 2001), from 0.2–0.9% to 0.6– 2.9% using 3.3 mM l-cysteine and 1 mM DTT (Paz et al. 2004) and from 0.2% to 5.9% using 3.3 mM l-cysteine (Zeng et al. 2004). Transformation efficiencies were reported to be, on average, as high as 16.4% when explants were co-cultivated with 1 mM DTT, 1 mM sodium thiosulfate and 8.8 mM l-cysteine when combined with hygromycin B selection (Olhoft et al. 2003). 3.3.1 Examples of Cotyledon-Node Transformation Method The transformation methods described follow the above-mentioned regeneration methods, plus an additional step after seed germination that includes explant wounding and inoculation with Agrobacterium, followed by a period of co-cultivation, usually 3–5 days for T-DNA transfer. 1. The methods reported in the patent by Townsend and Thomas (1994) incorporate various conditions that optimize Agrobacterium-mediated transformation and medium for rooting of transgenic plants of explants derived from hypocotyl or cotyledon nodes of a germinated soybean seedling. Cotyledon-node explants were prepared from 3- to 4-day-old seedlings by removing the hypocotyl 3–4 mm below the cotyledons, dividing the seed in half by cutting through the shoot apex, wounding the cotyledon node with a scapel and inoculating in Petri dishes containing Agrobacterium resuspended in 20 ml of liquid inoculation medium containing B5 salts, 2% sucrose, 44 μM BAP, 0.5 μM IBA, 100 μM acetosyringone, 10 mM 2-(Nmorphino)ethanesulfonic acid (MES), pH 5.5, for 30 min. After incubation, the explants were removed and placed abaxial side down on semi-solid inoculation medium with 0.2% Gelrite for 3 days. Explants were moved into

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liquid counter-selection medium (B5 salts, 2% sucrose, 3 mM MES, 5 μM BAP, 0.5 μM IBA, 200 mg l−1 vancomycin, 500 mg l−1 cefotaxime, pH 5.7) with slow agitation for 4 days. The explants were embedded in semi-solid selection medium with their adaxial sides down and cultured at 28 ◦ C with A 16-h photoperiod for 2 weeks. After 2 weeks, the explants were washed overnight in medium with kanamycin sulfate before embedding in selection medium for an additional 2 weeks. Healthy explants with green regenerating tissues were transferred to elongation medium containing B5 salts, 2% sucrose, 3 mM MES, 0.2% Gelrite, 3.3 μM IBA, 1.7 μM gibberellic acid, 100 mg l−1 vancomycin, 30 mg l−1 cefotaxime and 30 mg l−1 timentin at pH 5.7. Elongating shoots, each approximately 0.5 cm in size, were excised and placed in rooting medium (B5 salts, 15% sucrose, 3 mM MES, 0.2% Gelrite, 20 μM nicotinic acid, 900 mg l−1 pyroglutamic acid, 10 μM IBA, pH 5.7) for 10 days. After 10 days, the shoots were moved to rooting medium without IBA and pyroglutamic acid until the root systems was well established, prior to transfer to soil. 2. The Agrobacterium was grown overnight in YEP medium to an OD650 of 0.6–0.8 at 27 ◦ C (Zhang et al. 1999). The bacteria were centrifuged and the pellet resuspended in a liquid co-cultivation medium containing 10% B5 salts, 1.67 mg l−1 (7.5 μM) BAP, 0.25 mg l−1 (0.72 μM) GA3 , 200 μM acetosyringone, 20 mM MES and 3% sucrose at pH 5.4. After preparing the explants as described above, the area around the cotyledon node was wounded by cutting 7–12 times, from approximately 3 mm above the node and 1 mm below the node, using a sharp scalpel blade. The explants were then immersed in the liquid Agrobacterium suspension for 30 min before transfer to cocultivation plates containing semi-solidified co-cultivation medium (0.5% purified agar) atop a filter paper to prevent Agrobacterium overgrowth. Thiol compounds can be added to the semi-solid co-cultivation medium to increase the transformation efficiency (Olhoft and Somers 2001). Glufosinate selection was used in this paper to select for cells carrying the bar transgene. The authors reported transformation frequencies up to 3%, using a selection regime of 5 mg l−1 glufosinate during shoot initiation and 2 mg l−1 during shoot elongation. Using this method, T1 seeds were collected from primary transformants 7–10 months after inoculation of explants.

4 Other Regeneration and Transformation Methods The production of transgenic soybean plants is not limited to the embryogenic and organogenic approaches outlined earlier. Other DNA delivery, regeneration and transformation techniques, in combination with various explant tissues, have been attempted and some are regularly used across laboratories. One successful approach involves targeting the embryonic axes of immature or mature seeds using particle bombardment or Agrobacterium for DNA de-

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livery (McCabe et al. 1988; Aragao et al. 2000). This approach targets the apical meristem cells very early in development. The small target explant offers an advantage over the more bulky explants used in the cotyledon-node transformation methods and faster culturing than embryogenic transformation methods. The aforementioned regeneration and transformation methods all involve significant periods of tissue culture. These methods require researchers skilled in the art and are relatively labour-intensive. Transformation methods that use ‘in planta’ approaches (with few or no tissue culture steps) are desirable in that problems associated with extended culture times, like sterility and time to-plant-production, are minimized along with cost and experience needed to produce transgenic plants. As of today, research in developing an in planta transformation method for soybean using Agrobacterium delivery of foreign DNA via the pollen tube pathway or ovarian injection has not produced a successful method that is repeatable across laboratories (Hu and Wang 1999; Li et al. 2002). However, two in planta transformation approaches have been reported to produce transgenic plants that use either microinjection of Agrobacterium into imbibed seedlings or electroporation-mediated transformation of mature axillary buds (Chee et al. 1989; Chowrira et al. 1996). Although these reports are promising, the transformation frequencies were low and have not been confirmed in other laboratories to date. Besides nuclear transformation, chloroplast transformation is also used for expression of foreign proteins in plants (Maliga 2004). Recently, the generation of fertile transplastomic soybean plants was reported by Dufourmantel et al. (2004). The benefits of chloroplast transformation include transmission of the transgene through maternal tissues and possibly higher gene expression than nuclear transformation. 4.1 Embryo Axes Transformation Methods McCabe et al. (1988) reported the recovery of a chimeric regenerated plant from embryonic axes of immature seeds after bombardment with gold particles covered with DNA using a discharge of a high-voltage capacitor through a small water droplet. Explants used for particle bombardment were isolated embryonic axes from immature seeds. Prior to bombardment, the primary leaves and stipules were removed and each explant placed onto medium to expose the apical meristem. After bombardment, the explants were cultured on MS media supplemented with 3% sucrose, 13.3 μM BAP, 0.2 μM NAA, 5 μM thiamine and 12 mM proline in the dark for 1–2 weeks. The explants were then transferred to new MS medium containing 1.7 μM BAP and 0.2 μM 2,4-D, and cultured in the light to induce shoot formation at the apical and axillary meristems. Unlike the transformation experiments described previously, this protocol did not use selection to enrich for transgenic shoots developing from the explant and resulted in the production of chimeric plants. Instead, T0 and T1 plants were screened for GUS or NPTII expression to differentiate between

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the transformed and non-transformed regenerants and seeds. Sato et al. (1993) found that even under kanamycin selection pressure up to 75 mg l−1 , the regenerating plants were chimeric with most of the positive sectors primarily located in the epidermis and outer cortex. The chimeric nature of the shoots can be attributed to regeneration of soybeans from shoot apical meristems that have multiple cellular origins for one or more of the L1, L2 and L3 cell layers (Christou 1990). Besides targeting embryo axes of immature seeds, embryo axes of mature seeds have been targeted for particle bombardment and transgenic cells selected for, using imazapyr (Lee et al. 1991) resistance conferred by the introduction of a mutant ahas gene in the bombarded plasmid (Aragao et al. 2000). In this method, the embryo axes were removed from 18−20 h imbibed seeds and the apical meristems exposed by removing primary leaves. After bombardment, the axes were placed into a shoot induction medium consisting of MS salts, 22.2 μM BAP, 3% sucrose, and 0.6% agarose for 16 h in the dark. Selection of transgenic cells was then carried out by culturing the embryo axes on MS medium with 500 nM imazapyr and continued until the shoots elongated. The production of chimeric plants was virtually eliminated by applying imazapyr selection and the transformation frequency was increased up to 200-fold over other methods without selective pressure (Aragao et al. 2000). Transformation of embryo axes from mature seeds using Agrobacterium-mediated approaches has also been reported. Liu et al. (2004) prepared explants as described by Aragao et al. (2000), but after shoot induction for 24 h, the explants were incubated with a liquid suspension of A. tumefaciens strain EHA105 carrying a binary vector with an nptII gene driven by the 35S promoter for 20 h at OD600 = 0.5. The axes were then removed and co-cultivated on 50% MS salts/B5 vitamin medium containing 6 mg l−1 (26.6 μM) BAP and 100 μM acetosyringone for 5 days. After co-cultivation, explants were recovered on 50% MS salts/B5 vitamin medium containing 0.2 mg l−1 (0.89 μM) BAP, 0.2 mg l−1 (1 μM) IBA, and 300 mg l−1 cefotaxime. Transgenic cells were selected on recovery medium supplemented with 100 mg l−1 kanamycin until shoots elongated. Unlike the kanamycin selection reported by Sato et al. (1993), the embryo axes were found to be sensitive to kanamycin selection and putative transformed plants were non-chimeric, with transformation efficiencies as high as 15.8%. 4.2 In Planta Transformation Methods 4.2.1 Microinjection Another approach to transforming soybean plants is microinjecting Agrobacterium into seeds (Chee et al. 1989). Seeds were germinated for 18−24 h on sterile moistened paper towels in the dark. Seed coats were removed, followed by one cotyledon and the remaining cotyledon with plumule and cotyledon node was wounded with a needle containing 30 μl of Agrobacterium cells at

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OD600 = 0.5. The seeds were placed in the dark at 26 ◦ C for 4 h and then planted in soil for full plant development. Plants were screened for nptII enzyme activity to identify transgenic plants. Of 4,000 inoculated seeds, 2,200 R0 plants were regenerated, of which only ten plants were positive for the nptII transgene using Southern analysis (transformation efficiency of 0.03%). Transmission of the T-DNA to T1 progenies was detected in only a few progeny of one line, suggesting that the T0 plants were chimeric. 4.2.2 Electroporation-Mediated Gene Transfer The production of transgenic soybean plants has also been reported by electroporating circular plasmid DNA into axillary buds of mature plants (Chowrira et al. 1996). The apical portion of 3-week-old plants were prepared by cutting the apical portion of the plants close to the node of a fully expanded leaf and removing the stipules and petioles to expose the axillary bud. Plasmid DNA was suspended in a salt solution and injected into the nodal buds to a depth of 1 mm using a syringe. After 20 min, each plant was electroporated with two square pulses of 99 ms at 200 V by placing the nodal bud in a DNA containing solution with in a circular electrode. Plants were placed into the glasshouse after treatment and progeny were tested for transgene integration in seeds derived from shoots arising from electroporated buds. Southern hybridization confirmed transgene integration into T2 soybean progeny plants from chimeric T0 plants.

5 Conclusions Several soybean regeneration and transformation methods have been established over the years. Although soybean transformation is not as efficient as transformation methods for other major crops such as maize, advances in improved regeneration techniques, coupled with DNA delivery methods recently led to the development of several reliable transformation protocols that are repeatable across laboratories. Continued improvements will take place more than likely in new selectable marker strategies, in optimization of culture conditions in transformation protocols and in Agrobacterium strain and binary vector improvements. As soybean transformation becomes more efficient, there are more opportunities to use transgenic soybean lines as genetic, molecular and genomic tools. For example, increased transformation efficiencies lead to the development of transposon tagging projects, as well as the development of virus-induced gene-silencing systems (Stacey et al. 2004). In addition to advancing soybean genomics, introgression of value added traits via nuclear or chloroplast transformation, with subsequent elite line development by plant breeders, will no doubt continue to be of economic importance in the future.

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Ghazi TD, Cheema HV, Nabors MW (1986) Somatic embryogenesis and plant regeneration from embryogenic callus of soybean, Glycine max L. Plant Cell Rep 5:452–456 Hammatt N, Davey MR (1987) Somatic embryogenesis and plant regeneration from cultured zygotic embryos of soybean (Glycine max L. Merr.). J Plant Physiol 128:219–226 Hansen G, Wright MS (1999) Recent advances in the transformation of plants. Trends Plant Sci 4:226–231 Hartweck LM, Lazzeri PA, Cui D, Collins GB, Williams EG (1988) Auxin-orientation effects on somatic embryogenesis from immature soybean cotyledons. In Vitro Cell Dev Biol 24:821–828 Hazel CB, Klein TM, Anis M, Wilde HD, Parrott WA (1998) Growth characteristics and transformability of soybean embryogenic cultures. Plant Cell Rep 17:765–772 Hinchee MAW, Connor-Ward DV, Newell CA, McDonnell RE, Sato SJ, Gasser CS, Fischhoff DA, Re DB, Fraley RT, Horsch RB (1988) Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. Bio/Technology 6:915–922 Hofmann N, Nelson RL, Korban SS (2004) Influence of media components and pH on somatic embryo induction in three genotypes of soybean. Plant Cell Tissue Organ Cult 77:157–163 Hu C-H, Wang L (1999) In planta soybean transformation technologies developed in China: procedure, confirmation and field performance. In Vitro Cell Dev Biol Plant 35:417–420 Jackson SA, Rokhsar D, Stacey G, Shoemaker RC, Schmutz J, Grimwood F (2006) Toward a reference sequence of the soybean genome: a multiagency effort. Crop Sci 46:55–61 Kaneda Y, Tabei Y, Nishimura S, Harada K, Akihama T, Kitamura K (1997) Combination of thidiazuron and basal media with low salt concentrations increases the frequency of shoot organogenesis in soybeans [Glycine max (L.) Merr.]. Plant Cell Rep 17:8–12 Kim J, LaMotte CE, Hack E (1990) Plant regeneration in vitro from primary leaf nodes of soybean (Glycine max) seedlings. J Plant Physiol 136:664–669 Kim J, Hack E, LaMotte CE (1994) Synergistic effects of praline and inorganic micronutrients and effects of individual micronutrients on soybean (Glycine max) shoot regeneration in vitro. J Plant Physiol 144:726–734 Kimball SL, Bingham ET (1973) Adventitious bud development of soybean hypocotyl segments in culture. Crop Sci 13:758–760 Ko T-S, Korban SS (2004) Enhancing the frequency of somatic embryogenesis following Agrobacterium-mediated transformation of immature cotyledons of soybean [Glycine max (L.) Merrill.]. In Vitro Cell Dev Biol Plant 40:552–558 Ko T-S, Lee S, Krasnyanski S, Korban SS (2003) Two critical factors are required for efficient transformation of multiple soybean cultivars: Agrobacterium strain and orientation of immature cotyledonary explant. Theor Appl Genet 107:439–447 Ko T-S, Lee S, Farrand SK, Korban SS (2004) A partially disarmed vir helper plasmid, pKYRT1, in conjunction with 2,4-dichlorophenoxyacetic acid promotes emergence of regenerable transgenic somatic embryos from immature cotyledons of soybean. Planta 218:536–541 Komatsuda T, Kaneko K, Oka S (1991) Genotype x sucrose interactions for somatic embryogenesis in soybean. Crop Sci 31:333–337 Lazzeri PA, Hildebrand DF, Collins GBA (1985) A procedure for plant regeneration from immature cotyledon tissue of soybean. Plant Mol Biol Rep 3:160–167 Lazzeri PA, Hildebrand DF, Collins GB (1987) Soybean somatic embryogenesis: effects of hormones and culture manipulations. Plant Cell Tissue Organ Cult 10:197–208 Lee A, Gatterdam PE, Chiu TY, Mallipudi M, Fiala RR (1991) Plant metabolism. In: Shaner DL, O’Connor SL (eds) The imidazolinone herbicides. CRC, Boca Raton, pp 151–165 Li Z, Nelson RL, Widholm JM, Bent A (2002) Soybean transformation via the pollen tube pathway. Soybean Genet Newsl 29:1–11 Lippmann B, Lippmann G (1984) Induction of somatic embryos in cotyledonary tissue of soybean, Glycine max L. Merr. Plant Cell Rep 3:215–218 Liu H-K, Yang C, Wei Z-M (2004) Efficient Agrobacterium tumefaciens-mediated transformation of soybeans using an embryonic tip regeneration system. Planta 219:1042–1049 Liu W, Moore PJ, Collins GB (1992) Somatic embryogenesis in soybean via somatic embryo cycling. In Vitro Cell Dev Biol 28P:153–160

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Liu W, Torisky RS, McAllister KP, Avdiushko S, Hildebrand D, Collins GB (1996) Somatic embryo cycling: evaluation of a novel transformation and assay system for seed-specific gene expression in soybean. Plant Cell Tissue Organ Cult 47:33–42 Maliga P (2004) Plastid transformation in higher plants. Annu Rev Plant Biol 55:289–313 McCabe DE, Swain WF, Martinell BJ, Christou P (1988) Stable transformation of soybean (Glycine max) by particle acceleration. Bio/Technology 6:923–926 Meurer CA, Dinkins RD, Redmond CT, McAllister KP, Tucker DT, Walker DR, Parrott WA, Trick HN, Essig JS, Frantz HM, Finer JJ, Collins GB (2001) Embryogenic response of multiple soybean [Glycine max (L.) Merr.] cultivars across three locations. In Vitro Cell Dev Biol Plant 37:62–67 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497 Olhoft PM, Somers DA (2001) L-Cysteine increases Agrobacterium-mediated T-DNA delivery into soybean cotyledonary-node cells. Plant Cell Rep 20:706–711 Olhoft PM, Lin K, Galbraith J, Nielsen NC, Somers DA (2001) The role of thiol compounds in increasing Agrobacterium-mediated transformation of cotyledonary-node cells. Plant Cell Rep 20:731–737 Olhoft PM, Flagel LE, Donovan CM, Somers DA (2003) Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method. Planta 216:723–735 Olhoft PM, Flagel LE, Somers DA (2004) T-DNA locus structure in a large population of soybean plants transformed using the Agrobacterium-mediated cotyledonary-node method. Plant Biotechnol J 2:289–300 Padgette SR, Kolacz KH, Delannay X, Re DB, LaVallee BJ, Tinius CN, Rhodes WK, Otero YI, Barry GF, Eichholtz DA, Peschke VM, Nida DL, Taylor NB, Kishore GM (1995) Development, identification, and characterization of a glyphosate-tolerant soybean line. Crop Sci 35:1451– 1461 Palanichelvam K, Oger P, Clough SJ, Cha C, Bent AF, Farrand SK (2000) A second T-region of the soybean-supervirulent chrysopine-type Ti plasmid pTiChry5, and construction of a fully disarmed vir helper plasmid. Mol Plant Microbe Interact 13:1081–1091 Parrott WA, Clemente TE (2004) Transgenic soybean. In: Boerma HR, Specht JE (eds) Soybeans: improvement, production, and uses, 3rd edn. (Agronomy Monograph 16) American Society of Agronomy/Crop Science Society of America/Soil Science Society of America, Madison, pp 265–302 Parrott WA, Dryden G, Vogt S, Hildebrand DF, Collins GB, Williams EG (1988) Optimization of somatic embryogenesis and embryo germination in soybean. In Vitro Cell Dev Biol 24:817– 820 Parrott WA, Hoffman LM, Hildebrand DF, Williams EG, Collins GB (1989) Recovery of primary transformants of soybean. Plant Cell Rep 7:615–617 Parrott WA, Durham RE, Bailey MA (1995) Somatic embryogenesis in legumes. In: Bajaj YPS (ed) Somatic embryogenesis and synthetic seed II. (Biotechnology in agriculture and forestry, vol 31) Springer, Berlin Heidelberg New York, pp 199–227 Paz MM, Shou H, Guo Z, Zhang Z, Banerjee AK, Wang K (2004) Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explant. Euphytica 136:167–179 Ranch JP, Oglesby L, Zielinski AC (1985) Plant regeneration from embryo-derived tissue cultures of soybeans. In Vitro Cell Dev Biol 21:653–657 Reichert NA, Young MM, Woods AL (2003) Adventitious organogenic regeneration from soybean genotypes representing nine maturity groups. Plant Cell Tissue Organ Cult 75:273–277 Sairam RV, Franklin G, Hassel R, Smith B, Meeker K, Kashikar N, Parani M, Abed Al, Ismail S, Berry K, Goldman SL (2003) A study on the effect of genotypes, plant growth regulators and sugars in promoting plant regeneration via organogenesis from soybean cotyledonary nodal callus. Plant Cell Tissue Organ Cult 75:79–85 Saka H, Voqui-Dinh TH, Cheng T-Y (1980) Stimulation of multiple shoot formation on soybean stem nodes in culture. Plant Sci Lett 19:193–201

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Samoylov VM, Tucker DM, Parrott WA (1998) Soybean [Glycine max (L.) Merrill] embryogenic cultures: the role of sucrose and total nitrogen content on proliferation. In Vitro Cell Dev Biol Plant 34:8–13 Sato S, Newell C, Kolacz K, Tredo L, Finer J, Hinchee M (1993) Stable transformation via particle bombardment in two different soybean regeneration systems. Plant Cell Rep 12:408–413 Schenk RU, Hildebrandt AC (1972) Medium and techniques for induction of growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:166–204 Shetty K, Asano Y, Oosawa K (1992) Stimulation of in vitro shoot organogenesis in Glycine max (Merrill.) by allantoin and amides. Plant Sci 81:245–251 Simmonds DH, Donaldson PA (2000) Genotype screening for proliferative embryogenesis and biolistic transformation of short-season soybean genotypes. Plant Cell Rep 19:485–490 Sleper DA, Shannon JG (2003) Role of public and private soybean breeding programs in the development of soybean varieties using biotechnology. AgBio Forum 6:27–32 Stacey G, Vodkin L, Parrott WA, Shoemaker RC (2004) National science foundation-sponsored workshop report. Draft plan for soybean genomics. Plant Physiol 135:59–70 Tomlin ES, Branch SR, Chamberlain D, Gabe H, Wright MS, Stewart CN Jr (2002) Screening of soybean, Glycine max (L.) Merrill, lines for somatic embryo induction and maturation capability from immature cotyledons. In Vitro Cell Dev Biol Plant 38:543–548 Townsend JA, Thomas LA (1994) An improved method of Agrobacterium-mediated transformation of cultured soybean cells. Patent WO9402620 Trick HN, Finer JJ (1997) SAAT: sonication-assisted Agrobacterium-mediated transformation. Transgenic Res 6:329–336 Trick HN, Finer JJ (1998) Sonication-assisted Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue. Plant Cell Rep 17:482–488 Trick HN, Dinkins RD, Santarem ER, Di R, Samoylov V, Meurer C, Walker D, Parrott WA, Finer JJ, Collins GB (1997) Recent advances in soybean transformation. Plant Tissue Cult Biotechnol 3:9–26 Wilcox JR (2004) World distribution and trade of soybean. In: Boerma HR, Specht JE (eds) Soybeans: improvement, production and uses. (Agronomy Monograph 16, 3rd edn) American Society of Agronomy/Crop Science Society of America/Soil Science Society of America, Madison, pp 1–13 Wright MS, Koehler SM, Hinchee MA, Carnes MG (1986) Plant regeneration by organogenesis in Glycine max. Plant Cell Rep 5:150–154 Wright MS, Ward DV, Hinchee MA, Carnes MG, Kaufman RJ (1987a) Regeneration of soybean (Glycine max L. Merr.) from cultured primary leaf tissue. Plant Cell Rep 6:83–89 Wright MS, Williams MH, Pierson PE, Carnes MG (1987b) Initiation and propagation of Glycine max L. Merr.: Plants from tissue-cultured epicotyls. Plant Cell Tissue Organ Cult 8:83–90 Wright MS, Launis KL, Novitzdy R, Duesiing JH, Harms CT (1991) A simple method for the recovery of multiple fertile plants from individual somatic embryos of soybean [Glycine max (L.) Merrill]. In Vitro Cell Dev Biol 27P:153–157 Yan B, Srinivasa Reddy MS, Collins GB, Dinkins RD (2000) Agrobacterium tumefaciens-mediated transformation of soybean [Glycine max (L.) Merrill] using immature zygotic cotyledon explants. Plant Cell Rep 19:1090–1097 Yoshida T (2002) Adventitious shoot formation from hypocotyl sections of mature soybean seeds. Breed Sci 52:1–8 Zeng P, Vadnais DA, Zhang Z, Polacco JC (2004) Refined glufosinate selection in Agrobacteriummediated transformation of soybean [Glycine max (L.) Merrill]. Plant Cell Rep 22:478–482 Zhang Z, Xing A, Staswick P, Clemente T (1999) The use of glufosinate as a selective agent in Agrobacterium-mediated transformation of soybean. Plant Cell Tissue Organ Cult 56:37–46

I.2 Canola V. Cardoza and C.N. Stewart1

1 Introduction Canola (Brassica napus L.) is an important oil crop that ranks only behind soybean and palm oil in global production. The term ‘canola’ was adopted by Canada apparently as an acronym of the Canadian Oilseed Association in 1979, with the goal of branding, and to replace the terms oilseed rape and rapeseed. Unlike these, canola oil is defined as an oil that must contains less than 2% erucic acid and the solid component of the seed must contain 3 years old were subjected to leaf painting and shown to be resistant to Basta. Bombardment with six plasmid constructs, carrying different versions of green florescent protein (GFP) and driven by different promoters in embryogenic cultures, was also successful. Transient expression of GFP in embryogenic cultures was observed (Parveez et al. 2000). Immature embryos 11–12 weeks after anthesis were transformed with pCAMBIA 1301 using the sonication-assisted Agrobacterium-mediated transformation (SAAT) method (Santarem et al. 1998). Plasmid CAMBIA 1301 carried the transgenes gusA encoding β-glucuronidase and hpt encoding hygromycin under the control of the CaMV 35S promoter.

6 Transgenic Plants for Oil Palm Improvement Recent advances in the biochemistry of seed oil biosynthesis, coupled with identification of genes for oilseed modification, have paved the way for the genetic engineering of oilseed crops that produce “designer” plant seed oils tailored for specific applications. Future transgenic oilseeds producing elevated concentrations of novel fatty acids represent renewable sources of raw materials that may compete with, and eventually replace, some petrochemicals (Dyer and Mullen 2005; Murphy 2006). The main goal for genetic engineering of oil palm is to alter oil quality by increasing the oleic acid and lowering the palmitic acid content (Cheah et al. 1995; Kadir and Parveez 2000; Sambanthamurthi et al. 2000). Production of novel high-value products is also targeted, including increased stearic acid, palmitoleic acid and ricinoleic acid content and producing biodegradable plastics (Parveez et al. 2000). Shell thickness and resistance to fungal diseases and insect pests are other traits, albeit complex and mostly polygenic, that can be manipulated to increase the yield and quality of oil palm. The fatty acid biosynthetic pathway, which is common to all plants (Fig. 6), involves repeated incorporation of two-carbon units derived from malonylCoA to elongate the fatty acid chain to approximately 16 or 18 carbons (Stumpf 1994). Ketoacyl-ACP synthase (KAS) II activity is rate-limiting in the mesocarp, resulting in the accumulation of palmitic acid. Increasing KAS II activity would increase stearoyl ACP, which would subsequently be desaturated to oleic acid. The relationship between KAS II activity and unsaturation has

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Fig. 6. Fatty acid biosynthetic pathway in oil palm fruits

been studied in the mesocarp of E. guineensis and E. oleifera, as well as in E. guineensis x E. oleifera hybrids. It was found that there was a positive correlation between KAS II activity and both I.V and C18 unsaturated fatty acids (C18 : 1 + C18 : 2 + C18 : 3; Sambanthamurthi et al. 1996). In most plants, the major product of fatty acid biosynthesis in the plastid is oleic acid (Murphy 2006). A thioesterase highly active towards oleoyl ACP has been described which ensures that oleic acid is released from ACP and exported out of the plastids. Plants that accumulate medium-chain fatty acids express a mediumchain-specific acyl ACP thioesterase (Davies et al. 1991; Pollard et al. 1991). In oil palm, two approaches were considered for producing higher oleic acid yields. One approach was to stimulate KAS II activity by overexpressing the KAS II gene, while the other approach was to reduce thioesterase activity towards palmitoyl ACP by expression of an antisense RNA of the palmitoylACP thioesterase gene (Parveez et al. 2000). The aim is to increase the oleate content in the mesocarp, where the oil is being synthesized. Construction of the palmitoyl-ACP thioesterase, KAS II and Δ9 stearoyl-ACP desaturase gene transformation vectors driven by a mesocarp-specific promoter is in progress (Siti Nor Akmar et al. 2001). Four transformation vectors have been constructed driven by constitutive promoters, the latter being pUbiSADN (full-length Δ9 stearoyl-ACP desaturase gene driven by maize ubiquitin 1 promoter), p35SSADN (full-length 9 stearoyl-ACP desaturase gene driven by CaMV 35S promoter), pCB302-AT1 and pCB302-AT2 (full-length antisense palmitoyl-ACP thioesterase gene driven by CaMV 35S promoter with differ-

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ent enhancers). Transformation of the full-length antisense palmitoyl-ACP thioesterase and Δ9 stearoyl-ACP desaturase gene constructs is under way. Embryogenic and suspension cultures have been transformed with the constructs via co-bombardment; and transgenic embryogenic cultures are being selected for their resistance to Basta. Oil palm contains 9 stearoyl-ACP desaturase, which desaturates steoryl-ACP into oleoyl-ACP and finally into oleic acid (Fig. 6). Introducing an antisense copy of the 9 stearoyl-ACP desaturase gene should cause stearic acid to be accumulated, by reducing the conversion of stearate to oleate (Knutzon et al. 1992). The expression of the stearoyl-ACP desaturase gene, driven by a seedspecific promoter, increases the stearate content from 1.8% by weight (normal) to 39.8% in the seed of transgenic rapeseed plants with a concomitant reduction of oleate. Four transformation vectors have been constructed for the production of high stearate transgenic oil palm: pPAsUbiSADN (partiallength antisense 9 stearoyl-ACP desaturase gene driven by maize ubiquitin 1 promoter), pPAs35SSADN (partial-length antisense 9-stearoyl-ACP desaturase gene driven by CaMV 35S promoter), pAsUbiSADN (full-length antisense Δ9 stearoyl-ACP desaturase gene driven by maize ubiquitin 1 promoter) and pAs35SSADN (full-length antisense 9-stearoyl-ACP desaturase gene driven by CaMV 35S promoter). Transformation of the two partial-length antisense Δ9 stearoyl-ACP desaturase gene constructs is in progress. Embryogenic cultures have been transformed with the constructs and some Basta resistant cultures have been produced. Regeneration of resistant embryogenic cultures has been initiated. The construction of a full-length antisense Δ9 stearoyl-ACP desaturase gene transformation vector driven by the mesocarp-specific promoter is also in progress (Rival and Parveez 2004). Palmitoleic acid is produced by desaturation of palmitic acid. Δ9-StearoylACP desaturase, which acts mainly on stearic acid, can also use palmitic acid as a substrate to produce palmitoleic acid (Fig. 6). Therefore, increasing stearoylACP desaturase activity may result in the accumulation of palmitoleic acid, which has important pharmaceutical applications (Parveez et al. 2000). Oil palm protoplasts can synthesize up to 30% of palmitoleic acid in their total lipids (Sambanthamurthi et al. 1996). The construction has been achieved for two vectors, pUbiSADN and p35SSADN, both with the full-length 9-stearoylACP desaturase gene driven by the maize ubiquitin 1 promoter and the CaMV 35S promoter, respectively. Embryogenic and suspension cultures have been transformed and some Basta-resistant cultures have been produced (Siti Nor Akmar et al. 2001). In bacteria, polyhydroxybutyrate (PHB) is derived from acetyl-coenzyme A by a sequence of three enzymatic reactions. The first enzyme of the pathway, 3-ketothiolase, catalyses the reversible condensation of two acetyl-CoA moieties to form acetoacetyl-CoA. Acetoacetyl-CoA reductase subsequently reduces acetoacetyl-CoA to d-(–)-3-hydroxybutyryl-CoA, which is then polymerized by the action of PHB synthase to form PHB (Anderson and Dawes 1990). Introduction of these genes into oil palm could result in the accumula-

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tion of PHB (Poirier et al. 1992). Both PHB and related polyhydroxylkanoates (PHAs) are renewable sources of biodegradable thermoplastic materials. Each of the three genes involved in PHB synthesis is driven by a different promoter. A second construct, in which the phbA gene is replaced with the bktB gene, can result in the accumulation of PHBV rather than PHB (Rival and Parveez 2004). The plastid-targeting sequence from the oil palm ACP gene (Rasid et al. 1999) is being used to make the chimeric gene constructs, because studies in Arabidopsis suggested that a higher yield could be obtained when PHB production was confined to the plastids (Nawrah et al. 1994). Both constructs are in a binary vector, flanked by matrix attachment regions of tobacco and using maize ubiquitin, CaMV 35S and rice actin promoters. Selection has been initiated for Basta-resistant embryogenic cultures (Siti Nor Akmar et al. 2001).

7 Conclusion and Perspectives Progress in oil palm breeding will rely on the association of neutral and gene molecular markers with selected phenotypic characters for the implementation of marker-assisted selection (MAS). MAS will limit the time lapse between selected generations, speed-up genetic progress and thus directly improve the quality of commercial seeds delivered to end-users. Major applications will include the monitoring of variability (germplasm, combining ability groups), the genotyping for characters which are highly influenced by the environment (or costly to measure), the control and monitoring of recombination, the prediction of genotypic values for complex characters, as well as the prediction of the value of a cross from information about the parents. Automatic genotyping will be applied to outstanding parents and the survey populations will be integrated into the oil palm breeding scheme, as well as the marker-assisted checking and selection of genitors already used in commercial seed gardens. Biomolecular results, in terms of favourable gene detection and evaluation using molecular tools, will be integrated into breeding schemes. Recent results in micropropagation using embryogenic cell suspensions pave the way for multiplication strategies based on the ‘artificial seed’ concept for oil palm. The latter could be obtained from embryos produced at a high rate from embryogenic cell suspensions, which would then be properly treated to achieve maturation (enrichment in storage proteins). Studies on global methylation rates provided an insight on molecular changes associated with the mantled abnormality, which is consistent with the epigenetic characters observed, including reversion. By isolating oil palm genes underlying the mantled abnormality, early markers will help to elucidate epigenetic changes causing this somaclonal variation and enable the development of an early testing procedure for clonal conformity. Genetic engineering is emerging as a powerful tool for oil palm improvement, as it is continuously benefiting from rapid progresses in plant rege-

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neration, transformation technology and gene discovery. Oil palm transgenic plants with modified fatty acid biosynthesis are about to be field-tested (Rival and Parveez 2004). In the near future, the expected intensification of oil palm culture resulting from growing global needs for vegetable oil will generate a sustained demand for plant material designed to withstand suboptimal agro-ecological conditions. In this perspective, optimizing water use efficiency in oil palm is a target of paramount interest for breeders and biotechnologists (Sivamani et al. 2000; Sinclair et al. 2004). It is worth noticing that fertilizer cost, which may account for ca. 20% of total running costs of oil palm estates, remains one of the main financial inputs for growers. In the future, this cost is expected to rise due to the global increase in energy costs and the rarefaction of natural minerals. Besides economic issues, there are increasing environmental risks linked to the utilization of chemical fertilizers on a large areas. For example, the loss of fertilizer N may result from gaseous plant emission, soil denitrification, surface runoff, volatilization and leaching (Raun and Johnson 1999). Genetically engineered oil palms able to produce high oil yields with limited fertilizer input (N and/or P) will provide a long-term alternative for the sustainable culture of oil palm. Acknowledgements. The author was supported by a Marie Curie Outgoing International Fellowship from the Sixth Framework Program of the European Commission.

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I.5 Peanut P. Ozias-Akins1

1 Introduction Peanut (Arachis hypogaea L.) is a food crop grown in warm temperate, subtropical and tropical regions of the world. Groundnut is another common name for A. hypogaea that frequently is encountered in literature of Asian origin. The seeds are consumed after minimal processing, such as roasting or boiling, or are used for peanut paste/butter, in confectionary items, or for oil extraction. The biology of fruit production is novel compared with other primary crops in that the fruit develop underground. This geocarpic development also requires novel solutions for pest and pathogen control, as well as harvesting. For example, foliage-applied contact pesticides rarely provide sufficient control of a soilborne pathogen or underground pest. Soil incorporation of a chemical can be carried out mechanically or passively, but environmental conditions at the time of treatment may not be favorable for passive incorporation. Systemic pesticides have the added risk of accumulation in seeds. In addition to the specific pesticide issues related to peanut, there are the broader issues of environmental degradation and ecological impact on pest populations (e.g. pest resistance or toxicity to beneficial insects). An alternative to pesticide use is the development of genetic or host-plant resistance, which can be accessed from within the Arachis gene pool through traditional breeding or outside the gene pool using genetic engineering (Knauft and Ozias-Akins 1995; Holbrook and Stalker 2003). The transfer of desirable traits using both of these approaches for the development of improved cultivars of peanut is the subject of this review.

2 Applications of Molecular Markers Comprehensive reviews on the history of molecular marker development in peanut are provided by Stalker and Mozingo (2001) and Dwivedi et al. (2003), and readers are referred to these papers for information on non-DNA-based molecular markers, such as isozymes and seed proteins. 1 Department of Horticulture, The University of Georgia Tifton Campus, Tifton, GA 31793, USA,

e-mail: [email protected]

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2.1 Origin of Cultivated Peanut Cultivated peanut is an allotetraploid, one of only two tetraploid species in the genus Arachis, section Arachis. The geographical origin of peanut is in South America around the current borders of Brazil, northeastern Paraguay, Bolivia and northern Argentina. A. hypogaea is classified into two subspecies and six varieties (var. hypogaea and var. hirsuta in subsp. hypogaea; var. fastigiata, var. vulgaris, var. peruviana and var. aequatoriana in subsp. fastigiata). Peanut can readily cross with tetraploid A. monticola, a species that may be a “weedy” conspecific relative of peanut (Hilu and Stalker 1995) or which may have evolved as a weedy phenotype from A. hypogaea (Stalker and Simpson 1995; Jung et al. 2003). It is highly likely to have originated through the same or a similar hybridization event between two diploid species (Kochert et al. 1996). All other species in the section are diploid and cross with A. hypogaea with varying degrees of difficulty. Peanut (and A. monticola) most likely originated from an interspecific cross between A. duranensis, an A-genome diploid, and A. ipaensis, a B-genome diploid. Several lines of evidence supporting this hypothesis include archeological data (Simpson et al. 2001), the frequency of common molecular markers (Kochert et al. 1991, 1996), cytological characteristics (Seijo et al. 2004) and gene sequence data (Jung et al. 2003; Ramos et al. 2006), although recent molecular data identify other putative A-genome progenitor candidates (Milla et al. 2005). Even though the A- and B-genomes of peanut can be readily distinguished by molecular polymorphisms, the level of polymorphism among peanut cultivars and accessions is very low, suggesting a genetic bottleneck at the time peanut originated (Kochert et al. 1996). 2.2 Genetic Diversity Infrequent polymorphisms have hindered the development and application of genetic maps of peanut. Nevertheless, various types of molecular markers have been used for diversity studies and trait tagging (Table 1). While the first example of microsatellite discovery resulted in the development of six simple sequence repeat (SSR) primer pairs that detected polymorphism (Hopkins et al. 1999), more recent studies significantly expanded this number (Ferguson et al. 2004b; He et al. 2003, 2005; Moretzsohn et al. 2004, 2005). Ferguson et al. (2004b) tested 110 SSR primer pairs on a panel of 24 landraces that represented all six varieties of peanut. The most frequently encountered microsatellite repeats were (ATT)n and (GA)n, which revealed an allele frequency of 4.8 and 3.6, respectively. In their companion study of 188 peanut accessions sampled from the six botanical varieties and three continents, ten of the SSR primer pairs detected 7.4 alleles per locus on average (Ferguson et al. 2004a). Based on their analysis of genetic distance var. perviana and var. hirsuta were suggested to be elevated to subspecies level. The (GA)n repeat also was found to be most abundant by He et al. (2003), who observed an average allele frequency

Peanut

83

of 4.2 for 19 microsatellite loci after surveying 24 plant introductions, four from each variety of peanut. A third recent microsatellite study identified TTG as an abundant repeat, but of 67 SSR markers developed, only three were polymorphic in cultivated peanut even when representatives of all six varieties were tested (Moretzsohn et al. 2004). In subsequent work, Moretzsohn et al. (2005) developed an additional 271 SSR markers and showed that, of the 62 that were polymorphic in A. hypogaea, an average of 5.8 alleles per locus were detected. The development of microsatellite markers will be enhanced by DNA sequencing projects, particularly expressed sequence tag (EST) projects targeting gene sequences (Luo et al. 2005b; Proite et al. 2007). Such projects in peanut are relatively small in scope at this time, due to the limited funding available for genomics research in this crop. Marker types other than microsatellites detect less allelic diversity, but are the focus of considerable research (Table 1). These include restriction fragment length polymorphisms (RFLPs) using genomic and cDNA clones as probes (Halward et al. 1991; Kochert et al. 1991; Paik-Ro et al. 1992), random amplified polymorphic DNAs (RAPDs; Halward et al. 1992; Lanham et al. 1992; Creste et al. 2005) and amplification fragment length polymorphisms (AFLPs; He and Prakash 1997; Milla et al. 2005; Tallury et al. 2005). The AFLP and RAPD techniques are relatively more efficient at detecting polymorphisms than RFLPs, mainly because at least an order of magnitude more fragments can be generated for each experiment. For all marker types, however, it can be concluded that little genetic diversity is present in cultivated peanut even though extensive polymorphism has been observed between peanut and its diploid relatives. For detection of polymorphism within the cultivated peanut, the marker type of preference probably is SSRs because of codominance and therefore the ability to sample allelic diversity. The disadvantage of SSRs is the time and cost of the development phase. The second-most informative marker type is AFLPs because of the large number of loci that can be sampled in a short period of time. Its main disadvantage is the dominant nature of the markers. 2.3 Genetic Maps The small amount of genetic diversity observed at the molecular level has so far prevented the application of genetic mapping to peanut breeding except in cases where polymorphic chromosomal regions have been introgressed into A. hypogaea from diploid relatives. Introgression pathways were summarized by Simpson (2001). The first genetic map in the genus was an interspecific cross between the two diploid species, A. stenosperma x A. cardenasii (Halward et al. 1993). Genomic and cDNA clones were used as probes and 15/100 and 190/300, respectively, showed a polymorphism between the parents. Because of banding pattern complexity, only 132 markers could be analyzed for segregation and 117 of these were assigned to 11 linkage groups. This map was used for the selection of 73 RFLP probes that were used to screen a set of 46 intro-

Accessions

Marker

Polymorphism within species

Polymorphism between species

Reference

A. hypogaea: 27 accessions representing 4 varieties; 12 other species represented by 29 accessions A. hypogaea: 8 cultivars representing 3 varieties A. hypogaea: 27 accessions representing 4 varieties; 31 other species A. hypogaea: 1 accession, 4 other species

RFLP, RAPD

Abundant

Halward et al. (1991)

RFLP

None for A. hypogaea using 60 probes with 13 restriction enzymes and 10 RAPD primers Little to none

Kochert et al. (1991)

RAPD

None in A. hypogaea

RAPD

N/A

A. hypogaea: 15 accessions representing 3 varieties; 5 other species

RFLP

A. duranensis, A. stenosperma

RFLP

2/23 probes showed polymorphism in A. hypogaea; frequency of polymorphism within A. cardenasii and A. duranensis reached 80% N/A

Abundant; 7/21 probes detected polymorphism Abundant polymorphism with all 10 primers 49 polymorphic fragments from 26/60 primers in a cross between A. hypogaea and an amphidiploid Abundant polymorphism between species

A. hypogaea: 1 accession plus 46 introgression lines; 7 other species

RFLP, RAPD

Halward et al. (1992) Lanham et al. (1992)

Paik-Ro et al. (1992)

Halward et al. (1993) Garcia et al. (1995) P. Ozias-Akins

Polymorphism only where chromosomal fragment introgressed from A. cardenasii between A. hypogaea and A. cardenasii

132 polymorphic markers from 400 probes Abundant; 244 fragments from 270 primers polymorphic

84

Table 1. Summary of molecular marker studies for analyzing diversity in the genus Arachis, with emphasis on cultivated peanut. AFLP Amplified fragment length polymorphism (Vos et al. 1995), DAF DNA amplification fingerprinting (Caetano-Anolles et al. 1991), ISSR inter-simple sequence repeat (Zietkiewicz et al. 1994), RAPD random amplified polymorphic DNA (Welsh and McClelland 1990; Williams et al. 1990), RFLP restriction fragment length polymorphism (Botstein et al. 1980), rDNA ribosomal DNA, SSR simple sequence repeat (Weber 1990)

Accessions

Marker

Polymorphism within species

Polymorphism between species

Reference

A. hypogaea: 2 accessions; 8 other species represented by 25 accessions

RAPD

Abundant; 132 polymorphic fragments from 10 primers

Hilu and Stalker (1995)

A. hypogaea: 1 cultivar and 12 mutants

RAPD

N/A

Bhagwat et al. (1997)

A. hypogaea: 6 accessions representing 3 varieties

RAPD (DAF); AFLP

N/A

He and Prakash (1997)

A. hypogaea: 19 accessions representing 4 varieties; 3 other species A. hypogaea: 40 accessions plus 30 other genotypes representing 3 varieties A. hypogaea: 14 cultivars representing 3 varieties A. hypogaea: 1 cultivar and one synthetic polyploid; 3 other species

SSR

None in A. hypogaea; highest among A. cardenasii accessions 65/1,182 polymorphic fragments from 12 primers 63 polymorphic fragments from 17/559 primers; 111 polymorphic fragments from 28/64 primer pairs 6/27 primer pairs polymorphic in A. hypogaea 7/48 primers yielded 27/408 polymorphic bands

Similar level of polymorphism as A. hypogaea N/A

Hopkins et al. (1999)

RAPD

RAPD RFLP

A. hypogaea: 26 accessions representing 3 varieties

RAPD

A. hypogaea: 44 accessions representing 6 varieties; A. monticola: 3 accessions

AFLP

7/37 primers polymorphic (1 highly variable) 762/917 fragments polymorphic between cultivar and synthetic polyploid 176 markers from 8 primers revealed a similarity of 59–99% among accessions No polymorphism in A. hypogaea; 19/20 markers polymorphic in A. duranensis

Subramanian et al. (2000)

N/A

Bhagwat et al. (2001)

427–809/917 fragments polymorphic among species/genotypes N/A

Burow et al. (2001)

Extensive polymorphism between species; A- and B-genomes could be clearly distinguished

Peanut

Table 1. continued

Dwivedi et al. (2001)

He and Prakash (2001)

85

86

Table 1. continued

Marker

Polymorphism within species

Polymorphism between species

Reference

A. hypogaea: 13 accessions representing 2 subsp.; 15 other species

RAPD ISSR

All fragments polymorphic across species

Raina et al. (2001)

A. hypogaea: 1 accession plus 30 introgression lines; 8 other species represented by 28 accessions

RFLP

122 and 46/223 fragments were unique to A- and B-genomes, respectively

Gimenes et al. (2002a)

A. hypogaea: 9 genotypes; 19 other species

AFLP

406/408 polymorphic fragments

Gimenes et al. (2002b)

A. hypogaea: 39 accessions; 15 other species represented by 36 accessions A. hypogaea: 24 accessions representing 6 varieties A. hypogaea: 21 genotypes representing 2 subspp

RFLPrDNA

94/220 polymorphic fragments from 17/21 primers; 67/124 polymorphic fragments from 23/29 primer pairs 17/24 probes detected introgressed fragments in 26/30 lines; considerable polymorphism among accessions of A. cardenasii and of A. duranensis 6/94 polymorphic fragments from 3 primer pairs in A. hypogaea 1 fragment could distinguish between subsp. of A. hypogaea

A. hypogaea and A. duranensis were the most similar

Singh et al. (2002)

19/56 SSR primer pairs showed polymorphism 48/80 primer combinations yielded 90/3241 polymorphic fragments N/A

N/A

He et al. (2003)

N/A

Herselman (2003)

80 polymorphic fragments from 10/34 primers

Dos Santos et al. (2003)

A hypogaea: 2 accessions; 9 other species in section Arachis; species from 4 other sections

SSR AFLP

RAPD

P. Ozias-Akins

Accessions

Peanut

Table 1. continued

Accessions

Marker

Polymorphism within species

Polymorphism between species

Reference

A. hypogaea: 24 accessions representing 6 varieties A. hypogaea: 188 accessions representing 6 varieties A. hypogaea: 60 accessions representing 6 varieties; 36 accessions representing 27 other species in section Arachis A. hypogaea: 48 genotypes from var. fastigiata A. hypogaea: 1 cultivar; 15 accessions representing 11 other species in sections Arachis and Heteranthae A. hypogaea: 48 accessions representing 6 varieties A. hypogaea: 10 accessions representing 6 varieties; 98 accessions representing 25 other species in section Arachis A. hypogaea: 16 accessions representing 6 varieties; 2 diploid species

SSR

110/192 primer pairs showed polymorphism 10 SSR primer pairs revealed 89 alleles at 12 loci 3/67 SSR primer pairs yielded polymorphism in A. hypogaea

N/A

Abundant polymorphism between species

Ferguson et al. (2004b) Ferguson et al. (2004a) Moretzsohn et al. (2004)

N/A

Krishna et al. (2004)

88 polymorphic fragments from 9 primers

Creste et al. (2005)

N/A

He et al. (2005)

8 primer combinations generated 239 polymorphic fragments 113/229 were polymorphic between species

Milla et al. (2005), Tallury et al. (2005)

A. hypogaea: 16 accessions representing 6 varieties; 2 diploid species

SSR SSR

SSR RAPD

SSR AFLP

SSR

SSR

18 SSR primer pairs amplified an average of 6.9 alleles N/A

8/38 SSR primer pairs yielded variety-specific fragments Little within A. hypogaea

66/234 SSR primer pairs amplified polymorphisms in A. hypogaea 4/84 SSR primer pairs amplified polymorphisms in A. hypogaea

N/A

21/84 SSR primer pairs amplified polymorphisms between A. duranensis and A. stenosperma

Moretzsohn et al. (2005) Proite et al. (2007)

87

88

P. Ozias-Akins

gression lines originating from the cross A. hypogaea x A. cardenasii (Garcia et al. 1995). Approximately 30% of the A. cardenasii genome was found to be introgressed among these 46 lines. Most (88%) of the introgressed fragments replaced A-genome alleles, while a minor percentage (12%) replaced B-genome alleles. Application of molecular markers to identify alien chromosome segments in A. hypogaea continues to be useful for evaluation of the variation recovered through the triploid/hexaploid introgression route (Anderson et al. 2004; Garcia et al. 2006). A second A-genome map was developed from the cross A. duranensis x A. stenosperma that also consists of 11 linkage groups and a total map length of 1,231 cM determined by linkage analysis of 170 SSR markers (Moretzsohn et al. 2005). Currently there is only a single genetic map published at the tetraploid level, although this map was produced from a complex interspecific hybrid of A. hypogaea, A. cardenasii, A. batizocoi and A. diogoi (Burow et al. 2001). The map consists of 379 RFLP loci assigned to 23 linkage groups, for a total map length of 2,210 cM. In non-introgressed germplasm, it is difficult to find a sufficient number of polymorphic markers for mapping quantitative trait loci (QTL) in multiple crosses. An example of the limitations associated with mapping in A. hypogaea is provided by Stalker and Mozingo (2001) who crossed a peanut cultivar, NC7, with multiple plant introductions that had resistance to early leafspot. After screening 572 RAPD primers in the populations, only eight showed polymorphisms, although all polymorphic markers showed significant associations with one or more traits in two of the populations. It is anticipated that SSR markers will deliver denser genetic maps, even within the cultivated species. The potential now exists to assemble a physical map of peanut using large-insert bacterial artificial chromosome (BAC) clones (Yuksel and Paterson 2005) and to integrate the genetic and physical maps (Yuksel et al. 2005a). Development of peanut genomics databases will facilitate the dispersal of map information that can be adapted to breeding applications (Jesubatham and Burow 2006). 2.4 Tagged Traits The complex hybrid, from which derived the only tetraploid mapping population, was very useful for the development of nematode-resistant cultivars COAN (Simpson and Starr 2001) and NemaTAM (Simpson et al. 2003). RAPD and RFLP markers linked to nematode resistance were found in this material (Burow et al. 1996; Choi et al. 1999) as well as in independently developed interspecific hybrids from North Carolina State University (Garcia et al. 1996). In both cases for introgression of resistance genes, the nematode resistance was most likely to originate from A. cardenasii. Burow et al. (1996) used bulked segregant analysis on susceptible and resistant bulks from a BC4F2 population derived from Florunner x TxAG7. The same three diploid species as used for the tetraploid mapping population described above, A. cardenasii, A. batizocoi and A. diogoi, were potential sources of the nematode resistance gene(s) in TxAG7.

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From 540 RAPD primers tested, only three markers were found to be associated with nematode resistance. Statistical analysis of segregation data for nematode resistance supported the inheritance as a single gene that limited egg production. The three linked markers, RKN410, RKN440 and RKN229, showed 5.4%, 5.8% and 9% recombination, respectively, with the resistance gene. Two of the three originated from either A. cardenasii or A. diogoi, but RKN440 was present only in A. cardenasii and resistant genotypes. Church et al. (2000) used more advanced backcross materials derived from TxAG7 and RFLPs for markerassisted selection. The advantage of RFLP markers is their codominance versus the dominance of RAPD or AFLP markers, and thus their ability to more rapidly identify homozygous individuals. The disadvantage of RFLPs is the cost and time involved in the assay. Church et al. (2000) were able to determine the genotype of only 65–86% of the individuals attempted because of technical difficulties, such as the low quality or quantity of DNA, incomplete digestion of DNA, or poor hybridization or background on Southern blots. Nevertheless, they were able to genotype 500 individuals over a 2-month period. A PCR-based marker that amplifies size-distinguishable fragments from the resistant and susceptible genotypes recently was developed for the RKN440 locus and can greatly facilitate the throughput for marker-assisted selection (Chu et al. 2007). In contrast to the TxAG7-derived nematode resistance, introgression line GA6 derived from interspecific crosses carried out in North Carolina, segregated for two resistance genes: Mae that reduced egg number and Mag that reduced host galling. The two genes were shown to be tightly linked, however, in crosses between GA6 and susceptible A. hypogaea. Furthermore, one linked RAPD marker identified (Z3/265) could be assigned to LG1 of peanut, the same linkage group contributing nematode resistance from TxAG7 (Burow et al. 2001). Diploid species in section Arachis could provide an extensive resource for mining disease/pest resistance genes, although transfer of genes of diploid origin to tetraploid peanut by traditional breeding is a long-term process. Efforts are underway to characterize disease resistance homologs from A. hypogaea and its wild relatives (Bertioli et al. 2003; Yuksel et al. 2005b). Gene-based marker development may also become feasible through the identification of differentially expressed genes upon comparison of genotypes resistant and susceptible to pathogens (late leaf spot), Aspergillus, and drought (Guo et al. 2006; Luo et al. 2005a,c). A few examples of resistance traits in the primary gene pool of peanut and their linkage to molecular markers are emerging. One is aphid resistance found in an African breeding line that is an important trait for reducing infection by groundnut rosette virus which is transmitted by this insect vector. Herselman et al. (2004) identified linkage of a recessive aphid resistance gene with AFLP markers in a segregating population of 60 F2 individuals. Three hundred and eight AFLP primer combinations generated 986 polymorphic fragments from parental lines out of a total of 12,315. Nineteen polymorphic markers were screened on the total mapping population and 11 mapped to five linkage groups, one of which also carried the aphid resistance gene.

90

P. Ozias-Akins

Another trait for which polymorphisms within a genome were detected is high-oleic peanut. The high oleate trait is desirable because the oil is less prone to oxidation and development of rancidity. Gene-specific polymorphisms were discovered from gene sequence data for fatty acid desaturase genes (FAD1, FAD2) and could be clearly designated as allelic after the subgenomic assignment of these gene copies were determined (Jung et al. 2003). Intragenomic (allelic) polymorphism was shown for ahFAD2A, the A-genome homeolog for oleoyl-PC desaturase. One allele (ahFAD2A-1) gives rise to a much less active enzyme than the other allele (ahFAD2A-2) and is part of the basis for the higholeate trait in peanut (Jung et al. 2000). Alleles in the B-genome homeolog that vary by the position of a miniature inverted-repeat transposon (MITE) insertion and possibly single-nucleotide polymorphisms also have been described among high-oleate mutants (Lopez et al. 2000, 2002; Patel et al. 2004). These studies point out the need to determine sub-genomic assignment before considering a sequence polymorphism in allotetraploid A. hypogaea to be allelic. A similar type of sub-genomic assignment was carried out for the seed storage and allergen protein genes, ara h 2 and ara h 6, which now makes possible the study of allelic diversity (Ramos et al. 2006; Ozias-Akins et al. 2006).

3 Peanut Transformation Progress in tissue culture and transformation of peanut was discussed by OziasAkins and Gill (2001). This review focuses mainly on reproducible methods and progress made since 2001. Peanut transformation is carried out with both direct DNA (biolistic) and Agrobacterium-mediated gene transfer methods. The biolistic method uses embryogenic cultures as the target tissue, whereas Agrobacterium-mediated transformation relies primarily on shoot-regenerating cultures. Both methods have advantages and disadvantages. Although there is not much evidence for peanut, the evidence for other crops is overwhelming for the integration of a smaller number of transgene copies via Agrobacterium rather than by direct DNA transfer methods (Shou et al. 2004). The higher frequency of low-copy insertions with Agrobacterium-mediated transformation results in fewer complex integration events that have to be discarded due to instability in expression levels. The biolistic method for peanut, however, has been adapted to a wider range of cultivars, particularly those cultivated in the United States. So far, Agrobacterium transformation has been successful with only one United States cultivar, New Mexico Valencia A (Cheng et al. 1996, 1997). Improvements in protocols for Agrobacterium-mediated transformation of soybean (Olhoft et al. 2003; Somers et al. 2003) could be tested on peanut cultivars, and success would perhaps result in wider application of this technology. For commercial purposes, use of either biolistic or Agrobacterium-mediated transfer technology is complicated by freedom-to-operate issues.

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91

3.1 Biolistic-Based Gene Transfer The biolistic method was shown to be useful for the production of transgenic peanut (Ozias-Akins et al. 1993; Brar et al. 1994). In the earlier study, embryogenic tissues were bombarded (Ozias-Akins et al. 1993), whereas in the latter shoot meristems were bombarded (Brar et al. 1994). No subsequent reports demonstrating transformation using the biolistic method in peanut have used the shoot meristem as a target. This is probably because of the difficulty in preparing explants and the expense of screening a large number of plants resulting from non-selective conditions. For embryogenic tissues, the most effective selectable marker gene is hph or hpt (hygromycin phosphotransferase) which confers the ability of tissues to grow on medium containing 20 mg l−1 hygromycin (Ozias-Akins et al. 1993; Livingstone and Birch 1999). This concentration of hygromycin proves sufficient for eliminating escapes. Selection is carried out in either semi-solid or liquid medium (Ozias-Akins et al. 1993). The bottlenecks for peanut transformation using the biolistic method are: (1) embryogenic culture growth rate, (2) transformation frequency and (3) regeneration frequency. Establishment of embryogenic cultures does not appear to be a particularly serious bottleneck since such cultures have been obtained from numerous genotypes (Ozias-Akins et al. 1992; Little et al. 2000; Ozias-Akins and Gill 2001). Cultures can be established from immature cotyledons, epicotyls, or young leaves (Ozias-Akins and Gill 2001). Auxin is the only growth regulator required for initiation of embryogenic cultures. Both 2,4-dichlorophenoxyacetic acid (2,4-D) and picloram have been extensively tested, but picloram at concentrations of 3−30 mg l−1 has been more widely used in recent studies (Yang et al. 1998; Livingstone and Birch 1999; Jayabalan et al. 2004). Basal medium does not appear to have a large effect on the establishment or maintenance of embryogenic cultures, since both MS medium (Murashige and Skoog 1962) and FN-Lite medium (Samoylov et al. 1998) prove satisfactory for embryogenic cultures (Ozias-Akins et al. 1992; Magbanua et al. 2000). However, the addition of hemoglobin has been shown to be responsible for an increase in initiation frequency, the magnitude of the initiation response and an increase in culture fresh weight (Jayabalan et al. 2004). Frequently glutamine has been included in the culture medium, although its effect appears to be more qualitative than quantitative (Ozias-Akins et al. 1993). Repetitive embryogenic cultures can be maintained on the same medium in which they are initiated. Homogeneous cultures often are not obtained until 3–6 months after initiation. Although long-term cultures are capable of regeneration, transformed regenerants from such cultures often are infertile (Ozias-Akins et al. 1993; Singsit et al. 1997; Magbanua et al. 2000) and may not form flowers. Since peanut is a self-pollinator and propagated through seed, this is an undesirable outcome. It is recommended that only recently initiated embryogenic cultures are used as starting material for transformation.

92

P. Ozias-Akins

3.2 Agrobacterium-Mediated Gene Transfer Agrobacterium-mediated transformation has been successful with New Mexico Valencia A, A. hypogaea subsp. fastigiata (Cheng et al. 1996, 1997) and several Indian genotypes of the same subspecies (Sharma and Anjaiah 2000; Khandelwal et al. 2003). In the former case, freshly excised leaf explants were cultured on a high concentration of cytokinin (25 mg l−1 benzyladenine; BA) and a low concentration of auxin (1 mg l−1 naphthaleneacetic acid; NAA) for 2 days in the presence of Agrobacterium tumefaciens (EHA105 harboring binary plasmid pBI121). Antibiotic selection (150 mg l−1 kanamycin) began 7 days after the initiation of cultures. Putative transgenic shoots expressed the reporter gene gus encoding β-glucuronidase (GUS), rooted on 50 mg l−1 kanamycin; and leaf segments callused on medium with 2 mg l−1 NAA and 0.5 mg l−1 BA plus 200 mg l−1 kanamycin. A critical step in the transformation protocol was the incubation of Agrobacterium in extract from wounded tobacco leaves. This treatment presumably stimulated vir gene expression in a similar manner as acetosyringone (Cheng et al. 2004). However, acetosyringone could not replace tobacco extract in these experiments, which was concluded when transient expression was observed to increase significantly only in the presence of tobacco extract upon cocultivation with an intron-containing gus gene. Fertile transgenic plants were regenerated from ∼0.3% of the leaf explants. Several (6/9) transgenic lines showed Mendelian segregation for GUS expression. A Southern blot that unequivocally demonstrated integration of the transgenes into the peanut genome was not shown by Cheng et al. (1996), but the presence of GUS activity in progeny was the convincing evidence of transmission. Modifications of this transformation protocol subsequently were used to introduce a gene for tomato spotted wilt virus resistance into peanut (Li et al. 1997). A second transformation protocol using A. tumefaciens was developed at ICRISAT with a Spanish-type cultivar, JL-24 (Sharma and Anjaiah 2000). Cotyledons were excised from mature seeds and the embryo axis was removed, providing a wound site for Agrobacterium infection (strain C58 with binary plasmid pBI121). Shoots were induced from 3-day-cocultured, longitudinal cotyledon halves on medium containing BA (4.5 mg l−1 ) and 2,4-D (2.2 mg l−1 ) under non-selective conditions for 2 weeks. Selection then was carried out on the same medium containing 125 mg l−1 kanamycin. Shoots rooted in the absence of selection were allowed to set seed, and progeny were subjected to Southern blot analysis which showed that eight out of ten plants contained a single copy of the kanamycin resistance gene (nptII) integrated into the peanut genome. The same transformation method was used to introduce the coat protein gene of Indian peanut clump virus into peanut. Similarly, the majority of the transformants carried a single copy of the gene, and at least one of these was tested and shown to follow Mendelian segregation. Another transformation protocol using Agrobacterium also relied on shoot regeneration from a Spanish-type cultivar, TMV-2 (Khandelwal et al. 2003). The shoot-tip explant was excised from imbibed mature seeds and precultured

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93

for 2 days on 2 mg l−1 BA. The preculture step was considered to be important by Venkatachalam et al. (1998, 2000) who reported Agrobacterium-mediated transformation through shoot regeneration from excised seed tissues, but convincing evidence of stable transformation was not presented. In the study by Khandelwal et al. (2003), precultured explants were cocultured with Agrobacterium (strain EHA105 containing a modified pBI121 binary plasmid) for 2 days. Subsequently, shoot-tip explants were maintained on the same medium in the presence of 100 mg l−1 kanamycin. Surviving shoots were rooted under selective conditions. Since a gene for rinderpest virus hemagglutinin was introduced along with the selectable marker gene, assays of progeny plants for expression of this viral protein indicated expression only in transgenic and not in control plants. Several studies on peanut transformation with Agrobacterium showed incomplete molecular evidence for transformation. It is not appropriate to claim transformation when putative transformants are tested only by PCR. PCR is a very sensitive assay that can also amplify a few molecules of the gene from residual Agrobacterium. The convincing evidence for integration of a transgene and stable transformation is a Southern blot where the genomic DNA is digested with an enzyme that cuts at only one site or not at all within the introduced plasmid (Bhat and Srinivasan 2002). Transmission of the gene to progeny, demonstrated by the appropriate molecular assays including expression analyses, is convincing evidence for stable transformation as long as sufficient controls are included in the assays. 3.3 Enhanced Characters via Transgenes Table 2 summarizes the traits that were introduced into cultivated peanut by genetic engineering. Selectable markers (hph, nptII) and reporter genes (gus, gfp, luc) are essential for the development of plant transformation protocols (Miki and McHugh 2004), but they are not the objective for genetic improvement of the crops. Peanut suffers from numerous disease problems that vary with the region of cultivation, although virus and fungal diseases (both foliar and soilborne) cause the most significant losses worldwide. The major effort to develop transgenic peanut focuses on these diseases. 3.3.1 Virus Resistance Tospovirus infections of peanut occur in most growing regions of the world. The infection is caused by several similar but serologically distinct viruses, such as tomato spotted wilt virus, groundnut ringspot virus and peanut (groundnut) bud necrosis virus. Attempts have been made to use pathogen-derived resistance against tospoviruses to achieve field resistance. Transformation of peanut by microprojectile bombardment with the nucleocapsid protein (NP) gene of tomato spotted wilt virus resulted in the recovery of transgenic lines

Trait

Promoter

Selectable Transformation marker method

Progeny analysis

Reference

β-Glucuronidase (uidA) β-Glucuronidase

CaMV 35S CaMV 35S

hph None

Biolistic Biolistic

None N, P, S

Ozias-Akins et al. (1993) Brar et al. (1994)

CaMV 35S MAS CaMV 35S CaMV 35S

nptII nptII nptII nptII

Agrobacterium Agrobacterium Agrobacterium Agrobacterium

None P, PCR, S EA, P, S EA, ELISA, P, S

Eapen and George (1994) McKently et al. (1995) Cheng et al. (1996, 1997) Li et al. (1997)

CaMV 35S CaMV 35S GmVsp CaMV 35S CaMV 35S CaMV 35S CaMV 35S

hph nptII hph

Biolistic Agrobacterium Biolistic

S None None

Singsit et al. (1997) Egnin et al. (1998) Wang et al. (1998)

hph nptII hph

Biolistic Agrobacterium Biolistic

ELISA, N, P, RT-PCR Yang et al. (1998, 2004) None Venkatachalam et al. (1998, 2000) P Livingstone and Birch (1999)

CaMV 35S

hph

Biolistic

P, PCR, S

Magbanua et al. (2000)

CaMV 35S CaMV 35S

nptII nptII

Agrobacterium Agrobacterium

P, S PCR, S

Rohini and Rao (2000) Sharma and Anjaiah (2000)

Phosphinothricin resistance (bar) Nucleocapsid protein gene from TSWV β-Glucuronidase (uidA) β-Glucuronidase (uidA) β-Glucuronidase (uidA) Nucleocapsid protein gene from TSWV β-Glucuronidase (uidA) Bt cryIA(c) β-Glucuronidase (intron-containing uidA) β-Glucuronidase (uidA) Nucleocapsid protein gene from TSWV

β-Glucuronidase (uidA) β-Glucuronidase (uidA)

94

Table 2. Reports of stable transformation in cultivated peanut. Promoters: AtACT2 actin 2 promoter from Arabidopsis thaliana, CaMV 35S cauliflower mosaic virus 35S, GmVsp vegetative storage protein promoter from soybean, MAS mannopine synthase promoter from Agrobacterium tumefaciens, UBI3 potato ubiquitin. Progeny analysis: EA enzyme assay, ELISA enzyme-linked immunosorbent assay, N Northern blot, P phenotypic assay, PCR polymerase chain reaction, RT-PCR reverse-transcriptase PCR, S Southern blot, W Western blot

Luciferase (luc) P. Ozias-Akins

Nucleocapsid protein gene from TSWV β-Glucuronidase (uidA) β-Glucuronidase (uidA) β-Glucuronidase (uidA) Peanut clump virus coat protein

Peanut

Table 2. continued

Trait

Promoter

β-Glucuronidase (intron-containing uidA) CaMV 35S Tobacco chitinase Rice chitinase Alfalfa glucanase Nucleocapsid protein gene from TSWV Mercury resistance (merA) Rinderpest virus hemagglutinin Peanut stripe virus coat protein Green fluorescent protein Mercury resistance (merB)

CaMV 35S CaMV 35S CaMV 35S AtACT2 CaMV 35S CaMV 35S CaMV 35S UBI3

Selectable Transformation marker method

Progeny analysis

Reference

hph nptII hph

Biolistic Agrobacterium Biolistic

none EA, P, S EA, P, PCR

Deng et al. (2001) Rohini and Rao (2001) Chenault et al. (2002, 2003, 2005)

hph hph nptII hph hph

Biolistic Biolistic Agrobacterium Biolistic Biolistic

ELISA, PCR P, PCR ELISA, P, PCR, W N, PCR P, PCR, S, W

Chenault and Payton (2003) Yang et al. (2003) Khandelwal et al. (2003, 2004) Higgins et al. (2004) Joshi et al. (2005)

or none

95

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P. Ozias-Akins

containing a single copy of the NP gene that expressed at the protein level (Yang et al. 1998; Chenault and Payton 2003). Transgene expression was stable in progeny that inherited the gene in Mendelian fashion (Yang et al. 1998). Subsequent field trials showed that this transgenic line displayed more tolerance to the viral disease than non-transgenic checks (Yang et al. 2004). It was observed that the transgenic line performed better than the most widely cultivated TSWV-tolerant peanut cultivar (Georgia Green) under conditions of high disease pressure. Li et al. (1997), who inoculated the plants mechanically under glasshouse conditions, reported similar TSWV tolerance in transgenic peanut expressing the TSWV NP gene via Agrobacterium-mediated transformation. These authors observed that transgenic plants displayed only local infection after mechanical inoculation, whereas the non-transformed control plants or transgenic plants with only marker/reporter genes (positive control) were systemically infected and stunted 40 days after inoculation. In another study where the NP gene of TSWV was introduced into peanut in an antisense orientation, a fertile transgenic line that showed Mendelian inheritance of the NP gene was field-tested (Magbanua et al. 2000). Plants were observed up to 14 weeks after planting, at which time the transgenic line was shown to be more resistant to TSWV than the non-transgenic controls. Results of these studies showed that the expression of sense or antisense NP gene from TSWV could confer a delay in symptom development, but was not sufficient to provide complete resistance to the disease. Nevertheless, the resistance after extensive field-testing was considered to be as good or better than the peanut genotypes currently cultivated (Yang et al. 2004). Viruses other than TSWV are significant agents of peanut diseases in African and Asian countries. Two examples are Indian peanut clump virus and peanut stripe virus (PStV; bean common mosaic virus), both of which are being explored for pathogen-derived resistance in peanut (Sharma and Anjaiah 2000; Higgins et al. 2004). To date, natural resistance to PStV has not been found in the peanut gene pool, and the virus can be seed-transmitted. Progress has been made with transgenic resistance to PStV, where non-translatable or truncated but translatable versions of the coat protein gene were introduced by microprojectile bombardment (Higgins et al. 2004). Mechanical inoculation under glasshouse conditions demonstrated resistance conferred by both genes, but the resistance phenotype ranged from high to a delay in or recovery from symptoms, although all transgenic lines contained multiple copies of the transgenes. The mechanical inoculation technique was effective because 100% of control plants were infected. Since none of the transgenic plants expressed the transgene at the protein level, resistance was considered to be RNA-mediated. This is supported by the evidence showing the presence of small interfering RNAs homologous to the viral transgene. These small interfering RNAs are characteristic of the post-transcriptional gene silencing mechanism (Baulcombe 2004).

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3.3.2 Insect Resistance Most peanut pests are relatively easily controlled using contact pesticides. The exceptions are those that burrow underground to feed on and damage roots and pods. It is desirable to use integrated management tools including hostplant resistance for the control of insect pests in peanut (Lynch and Mack 1995). A pest of particular concern in the southeastern United States is the lesser cornstalk borer, a lepidopteran insect that feeds on stems and developing pods of peanut. This insect is susceptible to the cryIA(c) toxin from Bacillus thuringiensis as shown by in vitro feeding studies (Moar et al. 1995). Singsit et al. (1997) introduced the insecticidal crystalline protein gene, cryIA(c), into peanut by microprojectile bombardment and showed that expression of cryIA(c) could provide transgenic plants with resistance to the lesser cornstalk borer. It was observed that lesser cornstalk borer damage is associated with infection by Aspergillus flavus or A. parasiticus and subsequent aflatoxin production (Lynch and Wilson 1991; Bowen and Mack 1993). In view of this, research was aimed to determine whether insect resistance could contribute to aflatoxin elimination (Ozias-Akins et al. 2002b). 3.3.3 Fungal Resistance Apart from a reduction in fungal (Aspergillus) invasion as a consequence of targeting the “vector” (lesser cornstalk borer), a variety of putative antifungal genes have been explored for their efficacy against primarily Aspergillus (Weissinger et al. 2002; Ozias-Akins et al. 2002a) and other soilborne diseases (Chenault et al. 2002, 2003, 2005; Livingstone et al. 2005). These genes encode antimicrobial peptides, tomato anionic peroxidase, bacterial chloroperoxidase, ribosome inactivating protein, anti-apoptotic proteins, oxalate oxidase, chitinases and glucanases. Chitinase and/or glucanase genes were introduced into a Spanish-type peanut cultivar, Okrun, using microprojectile bombardment (Chenault et al. 2002). Field tests consisting of 3 years of evaluation of 32 transgenic lines, each with a single copy of the transgene were conducted (Chenault et al. 2005). The 3-year disease ratings showed that 14 of these lines were significantly more resistant to Sclerotinia blight than the parental cultivar, Okrun, and five were not statistically different from the resistant check. One transgenic line showed no disease incidence over the 3-year period. It is not yet clear whether this high level of resistance was due to expression of the rice chitinase transgene that had been introduced. Coincidentally, this line possessed an unexpected upright growth habit with open canopy similar to the characteristic of the resistant genotype, SW Runner. It is speculated that growth habit change may be attributed to the transgene insertion that disrupted or altered the expression of a growth-habit gene. Evidence indicated that glucanase activity was not necessary for resistance to Sclerotinia blight. A second gene very effective in targeting Sclerotinia blight is barley oxalate

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oxidase (Livingstone et al. 2005). The fungus secretes oxalic acid that causes tissue damage in infected plants. Oxalate oxidase-expressing plants are able to degrade the fungus-derived oxalic acid, thereby significantly enhancing resistance to the pathogen. 3.3.4 Other Traits Edible vaccines could be particularly beneficial in regions of the world, where infrastructure for storage and handling of labile vaccines is insufficient. Such a delivery method also would be more economical for animal vaccines required on a mass scale (Horn et al. 2004). Khandelwal et al. (2004) reported the development of an edible vaccine in peanut to target rinderpest virus in cattle. The surface glycoproteins of the rinderpest virus are highly immunogenic and the gene hemagglutinin, for a surface glycoprotein, was cloned and transformed into peanut (Khandelwal et al. 2003). Transgenic peanut that expressed hemagglutinin was recognized in T1 progeny by several antibodies specific for the protein, while there was no cross-reaction with plant proteins. Mice could be immunized by intraperitoneal injection of peanut extract or by oral ingestion (Khandelwal et al. 2004). It is anticipated that cattle could be immunized against rinderpest virus upon ingestion of peanut fodder. Down-regulation of allergen genes has been proposed as a means for the production of hypoallergenic peanut (Dodo et al. 2005). The most effective method for down-regulation using a transgenic approach will be through the RNA interference (RNAi) pathway (Baulcombe 2004). Only a portion of a coding sequence introduced in an inverted repeat orientation is required for effective knockdown of expression. The biggest challenge in peanut is that at least eight seed-expressed proteins, three of these being major seed storage proteins, are allergenic (Ozias-Akins et al. 2006; Ramos et al. 2006). Knockdown of the three most allergenic proteins eliminates the three most abundant seed storage proteins and is likely to severely alter the protein composition of peanut seed. 3.3.5 Regulatory Elements The cauliflower mosaic virus (CaMV) 35S promoter is used extensively in transgenic peanut, as in many other plants (Table 2). This promoter allows constitutive expression of a gene and often is chosen for the regulation of a selectable marker gene. It frequently is duplicated in a construct, regulating not only the selectable marker gene, but a gene of interest as well. Since duplication of sequences can promote gene silencing (Baulcombe 2004), more constructs are being designed to minimize this duplication. Two other promoters that function well in peanut to drive selectable marker genes are the nos (nopaline synthase) promoter that controls the nptII gene in pBI121 and the potato ubiquitin promoter (ubi3) that is used to express the hygromycin

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resistance gene (Yang et al. 2003). The CaMV 35S, MAS, NOS and ACT2 promoters are either constitutive or active in the majority of tissues/developmental stages of the plant. ACT2, however, does not drive strong expression in embryos/embryogenic tissues (Yang et al. 2003) and therefore is not suitable for regulating a selectable marker gene in these tissues. A promoter that is active in only specific tissues/organs and is inducible is the vegetative storage protein B gene promoter from soybean (Mason and Mullet 1990). This promoter was tested in peanut for its ability to drive expression of β-glucuronidase (GUS) in pod walls, but not seeds (Wang et al. 1998), a pattern characteristic of its expression in soybean. GUS was expressed in this organ-specific manner and also was modulated by the wounding-related hormone, methyl jasmonate.

4 Conclusions Cultivated peanut is an important food crop worldwide, but it has not received the research input of other grain legumes such as soybean. Nevertheless, progress has been made in the development of molecular markers for application to genetic studies and breeding. Application to breeding is hindered by the low levels of polymorphism and allopolyploidy of this species. Although AFLP and SSR markers have been or will be useful for tagging traits, marker discovery could be accelerated considerably by the availability of extensive DNA sequence information. Similarly, progress has been made with the development of transformation systems for this crop using either Agrobacterium or biolistics, but there is still room for improvement of transformation frequency and genotype range. Transformation systems in peanut are not adequate for high-throughput functional genomics projects, but currently they are useful for introduction of a small number of genes with specific target traits.

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Raina S, Rani V, Kojima T, Ogihara Y, Singh K, Devarumath R (2001) RAPD and ISSR fingerprints as useful genetic markers for analysis of genetic diversity, varietal identification, and phylogenetic relationships in peanut (Arachis hypogaea) cultivars and wild species. Genome 44:763–772 Ramos M, Fleming G, Chu Y, Akiyama Y, Gallo M, Ozias-Akins P (2006) Chromosomal and phylogenetic context for conglutin genes in Arachis based on genomic sequence. Mol Gen Gen 275:578–592 Rohini VK, Rao KS (2000) Transformation of peanut (Arachis hypogaea L.): a non-tissue culture based approach for generating transgenic plants. Plant Sci 150:41–49 Rohini VK, Rao KS (2001) Transformation of peanut (Arachis hypogaea L.) with tobacco chitinase gene: variable response of transformants to leaf spot disease. Plant Sci 160:889–898 Samoylov V, Tucker D, Parrott W (1998) Soybean [Glycine max (L.) Merill] embryogenic cultures: the role of sucrose and total nitrogen content on proliferation. In Vitro Cell Dev Biol 34:8–13 Seijo JG, Lavia GI, Fernandez A, Krapovickas A, Ducasse D, Moscone EA (2004) Physical mapping of the 5S and 18S-25S rRNA genes by FISH as evidence that Arachis duranensis and A. ipaensis are the wild diploid progenitors of A. hypogaea (Leguminosae). Am J Bot 91:1294–1303 Sharma KK, Anjaiah V (2000) An efficient method for the production of transgenic plants of peanut (Arachis hypogaea L.) through Agrobacterium tumefaciens-mediated genetic transformation. Plant Sci 159:7–19 Shou H, Frame B, Whitham S, Wang K (2004) Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation. Mol Breed 13:201–208 Simpson CE (2001) Use of wild Arachis species/introgression of genes into A. hypogaea L. Peanut Sci 28:114–116 Simpson CE, Starr JL (2001) Registration of ‘COAN’ peanut. Crop Sci 41:918 Simpson CE, Krapovickas A, Valls JFM (2001) History of Arachis including evidence of A. hypogaea L. progenitors. Peanut Sci 28:78–80 Simpson CE, Starr JL, Church GT, Burow MD, Paterson AH (2003) Registration of ‘NemaTAM’ peanut. Crop Sci 43:1561 Singh KP, Singh A, Raina SN, Singh AK, Ogihara Y (2002) Ribosomal DNA repeat unit polymorphism and heritability in peanut (Arachis hypogaea L.) accessions and related wild species. Euphytica 123:211–220 Singsit C, Adang MJ, Lynch RE, Anderson WF, Wang A, Cardineau G, Ozias-Akins P (1997) Expression of a Bacillus thuringiensis crylA(c) gene in transgenic peanut plants and its efficacy against lesser cornstalk borer. Transgenic Res 6:169–176 Somers DA, Samac DA, Olhoft PM (2003) Recent advances in legume transformation. Plant Physiol 131:892–899 Stalker HT, Mozingo LG (2001) Molecular markers of Arachis and marker-assisted selection. Peanut Sci 28:117–123 Stalker H, Simpson C (1995) Germplasm resources in Arachis. In: Pattee H, Stalker H (eds) Advances in peanut science. American Peanut Research and Education Society, Stillwater, OK, pp 14–53 Subramanian V, Gurtu S, Nageswara Rao R, Nigam S (2000) Identification of DNA polymorphism in cultivated groundnut using random amplified polymorphic DNA (RAPD) assay. Genome 43:656–660 Tallury SP, Hilu KW, Milla SR, Friend SA, Alsaghir M, Stalker HT, Quandt D (2005) Genomic affinities in Arachis section Arachis (Fabaceae): molecular and cytogenetic evidence. Theor Appl Genet 111:1229–1237 Venkatachalam P, Geetha N, Jayabalan N, Sita S, Sita L (1998) Agrobacterium-mediated genetic transformation of groundnut (Arachis hypogaea L): an assessment of factors affecting regeneration of transgenic plants. Plant Sci 111:565–572 Venkatachalam P, Geetha N, Shandelwal A, Shaila MS, Sita GL (2000) Agrobacterium-mediated genetic transformation and regeneration of transgenic plants from cotyledon explants of groundnut (Arachis hypogaea L.) via somatic embryogenesis. Curr Sci 78:1130–1136

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Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP – A new technique for DNA-fingerprinting. Nucleic Acids Res 23:4407–4414 Wang A, Fan H, Singsit C, Ozias-Akins P (1998) Transformation of peanut with a soybean vspB promoter-uidA chimeric gene I. Optimization of a transformation system and analysis of GUS expression in primary transgenic tissues and plants. Physiol Plant 102:38–48 Weber JL (1990) Informativeness of human (DC-DA)n.(DG-DT)n polymorphisms. Genomics 7:524–530 Weissinger A, Wu M, Liu Y-S, Ingram K, Rajasekaran K, Cleveland T (2002) Development of transgenic peanut with enhanced resistance against preharvest aflatoxin contamination. Mycopathologia 155:97 Welsh J, McClelland M (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 18:7213–7218 Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic-markers. Nucleic Acids Res 18:6531–6535 Yang H, Singsit C, Wang A, Gonsalves D, Ozias-Akins P (1998) Transgenic peanut plants containing a nucleocapsid protein gene of tomato spotted wilt virus show divergent levels of gene expression. Plant Cell Rep 17:693–699 Yang HY, Nairn J, Ozias-Akins P (2003) Transformation of peanut using a modified bacterial mercuric ion reductase gene driven by an actin promoter from Arabidopsis thaliana. J Plant Physiol 160:945–952 Yang H, Ozias-Akins P, Culbreath A, Gorbet D, Weeks J, Mandal B, Pappu H (2004) Field evaluation of Tomato spotted wilt virus resistance in transgenic peanut (Arachis hypogaea). Plant Dis 88:259–264 Yuksel B, Paterson AH (2005) Construction and characterization of a peanut HindIII BAC library. Theor Appl Genet 111:630–639 Yuksel B, Bowers JE, Estill J, Goff L, Lemke C, Paterson AH (2005a) Exploratory integration of peanut genetic and physical maps and possible contributions from Arabidopsis. Theor Appl Genet 111:87–94 Yuksel B, Estill J, Schulze S, Paterson A (2005b) Organization and evolution of resistance gene analogs in peanut. Mol Gen Gen 274:248–263 Zietkiewicz E, Rafalski A, Labuda D (1994) Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain-reaction amplification. Genomics 20:176–183

I.6 Cotton K.S. Rathore1

1 Introduction Cotton represents the world’s most important non-food agricultural crop. It has been under cultivation and serving the clothing needs of humanity for over seven millennia. This plant has played a major role in the economy, social structure, and history of many countries around the globe. Despite the availability of synthetic alternatives, it continues to be an important source of fiber for the textile industry. It is grown in over 80, mostly developing countries, where it serves as a cash crop for poor farmers. The top three cotton producers are China, the United States, and India. Amongst the commercially cultivated species, more than 90% of worldwide acreage is devoted to the tetraploid, Gossypium hirsutum, or upland cotton. Another tetraploid, G. barbadense or Egyptian cotton, is also grown in some parts of the world for its prized extralong staple. The two Old World, diploid species, G. arboreum and G. herbaceum, are still cultivated in some parts of Africa and Asia. However, they share a very small percentage of the total worldwide acreage devoted to cotton because of their low productivity and poor quality fiber. Although cotton is grown primarily for its fiber, the plant also produces large quantities of seeds, in the order of 1.65 kg of seed for every 1.0 kg of fiber. Cottonseed, in addition to providing oil, has the potential to be an important source of dietary protein, either directly as food or indirectly as feed for nonruminants, if it were not for the presence of toxic gossypol. A better utilization of this abundant but underutilized resource will help in meeting the challenges posed by the growing global population.

2 Importance of Genetic Engineering in Cotton The cotton plant is particularly susceptible to a wide variety of insect pests and nematodes. Because of this vulnerability and the fact that cotton is generally not considered a food crop, production of cotton has traditionally relied on the use of large amounts of toxic pesticides. Prior to the introduction of Bt cottons, 5–10 insecticide applications were needed each season to control damage from 1 Institute for Plant Genomics & Biotechnology, and Department of Soil and Crop Science, Texas

A & M University, College Station, TX 77843-2123, USA, e-mail: [email protected] Biotechnology in Agriculture and Forestry, Vol. 61 Transgenic Crops VI (ed. by E.C. Pua and M.R. Davey) © Springer-Verlag Berlin Heidelberg 2007

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insects. Some estimates suggest that, prior to the widespread adoption of GM cotton, nearly 25% of all insecticides used worldwide were for the production of cotton. Pesticides used on cotton are often broad-spectrum organophosphates and carbamates that are expensive and harmful to people, wildlife, and the environment. With the exception of a few developed countries, cotton is grown mostly in developing countries as a cash crop by resource-poor farmers. In some cases, their crops are destroyed by insects as some farmers cannot afford to buy pesticides. In most cases, they resort to the use of some highly toxic chemicals, leading to pesticide poisonings and deaths as they can rarely afford the correct equipment for safe chemical application (Mancini et al. 2005). The issues discussed above made cotton an ideal crop that could benefit greatly from genetic engineering. It was, therefore, no surprise that upon receiving approval for commercial planting, of all the GM crops, cotton was cultivated on the largest acreage for the first year in the United States (James 1997). The genetically modified cotton introduced in the United States was in the form of insect-resistant cotton (Bollgard cotton; Jones et al. 1996; Hardee and Herzog 1997), herbicide (Buctril)-resistant cotton (BXN 57; Panter et al. 1996), and herbicide (glyphosate)-resistant cotton (Roundup Ready cotton; James and Krattiger 1996). In the year 2004, GM cotton amounted to 80% of United States, 66% of Chinese, 80% of Australian, and 85% of South African cotton acreage (James 2004). Often, cotton is the first crop to receive approval for commercial planting in many other countries from their respective regulatory agencies. GM varieties continue to increase their share of global cotton acreage every year. This bodes well for future products that are likely to be generated by further genetic modification of cotton.

3 Modification of Cotton via Genetic Transformation A list of papers reporting transgenic modification of cotton is presented in Table 1. This list is by no means comprehensive. It includes most of the early papers, reports that describe the use of alternative methods for transformation, and investigations dealing with some interesting genes and traits.

4 Transformation Methods The first two reports on successful transformation of cotton appeared in 1987 (Firoozabady et al. 1987; Umbeck et al. 1987), only three years following the transformation of the model plant species, tobacco. However, progress in producing transgenic cotton, especially in academic laboratories, was rather slow until the turn of the present century. Although surprising considering

Transformation method

Cultivar

Target tissue/mode of transformant recovery

Transgenes

Comments

Reference

Agrobacterium

Coker 201

C/somatic embryogenesis

nptII and OCS

Agrobacterium

H/somatic embryogenesis

cat and nptII

Firoozabady et al. (1987) Umbeck et al. (1987)

Gene gun

Coker 310, 312, 5110 Coker 310

hpt

Agrobacterium

Coker 312

ESC/somatic embryogenesis H/somatic embryogenesis

Immunoblot and Southern for confirmation Enzyme assays and Southern for confirmation Southern for confirmation

Agrobacterium

Siokra 1–3

H/somatic embryogenesis

Cry1Ac, Cry1Ab and nptII nptII and gusA

Agrobacterium

Coker 312

H/somatic embryogenesis

nptII and tfdA

Agrobacterium

Coker 315

C/somatic embryogenesis

nptII, gusA and tfdA

Gene gun

DeltaPine 50, DeltaPine 90, Sea Island, Pima S-6 Coker 312

SAM from mature seed/shoot regeneration in culture

gusA

C/somatic embryogenesis

nptII, protease inhibitors

Agrobacterium

Western and bioassay for confirmation NPTII and GUS enzyme assays for confirmation 2,4-D monooxygenase activity, PCR, and 2,4-D resistance for confirmation Southern, GUS enzyme assay, and 2,4-D resistance for confirmation GUS histochemical analysis and Southern for confirmation

Western for confirmation; some protection against insects

Cotton

Table 1. Summary of studies on transgenic cotton. C Cotyledon, ESC embryogenic cell suspension, H hypocotyl, P cotyledonary petiole, SAM shoot apical meristem

Finer and McMullen (1990) Perlak et al. (1990) Cousins et al. (1991) Bayley et al. (1992)

Lyon et al. (1993)

McCabe and Martinell (1993)

Thomas et al. (1995) 109

110

Table 1. continued

Transformation method

Cultivar

Target tissue/mode of transformant recovery

Transgenes

Comments

Reference

Gene gun

Not stated

nptII

Western for confirmation

Chlan et al. (1995)

Gene gun

DeltaPine 50

Coker 312, DeltaPine 50, Sea Island

GUS assay, Northern, Western, and fiber quality analysis for confirmation GUS assay, Southern, Western, and biochemical analyses for confirmation

Gene gun

DeltaPine 50

SAM from mature seed/shoot regeneration in culture

Agrobacterium and Gene gun

Coker 315, Acala varieties

C, H, ESC/somatic embryogenesis

Fiber-specific E6 antisense and gusA Fiber-specific, FbL2A promoter driving phaB and phaC, and gusA Fiber-specific, E6 or FbL2A promoter driving phaB and phaC, and gusA nptII, mutant native AHAS genes

John (1996)

Gene gun

SAM from mature seed/shoot regeneration in culture SAM from mature seed/shoot regeneration in culture SAM from mature seed/shoot regeneration in culture

Agrobacterium

Coker 312

H/somatic embryogenesis

GUS assay, Southern, Northern, microscopic, and biochemical analyses for confirmation; fiber’s thermal properties were altered

John and Keller (1996); Chowdhury and John (1998)

Southern and resistance to herbicides, imidazolinone and sulfonylurea, for confirmation Southern, ELISA, and resistance to herbicide; glyphosate for confirmation

Rajasekaran et al. (1996)

Nida et al. (1996)

K.S. Rathore

nptII, CP4-EPSPS

Rinehart et al. (1996)

Transformation method

Cultivar

Target tissue/mode of transformant recovery

Transgenes

Comments

Reference

Gene gun

SAM from mature seed/shoot regeneration in culture

bar and gusA

C/somatic embryogenesis

nptII, glucose oxidase

Agrobacterium

CUBQHRPIS

SAM from seedling/shoot regeneration in culture

nptII, gusA

Gene gun

ESC/somatic embryogenesis H/somatic embryogenesis

nptII, gusA

Agrobacterium

Acala B1654, Coker 315 Coker 315

Southern, Northern and biochemical analyses, and resistance to herbicide, Basta, for confirmation Biochemical analyses for confirmation; some protection against a root pathogen but phytotoxicity observed Resistance to kanamycin and Southern for confirmation GUS histochemical analysis and Southern for confirmation Some protection against Verticillium wilt with each gene

Keller et al. (1997)

Agrobacterium

Coker 312, Delta-Pine 50, El Dorado, Pima S6 Coker 315

Agrobacterium

Coker 315

C/somatic embryogenesis

Ellis et al. (2000)

Agrobacterium

Coker 312

H/somatic embryogenesis

Biochemical and Western analyses; no increase in waterlogging tolerance PCR and Southern for confirmation; a stepwise and comprehensive account of transgenic cotton production

Tobacco basic chitinase, glucose oxidase, nptII Cotton Adh2, rice Pdc1, nptII nptII, gusA

Cotton

Table 1. continued

Murray et al. (1999)

Zapata et al. (1999)

Rajasekaran et al. (2000) McFadden et al. (2000)

Sunilkumar and Rathore (2001) 111

112

Table 1. continued

Transformation method

Cultivar

Target tissue/mode of transformant recovery

Transgenes

Comments

Reference

Agrobacterium

Coker 312

H/somatic embryogenesis

Mn-SOD, APX, GR, nptII

Kornyeyev et al. (2001, 2003)

Agrobacterium

Coker 312

H/somatic embryogenesis

Mn-SOD, APX, GR, nptII

Agrobacterium

Coker 312

H/somatic embryogenesis

Agrobacterium

Coker 312

H/somatic embryogenesis

Agrobacterium

Coker 315

C/somatic embryogenesis

Cotton α-globulin promoter driving gusA, nptII CaMV 35S promoter driving GFP gene, nptII Seed-specific RNAi of Cotton SAD-1 and Cotton FAD-1,nptII

Enzymatic assays confirmed overexpression; chilling-induced photoinhibition of photosystem II reduced Enzymatic assays confirmed overexpression; some protection of photosynthetic capacity during chilling Histochemical and biochemical analyses; seed-specificity of promoter demonstrated

Sunilkumar et al. (2002a)

Sunilkumar et al. (2002b)

Liu et al. (2002) K.S. Rathore

Fluorescence microscopy analysis; developmental- and tissue-specific activity of promoter demonstrated Southern, Northern, and biochemical analyses; seed oil with substantially higher stearic acid or oleic acid levels

Payton et al. (2001)

Cotton

Table 1. continued

Transformation method

Cultivar

Target tissue/mode of transformant recovery

Transgenes

Comments

Reference

Agrobacterium

MCU5, DCH32, Coker 310FR

Shoot tip from seedling/shoot regeneration in culture

gusA, nptII

Satyavathi et al. (2002)

Agrobacterium

Coker 312

C, H/somatic embryogenesis

Agrobacterium

Coker 312

H/somatic embryogenesis

Cotton β-tubulin promoter driving gusA, nptII Endochitinase gene from Trichoderma virens, nptII

Histochemical, PCR and Southern analyses and resistance of progeny to kanamycin for confirmation Histochemical analyses; preferential activity in fiber and root tip observed

Agrobacterium

Coker 315

C/somatic embryogenesis

Sense and antisense suppression of sucrose synthase, nptII

Southern, Northern, and biochemical analyses; protection against Rhizoctonia solani and Alternaria alternata observed Southern, immunolocalization, electron microscopy, and biochemical analyses; fiber development inhibited

Li et al. (2002)

Emani et al. (2003)

Ruan et al. (2003)

113

114

Table 1. continued

Transformation method

Cultivar

Target tissue/mode of transformant recovery

Transgenes

Comments

Reference

Agrobacterium

Not stated

H/somatic embryogenesis

nptII

Wilkins et al. (2004)

Gene gun

Coker 310FR

H-derived friable callus/somatic embryogenesis

Chloroplastspecific expression of aphA-6 and nptII

Agrobacterium

Coker 312

H/somatic embryogenesis

GF14λ, nptII

Agrobacterium

G007

Pollen grain/fertilized zygotic embryo

acsA, acsB, hpt, gusA

Pollen-tube pathway

Xin-Cai, Lv-9902, Lv-9903

Pollen tube/fertilized zygotic embryo

GAFP, bar

Agrobacterium

Coker 312

H/somatic embryogenesis

Seed-specific antisense of cotton FAD-2, nptII

Transformation and regeneration protocol provided PCR and Southern to confirm plastid genome transformation; strict maternal inheritance of kanamycin-resistance Northern and Western analyses; obtained moderate drought tolerance PCR, Southern, and Northern for confirmation; improvements in fiber quality reported Southern and RT-PCR for confirmation; resistance to Verticillium wilt reported Biochemical analysis; seed oil with higher oleic acid level

Kumar et al. (2004)

Yan et al. (2004)

Li et al. (2004)

Wang et al. (2004)

K.S. Rathore

Sunilkumar et al. (2005)

Transformation method

Cultivar

Target tissue/mode of transformant recovery

Transgenes

Comments

Reference

Agrobacterium

Coker 315

C/somatic embryogenesis

Southern, Northern, and Western analyses; no reduction in gossypol levels; induction of the target gene by bacterial blight was blocked

Townsend et al. (2005)

Agrobacterium

Coker 312

C, H/somatic embryogenesis

F846

Shoot tip from seedling/shoot regeneration in culture

Agrobacterium

Coker 312

H/somatic embryogenesis

Arabidopsis NHX1,nptII

Gene gun

7MH, CD-401, Antares, ITA94 Christina

SAM from mature seed/shoot regeneration in culture

Arabidopsis ahas gene, gusA

Pollen grains/fertilized zygotic embryo

hmgr and nptII

Southern, PCR and RT-PCR analyses; transgenic plants resistant to several fungal pathogens PCR and Southern for confirmation; some resistance to leaf curl virus reported PCR, Northern, and Western analyses; more biomass and more fiber produced under salt stress conditions Selection on imazapyr. PCR, Southern and histochemical analyses for confirmation PCR for confirmation

Rajasekaran et al. (2005)

Agrobacterium

Soybean lectin promoter or CaMV 35S promoter driving antisense cdn1-C4, nptII Synthetic antimicrobial peptide D4E1, nptII Antisense of AV2, nptII

Sanjaya et al. (2005)

He et al. (2005)

Aragao et al. (2005)

Gounaris et al. (2005)

115

Gene gun

Cotton

Table 1. continued

116

Table 1. continued

Transformation method

Cultivar

Target tissue/mode of transformant recovery

Transgenes

Comments

Reference

Agrobacterium

Coker 312

C, H/somatic embryogenesis

Histochemical analyses; preferential activity observed in the fiber; RNAi-inhibition of fiber elongation

Li et al. (2005)

Agrobacterium

Zhongmiansuo H/somatic embryogenesis 35

Southern and Western for confirmation; resistance to cotton aphid observed

Wu et al. (2006)

Agrobacterium

Coker 312

Cotton ACTIN1 promoter driving gusA, RNAi of ghACT1, nptII Phloem-specific promoter driving ACA gene, nptII Seed-specific RNAi of cotton δ-cadinene synthase, nptII

Southern, RT-PCR, Northern, and biochemical analyses for confirmation; over 98% reduction in the seed-gossypol level obtained

Sunilkumar et al. (2006)

H, P/somatic embryogenesis

K.S. Rathore

Cotton

117

the economic importance of cotton, the reasons for this dearth in reports of cotton transformation were the difficulties involved in producing transgenic cotton plants. A thorough investigation into various steps involved in generating transgenic cotton was undertaken in the author’s laboratory, and a comprehensive report unraveling the mystic surrounding cotton transformation was published in 2001 (Sunilkumar and Rathore 2001). Using green fluorescent protein (GFP) as a reporter gene, this study revealed that the Agrobacterium-mediated transfer of T-DNA to the cotyledonary cells of cotton was highly efficient. Also, the conversion of transient transformation events to stable events in cotyledons, hypocotyls, and petiole segments is quite efficient in cotton (Sunilkumar and Rathore 2001; Rathore et al. 2006). The difficulties in generating transgenic cotton are largely due to poor regeneration from cultured tissues. The recovery of transformed plants from cultured cotyledon-, hypocotyl-, and petiole-derived callus tissues requires careful selection of friable, embryogenic callus and several subcultures on various media under appropriate environmental conditions. Given the complexities of creating transgenic cotton, a simple yet robust method for cotton transformation was developed and described in detail (Rathore et al. 2006). Following this protocol, it is possible to generate transformed cotton plants (cv. Coker 312) in 8–10 months. It should be noted, however, that regeneration of cotton plants from cultured tissue via somatic embryogenesis still remains highly genotypedependent (Trolinder and Xhixian 1989). Information provided in Table 1 suggests that transformation of cells within hypocotyl or cotyledon segments via Agrobacterium-mediated gene delivery, the selection and proliferation of transformed cells in culture, followed by somatic embryogenesis, appears to be the most popular means of creating transgenic cotton. This seems to be the method of choice in industrial laboratories. Some investigators utilize particle bombardment (gene gun) or Agrobacterium to transform embryogenic suspension cells and then recover transformed cotton plants via somatic embryogenesis Finer and McMullen 1990; Rajasekaran et al. 1996). As described earlier, plant recovery systems based on somatic embryogenesis from cultured tissues are time- and resource-intensive and highly genotype-dependent. It is precisely these difficulties with regeneration that led many researchers to seek alternative procedures for the recovery of transgenic cotton plants following transformation. Some studies reported direct transformation of cells in the shoot apical meristem, either via the gene gun or Agrobacterium-based procedures. Since plants can be recovered from these tissues relatively easily (Gould et al. 1991), it was hoped that this system would make transformation of cotton more genotype-independent. Particle bombardment studies provided ample evidence showing that the method can transform either epidermal cells of the L1 layer or the germline progenitor cells in the L2 or L3 layer (McCabe and Martinell 1993; McCabe et al. 1998). The progeny from L1 transformants did not inherit the transgene, while the germline transformants (recovered from transformation of L2/L3 cells) were able to pass on the transgenic trait to subsequent generations. These studies

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K.S. Rathore

also suggested that the primary transformants recovered from these shoot apices are chimeric and the efficiencies of germline transformation events are extremely low. Production of transgenic cotton plants via Agrobacterium-mediated transformation of shoot apices was also reported by two different laboratories (Zapata et al. 1999; Satyavathi et al. 2002). These reports provided results from Southern analyses that suggest integration of transgenes with the plant genome. Transformation efficiencies as high as 60–70% were reported for three different cotton cultivars in the study conducted by Satyavathi et al. (2002). Although these rates are extremely high for cotton, it is unclear how these were calculated. Neither study investigated the type of cells being transformed in the shoot apex by Agrobacterium. This is an important issue, as the transformation of meristematic cells in the shoot apex remains controversial (Potrykus 1991). As mentioned previously, gene gun studies conducted at Agracetus showed that, when the cells within the shoot apex are transformed, the resultant plant following regeneration is chimeric (McCabe and Martinell 1993; McCabe et al. 1998). By careful tracking of the transgenic leaves and selective pruning of nontransgenic branches, they were able to obtain a uniformly transformed mericlonal plant. The same principles should apply when Agrobacterium is used for transformation of cells in the shoot apex. Even if Agrobacterium is somehow able to transform deeper cells within the L2 or L3 layers of the shoot meristem, a chimeric plant will be produced. Without precise pruning of such a plant to eliminate non-transgenic branches, it will not be possible to obtain T1 seeds that segregate in a Mendelian manner. Thus, it is important to demonstrate the cell types within the shoot apex that are transformed by Agrobacterium (GFP will make an excellent reporter gene here), provide a clear-cut proof of integrative transformation, and show Mendelian inheritance of the transgenic trait. Addressing these critical aspects of the transformation process will ensure widespread adoption of the method, because recovery of a normal, fertile plant from a shoot apex is relatively easy and genotype-independent.

5 Alternative Methods Used to Transform Cotton Li et al. (2004) described a method for directly cocultivating pollen grains of brown cotton with Agrobacterium and then using this pollen/Agrobacterium mixture for the pollination of emasculated flowers. Seeds obtained from plants pollinated in this manner were screened on antibiotic medium to select transformants. Given the fact that pollen from most commercial varieties of cotton is extremely susceptible to bursting in an aqueous environment, the general applicability of this method remains questionable. Wang et al. (2004) described a method that involved direct application of naked plasmid to the stigmatic surface, obtaining seeds from treated flowers, and screening of seedlings for resistance to the herbicide, Basta. Although the technique described is extremely

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119

simple and does not require a biological vector such as Agrobacterium, it is difficult to envisage how the transgenes are delivered into the target cells without the application of any physical or chemical treatments. The nature of the target cell(s) is also unclear in this system. Without proof of the transfer mechanism and gene integration, it is difficult to assess the suitability of these alternative methods for performing cotton transformation in a routine manner.

6 Selectable Marker Genes Used for Generating Transgenic Cotton The list of papers presented in Table 1 suggests that the neomycin phosphotransferase II (nptII) gene, the first selectable marker gene used to create transgenic cotton, remains a popular choice. Its widespread use in transforming cotton is due to the fact that kanamycin-based selection is relatively inexpensive and does not have adverse effects on the regeneration of plants from cultured tissues. Hygromycin phosphotransferase (hpt) has been used as a selectable marker gene in a few studies, suggesting its suitability in generating transgenic cotton (Finer and McMullen 1990; Li et al. 2004). There are two reports listed in the table involving incorporation of the bialaphos resistance (bar) gene into cotton. However, in both cases, this gene was not used in the initial selection of transformants (Keller et al. 1997; Wang et al. 2004). An interesting use of another herbicide resistance gene (Arabidopsis ahas) was described by Aragao et al. (2005), who used it in conjunction with gene gun-mediated transformation of shoot apical meristems of cotton. For routine cotton transformation, nptII will continue to be used as a selectable marker gene.

7 Reporter Genes Used in Cotton The information provided in Table 1 shows that β-glucuronidase (gusA) gene, developed as a reporter gene for plants (Jefferson et al. 1987), remains the gene of choice to evaluate different transformation methods as well as for the characterization of promoter activities in various tissues in cotton (Lyon et al. 1993; McCabe and Martinell 1993; Sunilkumar et al. 2002a; Li et al. 2005). In fact, in studies involving gene gun-mediated transformation of shoot apical meristems where the primary transformants recovered are transgenic chimeras, the selection of transformed tissues is based entirely on the monitoring of gusA activity and selective pruning of the non-transformed parts (McCabe and Martinell 1993). Its use for transgenic research in cotton will continue because of the simple and relatively inexpensive assays to monitor GUS activity. The more recently developed GFP marker gene, that allows non-invasive monitoring of its expression (Chalfie et al. 1994; Haseloff et al. 1997), is not

120

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widely utilized in cotton. Despite its superiority, the need for an expensive microscope with fluorescence capability discourages its widespread use. Nevertheless, the utility and versatility of this reporter gene was demonstrated elegantly by revealing the tissue- and development-specific activity of CaMV 35S promoter in cotton (Sunilkumar et al. 2002b). The use of GFP and other related fluorescence reporter genes will increase as fluorescence microscopy becomes more affordable.

8 Traits Introduced into Cotton Through Genetic Transformation Bollgard or Bt-cotton was one of the first commercially successful GM products introduced by Monsanto in 1996 (Perlak et al. 1990; Jones et al. 1996; Hardee and Herzog 1997). These plants expressed Cry1Ac gene from Bacillus thuringiensis (Bt) whose protein product (a delta-endotoxin) is toxic mainly to tobacco budworm and American bollworm. The success of this product provided a powerful demonstration of the power of biotechnology in addressing an important agronomical problem, whilst helping the environment by reducing our dependence on harmful chemicals. In fact, the problem of lepidopteran insect pests is so severe in all cotton-growing areas of the world that Bt-cotton technology is usually the first GM crop to receive approval from the respective regulatory agencies. Bollgard II, that in addition to Cry1Ac also contained Cry2Ab, became available in 2003 (Micinski et al. 2006; Robinson 2006). This second Bt gene extends the pest resistance to fall armyworm, beet armyworm, cabbage looper, and soybean looper. Syngenta recently developed a new generation of gene from B. thuringiensis called the vegetative insecticidal protein (VIP). The VIP gene encodes an exotoxin that is structurally, biochemically, and functionally different from the Bt delta-endotoxins and exhibits insecticidal activity against a variety of lepidopterans (Estruch et al. 1996; McCaffery et al. 2006). Another insect-resistant cotton was developed by Dow AgroSciences by combining Cry1F and Cry1Ac genes. This product, WideStrike cotton, also confers resistance to several lepidopteran pests (Bacheler et al. 2006; Micinski et al. 2006). A wider choice of more than one insect resistance genes, especially if they are stacked, will help delay the build-up of resistance in the target insects. Roundup Ready cotton, introduced in 1996 by Monsanto, confers resistance to glyphosate-based herbicide (Nida et al. 1996). This trait, that helps in the effective management of weeds, was also quickly adopted by cotton-growers in the United States. Traits for insect resistance and herbicide tolerance are also available in combination. Since their introduction a decade ago, GM cotton with these two traits, either combined or alone, is grown on 80% of the acreage devoted to cotton in the United States, and their share is rapidly increasing in other cotton-growing countries (James 2004). In addition to these two com-

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mercially successful traits, there are a number of reports from both industrial and academic laboratories on successful engineering of cotton with useful genes (Table 1). These include genes that confer resistance to three different types of herbicides, namely 2,4-D, phosphinothricin/bialaphos, and imidazolinone/sulfonylurea (Bayley et al. 1992; Lyon et al. 1993; Rajasekaran et al. 1996; Keller et al. 1997). Of these, only phosphinothricin/bialaphos-tolerant cotton, developed by Bayer CropScience and marketed by FiberMax under the name LibertyLink, is available commercially (Perkins 2004). There are some reports on the engineering of cotton to confer resistance to various fungal diseases (Murray et al. 1999; McFadden et al. 2000; Emani et al. 2003; Wang et al. 2004; Rajasekaran et al. 2005). Although these studies appear quite promising, in each case, the transgenic trait addresses only a limited spectrum of pathogens. A number of studies have been conducted that attempted to engineer cotton for resistance to abiotic stresses that include freezing, water-logging, salt-stress and drought (Ellis et al. 2000; Kornyeyev et al. 2001, 2003; Payton et al. 2001; Yan et al. 2004; He et al. 2005). However, these investigations had varying degrees of success in making cotton plants tolerant to the intended environmental stresses. Cotton is grown mainly for its fiber, and therefore, there is considerable interest in applying biotechnology to improve or modify it. The desired properties of cotton fiber include strength, fineness, and length. The number of genes involved in controlling these traits is likely to be extensive and the mechanism for the control of each trait is expected to be complex. As more of the genes involved in fiber initiation, elongation, and development become available, their coding sequences and promoters will be used in the future for engineering cotton to address issues related to the improvement of fiber quality. Nevertheless, some interesting work to modify cotton fiber has been already conducted by scientists at Agracetus. This research involved the expression of genes from Alcaligenes eutrophus in developing cotton fibers that resulted in the deposition of poly-d-(–)-3-hydroxy-butyrate (PHB) in their lumens (John and Keller 1996; Rinehart et al. 1996). This modification resulted in a fiber with altered thermal properties, such that its insulating characteristics were enhanced (Chowdhury and John 1998). Although this modified-fiber cotton has not been commercialized, this successful engineering feat demonstrates the feasibility of improving cotton fiber in ways that are not possible by traditional breeding methods. An important and abundant byproduct of fiber production is cottonseed. It contains 21% oil and a substantial portion of the global cottonseed output is used for edible oil extraction. Gene-silencing technologies have been used to alter cottonseed oil fatty acid composition in favor of higher oleic acid. Sunilkumar et al. (2005) used antisense technology to double the oleic acid from a wild-type level of ∼15% to ∼30%, while reducing the linoleic acid level from ∼55% to about ∼35%. A more powerful RNAi-mediated silencing of the same target gene resulted in a 5-fold increase in oleic acid level and a concomitant reduction in linoleic acid (Liu et al. 2002). In the same study, RNAi-mediated downregulation of the SAD-1 gene resulted in a >10-fold increase in stearic acid

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in cottonseed oil. These results show the possibility of improving the quality of cottonseed oil by modifying a biosynthetic pathway in a tissue-specific manner. In addition to oil, cottonseed contains 23% protein that is of relatively high quality. Worldwide cottonseed output can potentially meet the protein requirements of 500 million people. However, the ability to utilize this protein-rich resource for food is hampered by the presence of toxic gossypol that is unique to the tribe Gossypieae. This cardio- and hepatotoxic terpenoid, present in cottonseed glands, renders the seed unsafe for human and monogastric animal consumption. Since traditional breeding methods may not be able to bring about a significant reduction in gossypol in a seed-specific manner, biotechnological approaches are being tested in many laboratories around the world to solve this long-standing problem of cottonseed toxicity. Most of these attempts over the past decade have been unsuccessful (see Townsend et al. 2005 and references therein). However, in a recent breakthrough, the feat of selective and significant reduction of gossypol concentration in cottonseed through metabolic engineering was finally achieved (Sunilkumar et al. 2006). This study utilized RNAi to disrupt gossypol biosynthesis in cottonseed tissue by interfering with the expression of the δ-cadinene synthase gene during seed development. Some of the RNAi lines obtained showed a 98% reduction in the concentrations of gossypol in the seed, while maintaining this and other protective terpenoids in all other parts of the plant at the wild-type concentrations where they provide protection against insects and diseases. The results from oil and gossypol studies suggest that the cotton plant, in addition to meeting clothing requirements, can also play an important role in meeting nutritional requirements of humanity.

9 New Technological Advances and Their Role in Cotton Improvement Stable transformation of the plastid genome was reported for cotton recently (Kumar et al. 2004). Compared with nuclear transformation, chloroplast transformation is more difficult and less efficient, but it offers many advantages, including transgene containment because of maternal inheritance and a high level of consistent expression. By expressing two selectable marker genes to detoxify kanamycin in the green and non-green tissues, Kumar et al. (2004) were able to regenerate cotton plants from cells containing plastids that were stably transformed. These transformants were fertile and, more importantly, showed maternal inheritance of the transgene. Because of the lower efficiency and the complexity of the plastid transformation system, widespread adoption of this technology is currently unlikely. However, it will play an important role in cases where a high level of transgene expression and/or its containment are desired. Modification of endogenous genomic sequences by homologous recombination or gene targeting has considerable potential in plant improvement.

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Although efforts are being made to improve gene targeting in plants, it remains inefficient in comparison with what has been achieved in animal systems (Iida and Terada 2005). However, in combination with a technology based on the use of designed zinc finger nucleases that can efficiently create double-stranded breaks in a desired portion of the DNA, the efficiency of the targeting process can be improved substantially (Bibikova et al. 2003). Recent successes with model plant species raise the possibility that the cotton genome can also be modified using this novel technology (Wright et al. 2005). Another strategy for genome modification makes use of self-complementary, chimeric RNA/DNA oligonucleotides to create specific 1−2 bp alterations. This technology has been successfully used to mutate a gene encoding an enzyme of the branched-chain amino acid pathway in various plant systems, to confer resistance against either imidazolinone or sulfonylurea herbicides (Beetham et al. 1999; Zhu et al. 1999; Kochevenko and Willmitzer 2003). Although promising, the utility of each of these technologies, especially to introduce recessive modifications, may be complicated by the fact that the majority of commercial cotton varieties grown today are tetraploid. The two examples of RNAi-mediated modification of seed-oil fatty acid composition and gossypol elimination show clearly that this newly discovered gene-suppression mechanism will play an important role in the future improvement of cottonseed. Once the genes involved in controlling various aspects of fiber growth and development are identified, RNAi will be an extremely valuable tool in engineering the desired characteristics in the fiber.

10 Future Perspective It is 20 years since the first reports on cotton transformation were published. Since its introduction in 1996, cotton has become the most widely planted GM crop around the world. However, these cotton varieties carry only the input traits which benefit largely the cotton growers. The research described in this chapter shows the efforts being made to engineer a number of useful output traits. As new genes become available from cotton and other species, and as the genetic modification technologies are refined and improved, it will become possible to incorporate novel input and output traits into this important crop to benefit growers, consumers, and the environment. Acknowledgements. Research in the author’s laboratory is supported by funds from the Texas Cotton Biotechnology Initiative (TxCOT), Cotton Incorporated, the Texas Higher Education Coordinating Board (Advanced Research Program), the Texas Food and Fibers Commission, and the Texas Agriculture Experiment Station. The author thanks Dr. G. Sunilkumar and Ms. LeAnne M. Campbell for their contribution to the cotton-related research described in this chapter.

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He C, Yan J, Shen G, Fu L, Holaday AS, Auld D, Blumwald E, Zhang H (2005) Expression of an Arabidopsis vacuolar sodium/proton antiporter gene in cotton improves photosynthetic performance under salt conditions and increases fiber yield in the field. Plant Cell Physiol 46:1848–1854 Iida S, Terada R (2005) Modification of endogenous natural genes by gene targeting in rice and other higher plants. Plant Mol Biol 59:205–219 James C (1997) Global status of transgenic crops in 1997. ISAAA Briefs 5:12 James C (2004) Global status of commercialized biotech/GM crops: 2004. ISAAA Briefs 32:v James C, Krattiger AF (1996) Global review of the field testing and commercialization of transgenic plants, 1986 to 1995: the first decade of crop biotechnology. ISAAA Briefs 1:27 Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907 John ME (1996) Structural characterization of genes corresponding to cotton fiber mRNA, E6: reduced E6 protein in transgenic plants by antisense gene. Plant Mol Biol 30:297–306 John ME, Keller G (1996) Metabolic pathway engineering in cotton: biosynthesis of polyhydroxybutyrate in fiber cells. Proc Natl Acad Sci USA 93:12768–12773 Jones K, Kerby T, Collins H, Wofford T, Bates M, Presley J, Burgess J, Beuhler B, Deaton R (1996) Performance of NuCotn with Bollgard. In: National Cotton Council (ed) Proceedings of the beltwide cotton conference 1996, vol 1. National Cotton Council, Memphis, pp 46–47 Keller G, Spatola L, McCabe D, Martinell B, Swain W, John ME (1997) Transgenic cotton resistant to herbicide bialaphos. Transgenic Res 6:385–392 Kochevenko A, Willmitzer L (2003) Chimeric RNA/DNA oligonucleotide-based site-specific modification of the tobacco acetolactate synthase gene. Plant Physiol 132:174–184 Kornyeyev D, Logan BA, Payton P, Allen RD, Holaday AS (2001) Enhanced photochemical light utilization and decreased chilling-induced photoinhibition of photosystem II in cotton overexpressing genes encoding chloroplast-targeted antioxidant enzymes. Physiol Plant 113:323– 331 Kornyeyev D, Logan BA, Payton P, Allen RD, Holaday AS (2003) Elevated chloroplastic glutathione reductase activities decrease chilling-induced photoinhibition by increasing rates of photochemistry, but not thermal energy dissipation, in transgenic cotton. Funct Plant Biol 30:101–110 Kumar S, Dhingra A, Daniell H (2004) Stable transformation of the cotton plastid genome and maternal inheritance of transgenes. Plant Mol Biol 56:203–216 Li X, Wang XD, Zhao X, Dutt Y (2004) Improvement of cotton fiber quality by transforming the acsA and acsB genes into Gossypium hirsutum L. by means of vacuum infiltration. Plant Cell Rep. 22:691–697 Li X-B, Cai L, Cheng N-H, Liu J-W (2002) Molecular characterization of the cotton ghTUB1 gene that is preferentially expressed in fiber. Plant Physiol 130:666–674 Li X-B, Fan X-P, Wang X-L, Cai L, Yang WC (2005) The cotton ACTIN1 gene is functionally expressed in fibers and participates in fiber elongation. Plant Cell 17:859–875 Liu Q, Singh SP, Green AG (2002) High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated post-transcriptional gene silencing. Plant Physiol 129:1732–1743 Lyon BR, Cousins YL, Llewellyn DJ, Dennis ES (1993) Cotton plants transformed with a bacterial degradation gene are protected from accidental spray drift damage by the herbicide 2,4dichlorophenoxyacetic acid. Transgenic Res 2:162–169 Mancini F, Van Bruggen AHC, Jiggins JLS, Ambatipudi, AC, Murphy H (2005) Acute pesticide poisoning among female and male cotton growers in India. Int J Occup Environ Health 11:221–232 McCabe DE, Martinell BJ (1993) Transformation of elite cotton cultivars via particle bombardment of meristems. Bio/Technology 11:596–598 McCabe DE, Martinell BJ, John ME (1998) Genetic transformation of cotton through particle bombardment. In: Bajaj YPS (ed) Cotton. (Biotechnology in agriculture and forestry, vol 42) Springer, Berlin Heidelberg New York, pp 263–273

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Sanjaya, Satyavathi VV, Prasad V, Kirthi N, Maiya SP, Savithri HS, Lakshmi Sita G (2005) Development of cotton transgenics with antisense AV2 gene for resistance against cotton leaf curl virus (CLCuD) via Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 81:55–63 Satyavathi VV, Prasad V, Gita Lakshmi B, Lakshmi Sita G (2002) High efficiency transformation protocol for three Indian cotton varieties via Agrobactrium tumefaciens. Plant Sci 162:215– 223 Sunilkumar G, Rathore KS (2001) Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regeneration. Mol Breed 8:37–52 Sunilkumar G, Connell JP, Smith CW, Reddy AS, Rathore KS (2002a) Cotton a-globulin promoter: isolation and functional characterization in transgenic cotton, Arabidopsis, and tobacco. Transgenic Res 11:347–359 Sunilkumar G, Mohr L, Lopata-Finch E, Emani C, Rathore KS (2002b) Developmental and tissuespecific expression of CaMV 35S promoter in cotton as revealed by GFP. Plant Mol Biol 50:463–474 Sunilkumar G, Campbell LM, Hossen M, Connell JP, Hernandez E, Reddy AS, Smith CW, Rathore KS (2005) A comprehensive study of the use of a homologous promoter in antisense cotton lines exhibiting a high seed oleic acid phenotype. Plant Biotechnol J 3:319–330 Sunilkumar G, Campbell LM, Puckhaber L, Stipanovic RD, Rathore KS (2006) Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proc Natl Acad Sci USA 103:18054–18059 Thomas JC, Adams DG, Keppenne VD, Wasmann CC, Brown JK, Kanost MR, Bohnert HJ (1995) Protease inhibitors of Manduca sexta expressed in transgenic cotton. Plant Cell Rep 14:758– 762 Townsend BJ, Poole A, Blake CJ, Llewellyn DJ (2005) Antisense suppression of a (+)-δ-cadinene synthase gene in cotton prevents the induction of this defense response gene during bacterial blight infection but not its constitutive expression. Plant Physiol 138:516–528 Trolinder NL, Xhixian C (1989) Genotype specificity of the somatic embryogenesis response in cotton. Plant Cell Rep 8:133–136 Umbeck P, Johnson G, Barton K, Swain W (1987) Genetically transformed cotton (Gossypium hirsutum L.) plants. Bio/Technology 5:263–266 Wang YQ, Chen DJ, Wang DM, Huang QS, Yao ZP, Liu FJ, Wei XW, Li RJ, Zhang ZN, Sun YR (2004) Over-expression of Gastrodia anti-fungal protein enhances Verticillium wilt resistance in coloured cotton. Plant Breed 123:454–459 Wilkins TA, Mishra R, Trolinder NL (2004) Agrobacterium-mediated transformation and regeneration of cotton. J Food Agric Environ 2:179–187 Wright DA, Townsend JA, Winfrey RJ, Irwin PA, Rajagopal J, Lonosky PM, Hall BD, Jondle MD, Voytas DF (2005) High-frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J 44:693–705 Wu J, Luo X, Guo H, Xiao J, Tian Y (2006) Transgenic cotton expressing Amaranthus caudatus agglutinin, confers enhanced resistance to aphids. Plant Breed 125:390–394 Yan J, He C, Wang J, Mao Z, Holaday SA, Allen RD, Zhang H (2004) Overexpression of the Arabidopsis 14-3-3 protein GF14l in cotton leads to a “Stay-Green” phenotype and improves stress tolerance under moderate drought conditions. Plant Cell Physiol 45:1007–1014 Zapata C, Park SH, El-Zik KM, Smith RH (1999) Transformation of a Texas cotton cultivar by using Agrobacterium and the shoot apex. Theor Appl Genet 98:252–256 Zhu T, Peterson DJ, Tagliani L, St. Clair G, Baszczynski CL, Bowen B (1999) Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides. Proc Natl Acad Sci USA 96:8768–8773

I.7 Flax A. Preˇtová, B. Obert, and Z. Bartoˇsová1

1 Introduction Flax (Linum usitatissimum L.) is one of the oldest cultivated plants (the name usitatissimum means most useful), but still has an important impact on world economy. It is the only agriculturally important species in the family Linacea, which consists of 13 genera and 300 species (Heywood 1978). The plant originated from the Mediterranean and Southwest Asian regions. Traditionally, it is cultivated for the main products – fibre and seed oil – but it has gained a new interest in the emerging market of functional food due to its high content of fatty acids, mainly α-linolenic acid, and lignan oligomers. In addition, flax fibre is a valuable component of modern composite materials used in the automobile industry. Flax is also considered as an important diversification crop on set-aside land. Flax is, to a high degree, a self-pollinated species. Conventional breeding methods for flax are time-consuming and are hampered by limited genetic resources and a small number of close relatives amenable to crossing. Therefore, there is an increasing demand for flax improvement using biotechnological approaches. Tissue culture and gene transfer technologies can facilitate the breeding program for flax improvement by the transfer of genes responsible for desirable new traits into flax cultivars. These traits include resistance to fungi diseases, improved fiber and oil quality and tolerance to herbicides (Preˇtová et al. 2001). To date, the biotechnological approaches to flax include controlled in vitro regeneration via organogenesis and somatic embryogenesis, protoplast and suspension cultures, the production of haploids via androgenesis and gynogenesis, gene transfer techniques and application of molecular markers for genetic identification. Flax has a relatively small genome (2n = 30 chromosomes) and is considered as excellent material for gene tagging and genetic manipulation (Rakousky et al. 1999).

2 Tissue and Organ Culture Flax has a long history of research through tissue culture. The first report on the capacity of the species to initiate buds from decapitated hypocotyl sections 1 Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, P.O.

Box 39A, 95007 Nitra, Slovak Republic, e-mail: [email protected] Biotechnology in Agriculture and Forestry, Vol. 61 Transgenic Crops VI (ed. by E.C. Pua and M.R. Davey) © Springer-Verlag Berlin Heidelberg 2007

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was given by Link and Eggera (1946). In 1957, an in vitro approach was employed to study the pathogen Melanospora linii (Pers) H. Lev. on flax (Turel and Ledingham 1957). The culture conditions for this species were improved by Ibrahim (1971), enabling him to investigate the phenolic compounds in flax tissue cultures (Liau and Ibrahim 1973). An article published by Gamborg and Shyluk (1976) contributed significantly to the tissue culture applications in flax. Since then, flax has been regenerated from various tissues/organs, including hypocotyl-, cotyledon- and leaf-derived calli (McHughen 1990), mature and immature zygotic embryos (Preˇtová and Williams 1986), microspores (Nichterlein and Friedt 1993), anthers (Chen et al. 1998b; Chen and Dribnenki 2002; Obert et al. 2004a, b), ovaries (Bartoˇsová et at. 2003; Obert et al. 2004a) and protoplasts (Ling and Binding 1987, 1992). Flax tissue cultures have been shown to be responsive to a wide range of growth regulators, including thidiazuron (Bretagne et al. 1994; Dedicova et al. 2000; Jain and Rashid 2001). Further studies revealed that the carbon source in the medium can also modulate morphogenic response (Millam et al. 1992) and that there is an interaction between auxin and carbohydrate (Millam and Davidson 1993). In addition, the concentration of hydroxyl radicals formed by cultured explants has been shown to play a role in the formation of shoot, roots and embryo-like structures (ELS) in flax (Obert et al. 2005). The use of tissue culture techniques can broaden the genetic variability that is important for flax breeding programs. These techniques, coupled with induced mutations and recombinant DNA technologies, may help to enhance the diversification of genetic resources for plant breeders. Somaclonal variability is associated with point mutations and chromosomal rearrangements and recombination, DNA methylation, altered sequence copy number and transposable elements. Its occurrence is influenced by plant genotype, explant type, culture conditions, age of donor plants, time in culture and application of growth regulators (Veilleux and Johnson 1998; Jain 2001). The presence of plant growth regulators in the medium can induce dedifferentiation and differentiation, depending on type, administration and concentration. This re-programming can cause a wide range of epigenetic variation in plants regenerated from cultured cells and tissues. Although somaclonal variation can occur during the tissue culture process as the latter may act as a mutagenic system, it can also be induced by application of chemical mutagens or irradiation. Somaclonal variation can be used for screening and selection of cell lines or regenerants with desirable traits, such as tolerance to some fungal toxins, heat and salt, by applying the toxins or other selection agents directly to the media. This approach is less time-consuming and labour-intensive in developing lines with selected traits. Furthermore, it can be performed in the winter independent of field conditions. The selected genetically stable somaclones can be used for plant breeding. A tissue culture-derived flax somaclone of the cv. McGregor with salt and heat tolerance was released as the cv. ANDRO (O’Connor et al. 1991). A flax mutant “zero” containing a very low concentration of linolenic acid in seed oil was induced by methanesulfonate (Green 1986).

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This mutant was the source of the future edible flax cvs. Linola 947 (Dribnenki and Green 1995) and Linola 989, Linola 1084 and Linola 2047 (Dribnenki et al. 1996, 1999, 2003), and was also involved in the breeding of the cvs. Wallaga, Eyre and Ergylea (Ahloowalia et al. 2004). Flax has been exploited for a number of fundamental studies using embryo culture. One of the first plant embryos cultured in vitro was derived from flax (Dietrich 1924; Laibach 1925). These excised flax embryos in vitro were used for studying growth and development (Erdelska et al. 1973; Preˇtová 1974, 1983, 1986). A comparative study on the development, pigment content and deposition of reserve materials in flax embryos under in situ and in vitro conditions was also conducted (Preˇtová 1990). It is clear that the early flax embryo culture work facilitated the evolution of plant tissue culture as a science and exerted a considerable impact on modern methodologies of embryo culture, embryo rescue and studies on somatic embryogenesis and transformation in flax and other plant species (Millam et al. 2005).

3 Somatic Embryogenesis Somatic embryo development is a significant and potentially effective morphogenic pathway for plant regeneration. The process of somatic embryogenesis in flax was first reported using immature zygotic embryos excised in the late heart or early torpedo stage of the cv. Glenelg (Preˇtová and Williams 1986). Direct formation of somatic embryo was also achieved by culturing 2-mm hypocotyl segments from 6-day-old flax seedlings (cv. Szegedi 30) in liquid MS medium (Murashige and Skoog 1962) supplemented with 2 mg l−1 2, 4-dichlorophenoxyacetic acid (2,4-D) for 2 weeks. After the first subculture to a hormone-free medium, ELS were formed at the cut ends of the segments. More ELS were observed on the segments proximal to the shoot and less on their distal ends. The structures formed were liberated from the primary tissues after 3 weeks when they reached the heart stage, and floated freely in the medium (Preˇtová and Obert 2007). The formation of secondary embryos was also observed in the culture system. Similar secondary somatic embryo formation in flax was also reported by Tejavathi et al. (2000). Studies on flax regeneration in vitro have shown that the hypocotyl is the most responsive of explants (Kaul and Williams 1987; Millam and Davidson 1993; Bretagne et al. 1994; Cunha and Ferreira 1996; Dedicova et al. 2000; Mundhara and Rashid 2002). It was reported that hypocotyl explants with 2,4-D pre-treatment showed variable effects on somatic embryo formation of the oilseed flax cv. Szegedi 30 grown on Mo medium (Monnier 1978). Most ELS were obtained after a short pre-treatment with 2,4-D (5 mg l−1 ) and subsequent culture on medium supplemented with zeatin (2 mg l−1 ). There was a correlation between auxin concentration and pre-treatment duration, with respect to ELS formation (Dedicova et al. 2000).

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The globular and heart-shaped ELS had an epidermis on their surfaces, two or three sub-epidermal layers of parenchyma cells and meristematic tissues internally. The bipolar structures possessed elongated hypocotyls and two small polarly located regions interconnected with the vascular tissue. The vascular strands showed a deviation on the top of the “embryo-like” structure typical for entering of the vascular strands to cotyledons (Dedicova et al. 2000). Often, the organization of the shoot and root poles was abnormal. Occasionally, the root meristem did not develop a normal root cap, while the shoot meristem was not located on the top of the structure and normal leaf primordia failed to develop. Sometimes ELS were poorly defined, or the cotyledons were fused and shoot apices were not clear (Dedicova et al. 2000). Such structures were described as horn-shaped embryos in soybean (Lazzeri et al. 1987) and, more recently, as stm mutants of ELS (Mordhorst et al. 1998). As a consequence of the structural abnormalities, these arrested ELS were unable to produce mature embryos and complete plants (Dedicova et al. 2000). Sometimes additional post-embryogenic shoot apices were formed on the top of these ELS. Auxin transport can influence the initiation of cotyledons and bilateral symmetry of the early embryos. Even in globular zygotic flax embryos grown in vitro, the formation of cotyledons was a critical stage in their development (Preˇtová 1986, 1990). It can be assumed that there was a disturbance of polar auxin transport during somatic embryo formation from hypocotyl segments, probably due to the addition of exogenous auxin. Additional evidence on abnormal embryo development associated with 2,4-D was observed. The presence of high concentrations of 2,4-D in the induction medium inhibited further development of somatic embryos and caused abnormal development of apical meristems in carrot (Halperin and Wetherell 1964). Zauddin and Kasha (1990) observed that the extent of abnormalities increased with the concentration and time of exposure to the growth regulator. In pecan (Carya illnoinensis), the presence of 2,4-D in the induction medium promoted callus formation and embryos with a weak apex or lacking an apex (Rodriguez and Wetzstein 1994). A “cumulative” effect of 2,4-D on cell totipotency was observed by Preˇtová and Obert (2007). Nearly all callus cells derived from flax hypocotyl segments became embryogenic after 2 weeks of 2,4-D treatment (Preˇtová and Obert 2006, 2007). However, most embryos possessed weakly differentiated apical meristems, or lacked meristems, which was similar to stm mutants of Arabidopsis thaliana (Mordhorst et al.1998). They did not germinate. Stm somatic embryos prevented embryogenic and post-embryogenic meristem formation (Barton and Poethig 1993). The transition from globular to heart-shaped stage is an embryogenesis check-point and numerous embryogenesis mutants are blocked at this stage (Mayer et al. 1998). During somatic embryogenesis in flax, a wide range of bipolar structures that can be classified as ELS have been observed (Preˇtová and Obert 2006). This observation indicates that the morphogenic response in culture possesses much more plasticity than the cells within the intact plant body under strict

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control and cell to cell coordination, with regard to the extent of cell, tissue and organ differentiation. Indirect formation of somatic embryos in flax can be induced from nearly mature (28-day-old) zygotic embryos, in which the accumulation of reserve materials is in progress (Preˇtová 1978, 1990). These zygotic embryos are unable to express their totipotency. The presence of auxin can promote cell divisions of those zygotic embryos that undergo dedifferentiation resulting in callus formation. Dedifferentiation is thought to be required for cells to set a new developmental program. It is speculated that a certain degree of dedifferentiation may be required for direct somatic embryo development, even when no visible callus is formed. During somatic embryogenesis in flax, it was important to maintain tight cohesion between callus cells. Loose calli did not form somatic embryos because cell to cell coordination was disturbed. The more cohesive the cell clusters at the moment of application of external stimuli, the greater was the yield of somatic embryos (Preˇtová and Obert, unpublished data). Different morphogenic responses in flax may also be associated with changes in oxidative status of the initiating tissue during culture. Significant differences in hydroxyl radical generation were found to be related to the type of induced morphogenic pathway. Lower concentrations of hydroxyl radicals were observed during shoot regeneration, while the highest level was detected during ELS induction (Obert et al. 2005). Exogenous application of hydrogen peroxide also increased the number of ELS formed on flax hypocotyl explants (Takac and Preˇtová 2004). Results from these studies indicate that the occurrence of somatic embryogenesis is a stress response and that it is a way in which the plant cell realises its survival strategy under completely changed and unusual conditions, using its unique feature of totipotency (Preˇtová and Obert 2003). Under culture conditions, cells are exposed to sub-optimal nutrient and different hormone supply that generates a significant degree of stress. Oxidative stress responses may be linked to auxin signaling and cell cycle regulation through mitogen-activated protein kinase (MAPK) phosphorylation cascades (as reviewed by Hirt 2000). In order to investigate somatic embryogenesis in flax, protoplast cultures were employed initially (Ling and Binding 1992), followed by the more detail study reported by Cunha and Ferreira (1996). Cunha and Ferreira (1997) showed that the induction of somatic embryos and shoot organogenesis were associated with an increase in total sterols in the competent calli and a greater ratio of stigmasterol to β-sitosterol in callus-derived embryos. In addition, the high rate of sterol biosynthesis was correlated with an active membrane biogenesis. Other factors affecting somatic embryogenesis from hypocotyl explants of flax were also reported, including carbon source, total inorganic nitrogen, balance between ionic forms and interaction between calcium and zeatin (Cunha and Fernandes-Ferreira 1999). The composition and distribution of n-alkanes were shown to affect the development of flax somatic embryos (Cunha and Fernandes-Ferreira 2001). Furthermore, the content of free and esterified fatty acids during somatic embryo development was also investigated (Cunha and Fernandes-Ferreira 2003). The study was based on the findings that changes

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in lipid content and/or composition occurred not only during plant ontogenic development, but also in response to stress and other challenging conditions (Nishida and Murata 1996; Aziz and Larher 1998; Lauriano et al. 2000). The genetic basis of embryogenic competence is not well defined. In this respect, some molecular studies in flax were performed. The two homologs to the embryogenic cell protein (ECP) genes (ECP 31, ECP 63) in the flax genome were detected. These genes were shown to express in carrot somatic embryogenesis (Kiyosue et al. 1992; Yang et al. 1996, 1997). In flax, two ECP 63 transcripts were detected in 6-day-old seedlings, and one ECP 31 transcript in seeds (Hajduch et al. 1997). Attempts have been made to identify the signal molecules or markers for embryogenic potential in flax. It was reported that flax protoplasts derived from hypocotyl segments entrapped in either agarose or Ca-alginate, secreted basic chitinases (Roger et al. 1998). This prompted speculation that chitinases associated with cells expressing a morphogenic response (ELS) after their entrapment in Ca-alginate might generate rod-like oligosaccharides that served as signals for cell differentiation in flax. Identification of the potential substrates for these chitinases may shed light on the functions for these proteins in morphogenic processes in vitro. Preˇtová et al. (2004) also reported chitinase activity (acidic chitinase of 25 kDa) in flax suspension culture forming ELS. In carrot, chitinase genes encoding proteins secreted into the medium of wild-type embryogenic cell cultures were associated with the morphogenic response, and one chitinase gene was shown to promote the transition from globular to heart-stage embryos of the temperature-sensitive carrot mutant ts11 (Kragh et al. 1996). In conclusion, it can be stated that the embryogenic potential in flax is rather low and very often the somatic embryos are formed together with shoots (organogenesis). Indeed, it is hard to distinguish between the two. Both morphogenic processes, somatic embryogenesis and organogenesis, are considered to be the result of either fully or partially expressed totipotency of plant cells. Because of the lack of discriminating the morphological and/or histological features, it is imperative to develop molecular markers. A few studies have been undertaken to characterize the differences between histological aspects of embryonic tissues and their potential for regeneration at the molecular level (e.g. Stirn et al. 1995; Charbit et al. 2004). Attention should also be given to functional genomics in future studies, as ELS in flax provide a striking resemblance to stm mutants in A. thaliana (Mordhorst et al. 2002).

4 Protoplast and Cell Suspension Cultures The isolation of protoplasts from flax tissues was first reported in 1987 by Ling and Binding. The system was further developed to regeneration via somatic embryogenesis (Ling and Binding 1992). The morphogenic response of flax

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protoplasts was studied in relation to the regeneration of complete plants and cell wall composition (David et al. 1994). The formation of ELS from flax protoplasts was reported (Roger et al. 1998). Flax protoplasts were also used in a number of biochemical and physiological studies, as in the case of proteins in developing cell walls (David et al. 1995), the correlation between ionically bound cell-wall proteins and the morphogenic response (Roger et al. 1998), basic chitinase content and morphogenesis (Roger et al. 1998), water deficit and the water permeability of isolated flax root protoplasts (Morillon and Lassalles 2002). Protoplast cultures, as single-cell systems, are an important tool for the study of gene expression, for the transient analysis of genes and for early screening of novel promoters or genes of interest in flax (Millam et al. 2005). Suspension cultures with a high degree of homogenicity and rapid multiplication rates provide the ideal system for biochemical and molecular studies. Flax suspension cultures have been used for study of RNA profiling and nucleotide composition (Ibrahim et al. 1975), chloroplast ultrastructure and flavonoid synthesis (Ibrahim and Phan 1977), phenolic synthesis (Ibrahim and Phan 1978), pectin manipulation (Schaumann et al. 1993), and the effect of light on proteins and peroxidase activities (Bruyant et al. 1996). The activity of pectin methyltransferase (PMT) in microsomes and in cell walls was also investigated using a flax suspension (Schaumann et al. 1993, Bourlard et al. 2001). In addition, enzymes with galactan synthase activities were localized, isolated and characterized in flax suspension cultures (Goubert and Morvan 1993, 1994), where Peugnet et al. (2001) showed the transfer of a galactosyl residue from UDP-galactose to a polysaccharide acceptor.

5 Anther, Microspore and Ovary Cultures Anther and microspore cultures have been used for the production of haploid plants and homozygous lines for flax breeding and, more recently, for molecular marker studies (Millam et al. 2005). Currently, anther culture is the most successful method for the production of doubled haploids (DH) in flax (Sun and Fu 1981; Friedt et al. 1995; Bergmann and Friedt 1997; Chen et al. 1998a; Rutkowska-Krause et al. 2003; Obert et al. 2004a, b). The process of induction and regeneration of DH flax, via androgenic or gynogenic embryogenesis, is a complex process involving a number of factors common for most plant species. Environmental factors affecting the regeneration capacity of linseed anther culture were studied and plants were regenerated indirectly from antherderived callus (Nichterlein et al. 1991). Cytological examination of regenerated plants indicated that they were of microspore origin. In flax microspore culture, microspores underwent cell division which led to the formation of either

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microcallus or embryos (Nichterlein and Friedt 1993). Regenerated plants were transferred to soil. The superiority of DH lines generated from F-1 hybrids using an anther culture technique was demonstrated in field trials. There was considerable variation in the fatty acid composition and fat content amongst these DH plants. Some DH lines were shown to contain more fat and linolenic acid than their parental lines (Friedt et al. 1995). Factors affecting flax anther cultures were discussed by Tejklova (1996) and Steiss et al. (1998). Anthers pre-treated for 1 day at 35 ◦ C (Chen et al. 1998a), or the same pre-treatment combined with a 3-day treatment at 4 ◦ C (Bartoˇsová and Preˇtová 2004), resulted in high frequency regeneration of haploid plants. Chen et al. (1999, 2001) studied the production of DH plants, inheritance of rust resistance genes and molecular markers in microspore-derived flax populations. Other studies on flax anther culture include the association of some parameters with androgenic response (Chen and Dribnenki 2002), and the effect of sucrose concentration on shoot elongation (Chen et al. 2003). The effects of pre-treatment and media on the androgenic response in several flax genotypes were studied in an attempt to identify responsive lines (Preˇtová and Obert 2000; Obert et al. 2004b). In the practical application of flax anther culture, plants were reported with increased resistance to Fusarium oxysporum, the main fungus pathogen this plant (Rutkowska-Krause et al. 2003). Apart from anther and microspore cultures, DH flax plants were regenerated from cultured unfertilized ovaries (Bartoˇsová et al. 2003; Obert et al. 2004a). Somaclonal variation was also observed in ovary-derived calli and plants (Bartoˇsová et al. 2005). It was found that the level of ploidy in these tissues increased with time of culture.

6 Gene Transfer in Flax The pre-requisite of a successful plant transformation system is the availability of an efficient plant regeneration system. In early transformation studies, Basiran et al. (1987) described the regeneration of transformed shoots mediated by Agrobacterium tumefaciens via a callus stage, while Zhan et al. (1988) reported flax transformation using A. rhizogenes. The first successful verified transformation of flax using A. tumefaciens and consequent production of glyphosate-tolerant flax plants were reported by Jordan and McHughen (1988a, b). Escapes (plants that survive antibiotic or herbicide selection, without molecular analysis detecting introduced DNA sequences) were also observed. The efficiency of flax transformation was further improved by pre-culturing the explants prior to transformation (McHughen et al. 1989). Although the pattern of transformation intensity on flax explants varied (Dong and McHughen 1991), the use of a high kanamycin concentration (200 mg l−1 ) as a selection marker resulted in increased efficiency of transformation, with less escapes, but chimeric regenerants were still present (Dong and McHughen 1993a, b).

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Mlynarova et al. (1994) reported an improved, highly efficient transformation system for flax with few escapes, based on early detached calli derived from hypocotyl segments. However, T-DNA-induced mutations were also reported (Rakousky et al. 1998). A field trial for glyphosinate ammonium-tolerant transgenic flax was conducted (McHughen and Holm 1995). This transformation was based on the GM flax variety “Triffid” derived from the non-GM variety Norlin. However, this flax variety was soon removed from the market because of a major concern about the use of the kanamycin resistance gene as a selection marker. Rakousky et al. (1999) reported flax transformation using hygromycin selection. In most transformation events, flax hypocotyl segments were co-cultivated with Agrobacterium. Ling and Binding (1997) reported the regeneration of transgenic flax plants using a direct gene transfer approach involving the uptake of DNA into protoplasts in the presence of polyethyleneglycol (PEG). In addition, flax could be transformed using particle bombardment (Wijayanto and McHughen 1999). This approach was also adopted for transient expression studies of various seed-specific promoters (Drexler et al. 2003). In connection with the high variability in transgene expression caused by either position effect or gene silencing (Mlynarova and Nap 1997), plasmids containing short DNA sequences (matrix-associated regions; MARs) were used. This approach resulted in a high stability and reproducibility of transgenes in flax (Preˇtová et al. 2001; Hricova et al. 2002a, b). Flax transformation has also been reviewed (McHughen 1999, 2002; Rakousky et al. 2003; Wang et al. 2004; Millam et al. 2005).

7 Potential Applications of Transgenic Flax More applied transformation targets were reported by Ayliffe et al. (2002), who investigated up-regulation of gene expression in response to rust infection, and Drexler et al. (2003), who evaluated putative seed specific promoters. A chlorsulfuron-resistant gene was introduced into flax plants; the presence of the transgene did not alter the agronomic character of the plant (McHughen and Rowland 1991). Progress in transformation and regeneration protocols and increasing molecular and biochemical information derived from genomic and gene identification programs have increased considerably the potential for manipulating quality traits in flax. As flax is an important fiber-bearing plant, lignin, pectin and cellulose synthesis have been studied extensively in this plant. These components, as well as cell wall formation, are closely associated with fiber quality. Cinnamyl alcohol dehydrogenase (CAD) is an important enzyme of the phenylpropanoid pathway that catalyzes the formation of monolignans, the last step in lignin synthesis (Walter et al. 1988). In tobacco, the down-regulation of CAD resulted in reduced lignin content in fibers (Chabannes et al. 2001). Attempts are being

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made to transform flax with the CAD gene, with the aim to alter lignin quality (Valachyova et al. 2001, 2003). In addition to lignin, Lacoux et al. (2003) reported modified pectin in transgenic flax calli. Transgenic flax plants have been produced to synthesize polyhydroxybutyrate (PHB), which is manufactured conventionally by bacterial fermentation (Wrobel et al. 2004). These results demonstrated the feasibility of producing environmentally friendly, biodegradable and renewable polymers in flax. The use of flax in phytoremediation is also possible, as the plant is known to be a heavy metal chelator. Another application of transgenic flax is to improve oil composition and content, as many genes encoding key enzymes of fatty acid metabolism have been cloned and characterized. The results of a recent study show that it is possible to produce very-long-chain polyunsaturated fatty acids by encoding some very specific enzymes (acyl desaturases and elongases; Abbadi et al. 2004) in tobacco and flax.

8 Molecular Markers Different types of molecular markers have been developed for flax in the past few years. Random amplified polymorphic DNA (RAPD) has been used to study flax land races, to determine the taxonomic status of some Linum representatives and the extent of inter- and intraspecific genetic polymorphism (Lemesh et al. 2001). Microsatelite (SSR) markers for flax genome fingerprinting are being developed to disseminate and identify flax and linseed cultivars (Rakousky et al. 2001). The cultivar-specific SSR sequences are generated by microsatelite-directed polymerase chain reaction (PCR) and by screening microsatelite-enriched libraries within the framework of an International Flax Collaboration Consortium coordinated from Canada. Molecular markers for different characters, based on DNA polymorphism, have been reported for microspore-derived flax plants (Chen et al. 1998b, 1999, 2001). The polymorphism of isozymes amongst tissue culture-derived flax plants that have been generated via androgenensis and gynogenesis (Preˇtová and Bartoˇsová 2004; Bartoˇsová et al. 2006) has been exploited to estimate genetic diversity in flax cultivars (Yurenkova 2001).

9 Concluding Remarks and Further Prospects The use of biotechnological approaches for flax improvement has progressed well over several decades. It is anticipated that advances in cell and molecular biology will permit more rapid and targeted improvements of flax for a range of medical, industrial or environmental applications (Millam et al. 2005).

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The enhanced interest in dietary intake and nutritional value of flax also brings this species into the public awareness. This fact might lead to more opportunities for increased production as well as research into exploiting the wide range of flax germplasm that exists. Furthermore, the most valuable plant biotechnology approach in flax may be the use of haploid material in breeding programs in combination with several molecular markers for rapid screening and discrimination for gamete-derived regenerants. The use of flax in gene expression experiments has not yet been widely reported, certainly not to the same extent as Arabidopsis or Nicotiana species but this is an area that may be of future application. To our knowledge, there are no reports of gene silencing or RNAi applied to this species, but this may also be an area of substantial further utility (Millam et al. 2005).

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Preˇtová A, Obert B (2007) Hypocotyl segments are a good source for somatic embryo production in flax. J Plant Physiol (in press) Preˇtová A, Obert B (2006) Some aspects of embryo development in vitro. Acta Horticulturae 725:83–88 Preˇtová A, Williams EG (1986) Direct somatic embryogenesis from immature zygotic embryos of flax (Linum usitatisimum L.). J Plant Physiol 126:155–161 Preˇtová A, Obert B, Hricova A (2001) Improvement of flax using innovative biotechnology approaches. Natural fibers: bast plants in the new millennium. Second Global Workshop, Borovets, Bulgaria Rakousky S, Tejklova E, Wiesner I, Wiesnerova D, Kocabek T, Ondrej M (1998) T-DNA induced mutations and somaclonal variants of flax. Natural Fibers 2/1998: pp 244–246 Rakousky S, Tejklova E, Wiesner I, Wiesnerova D, Kocabek T, Ondrej M (1999) Hygromycin B – an alternative in flax transformant selection. Biol Plant 42:361–369 Rakousky S, Tejklova E, Wiesner I (2001) Recent advances in flax biotechnology in the Czech Republic. In: Kozlowski R, Atanasov A (eds) Natural fibers: bast plants in the new millennium. (Second global workshop, 3–6 June 2001) Borovets, Bulgaria, pp 151–156 Rakousky S, Tejklova E, Ohnoutkova L, Kocabek T, Serhantova V, Halamkova E (2003) Transformation for flax breeding. In: Salaj J, Obert B, Salaj T (eds) Plant biotechnology – progress and developments, Stara Lesna, High Tatras, Slovak Rep. (Fifth international symposium in the series: recent advances in plant biotechnology) Institute of Plant Genetics and Biotechnology/Slovak Academy of Sciences, Nitra, p. 78 Rodriguez APM, Wetzstein HY (1994) The effect of auxin type and concentration on pecan (Carya illinoinensis) somatic embryo morphology and subsequent conversion into plants. Plant Cell Rep 13:607–611 Roger D, Gallusci P, Meyer Y, David A, David H (1998) Basic chitinases are correlated with the morphogenic response of flax cells. Physiol Plant 103:271–279 Rutkowska-Krause I, Mankowska G, Lukaszewicz M, Szopa J (2003) Regeneration of flax (Linum usitatissimum L.) plants from anther culture and somatic tissue with increased resistance to Fusarium oxysporum. Plant Cell Rep 22:110–116 Schaumann A, Bruyantvannier MP, Goubert F, Morvan C (1993) Pectic metabolism in suspension cultured cells of flax Linum usitatissimum. Plant Cell Physiol 34:891–897 Steiss R, Schuster A, Friedt W (1998) Development of linseed for industrial purposes via pedigree selection and haploid technique. Indian Crop Prod 7:303–309 Stirn S, Mordhorst AP, Fuchs S, Lorz H (1995) Molecular and biochemical markers for embryogenic potential and regenerative capacity of barley (Hordeum vulgare L.). Plant Sci 106:195–206 Sun H, Fu W (1981) Induction of pollen plants in flax (Linum usitatissimum L.) and preliminary observations on performance of their progenies. Acta Genet Sin 8:369–374 Takac T, Preˇtová A (2004) The effect of hydrogen peroxide on flax embryo-like structure regeneration. Proc Plant Physiol Days 10:34 Tejavathi DH, Sita GL, Sunita AT (2000) Somatic embryogenesis in flax. Plant Cell Tissue Organ Cult 63:155–159 Tejklova E (1996) Some factors affecting anther culture in Linum usitatissimum L. Rost Vyroba 42:249–260 Turel FLM, Ledingham GA (1957) Production of aerial mycelium and uredospores by Melampsora lini (Pers) H. Lev on flax leaves in tissue culture. Can J Microbiol 3:813 Valachyova A, Libantova J, Obert B, Pretová A (2001) Genetic transformqtion of flax (Linum usitatissimum L.) by Agrobacterium tumefaciens. In: Bezo M (ed) Biotechnological methods in plant breeding 2001, Nitra. Slovak Agriculture University, Nitra, pp 102–103

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Section II Medicinal Crops

II.1 Ginseng Y.E. Choi1

1 Introduction Ginseng (Panax ginseng C.A. Meyer) is a perennial herbaceous species, belonging to the family Araliaceae. This species has been used as an important oriental medicine since ancient times. Ginseng roots, one of the most famous and expensive crude drugs, have been commonly used to promote the quality of life (Ellis and Reddy 2002; Coleman et al. 2003). Immune system modulation, anti-stress activity, anti-cancer and anti-diabetic activities are the most notable features of ginseng in laboratory and clinical trials (Vogler et al. 1999; Shibata 2001; Yun 2001; Dey et al. 2003; Kiefer and Pantuso 2003). About 12 ginseng species are distributed around the world (Table 1; Wen and Zimmer 1996; Zhu et al. 2004). The most commonly used species are P. ginseng, P. japonicus, P. notoginseng, P. quinquefolius, and P. vietnamensis. While P. ginseng is distributed in Northeast Asia such as Korea, China and Russia, P. quinquefolium is commonly found in North America, P. japonicus in Japan, P. vietnamensis in Vietnam and P. notoginseng in China (Baranov 1982). Recently, the medicinal value of ginseng became widely recognized in the Western world, through evidence from the increased international consumption and cultivation of ginseng (Vogler et al. 1999). The cultivation of ginseng requires a minimum of 4 years before roots can be harvested. Ginseng should be cultivated under special conditions, where direct sunlight is blocked (Fig. 1). Recurrent cultivation of ginseng in the same field is impossible. Therefore, farmers must prepare new ginseng fields for continuous cultivation. Moreover, various kinds of disease pose serious problems to ginseng cultivation. Agrochemical treatment to prevent disease is a serious problem for the international ginseng trade. The above problems emphasize the importance of ginseng breeding to improve its genetic characters. Wild ginseng is a valuable source for breeding programs, but is scarce due to over-exploitation. Conventional breeding of ginseng is also difficult because of unconserved germplasm and is impractical since the procedure usually takes more than 50 years. Until now, only limited success has been achieved for new variety development by line selection in the field (Kwon et al. 2000). In view of these facts, biotechnological applications have been considered as an alternative approach for ginseng improvement and propagation 1 Division of Forest Resources, College of Forest and Environmental Sciences, Kangwon National

University, Chunchon 200-701, Korea, e-mail: [email protected] Biotechnology in Agriculture and Forestry, Vol. 61 Transgenic Crops VI (ed. by E.C. Pua and M.R. Davey) © Springer-Verlag Berlin Heidelberg 2007

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Table 1. Species of ginseng (Panax)

Number

Species designation

Common name

1 2 3 4 5 6 7 8 9 10 11 12

P. ginseng C.A. Meyer P. japonicus C.A. Meyer P. major Ting P. notoginseng (Burkill) F.H. Chen P. pseudoginseng Wallich P. quinquefolius L. P. sinensis J. Wen P. stipuleanatus H.T. Tsai and K.M. Feng P. trifolius L. P. wangianus Sun P. zingiberensis C.Y. Wu and K.M. Feng P. vietnamensis Ha et Grushv

Korean ginseng Japanese ginseng Sanchi ginseng American ginseng

Dwarf ginseng

Vietnamese ginseng

Fig. 1. Panax ginseng cultivated in field under shade structure. Bar 7 cm

and the production of raw materials for medicinal use. Research in ginseng biotechnology has been extensive in recent years. Here, recent advances in ginseng biotechnology are discussed, including cell, tissue and organ culture, plant regeneration and genetic transformation, metabolic engineering and genomics.

2 Cell Culture of P. ginseng 2.1 Induction and Maintenance of Cells The growth of ginseng roots is very slow, as 6-year-old roots of individual plants produce less than 100 g of fresh weight. The use of plant cell culture has been considered as an alternative for the efficient production of ginseng and

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its active ingredients (ginseng saponin). The representative secondary compound in ginseng species is triterpenoid saponin, so-called ginsenoside. The production of plant materials by cell culture is more advantageous because they are free from natural limitations, such as seasonal climate and geographical environment. The production of ginseng cells in culture was first reported by Furuya et al. (1983a). Callus was derived from the petiole of 2-year-old ginseng grown on MS agar medium (Murashige and Skoog 1962) containing 0.1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D). It was found that the saponin content in ginseng callus was less (about 0.1–1.0%) than in naturally cultivated ginseng roots (about 4.0%). For optimal biomass growth and saponin yield, the medium composition and culture environment were modified. Auxin treatment enhanced both the growth and saponin content of callus (Furuya et al. 1983a). Choi et al. (1982) reported that the most suitable concentration of 2,4-D for callus growth was in the range 1−5 mg l−1 , while a much lower concentration, e.g. 0.1 mg l−1 , was effective for saponin synthesis. In suspension cultures of P. quinquefolium, Zhong et al. (1996) demonstrated that not only the individual phytohormone concentration but also the combination of growth regulators showed significant influences on cell growth and ginsenoside accumulation. The ginsenoside content in cell or callus was lower than in naturally cultivated ginseng roots (Furuya et al. 1983a; Asaka et al. 1993a; Choi and Jeong 2003). Some attempts have been made to increase the saponin yield through metabolic regulation in ginseng tissue culture, i.e. with the use of elicitors and precursors of saponin synthesis. Furuya et al. (1983b) tested a number of intermediates in saponin biosynthesis. Precursor treatment, such as the use of mevalonate or farnesol, was effective for increasing saponin production (Furuya et al. 1983b). Furthermore, elicitor treatments, yeast extract and methyl jasmonate also significantly improved saponin production (Lu et al. 2001). The highest additive level of the seven ginsenosides tested was 2.07% (dry weight basis), which was 28-fold higher than that in the control. 2.2 Bioreactor Culture Two basic types of bioreactors have been used for plant cell suspension cultures, namely stirred-tanks and air-lifts. In the culture of ginseng cells using the turbine type bioreactor, a kind of stirred-tank type, the turbine type and turning speed have important effects on the growth of cells and saponin content (Furuya et al. 1984). In stirred-tank bioreactors, the agitator design and agitation rate are the major factors affecting cell growth and ginseng saponin production. Furuya et al. (1984) tested the effects of impellor design and agitation speeds on ginseng cell culture in a 30-l stirred-tank fermentor. Of the three different impellors tested (flat-blade, angled-blade disc turbine, anchor impeller), the angled-blade disc impeller resulted in the highest growth ratio. Asaka et al. (1993a) used an air-lift bioreactor and a stirred-tank fermentor

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with two different types of impellors, a flat-blade turbine and a paddle impeller, for the culturing of embryogenic tissues induced from ginseng leaves. They achieved >3-fold higher biomass growth ratio and a 2-fold higher saponin productivity in the air-lift bioreactor than in the stirred-tanks. 2.3 Hormone-Independent Callus Culture Synthetic auxin such as 2,4-D is possibly toxic to animals (Kaioumova et al. 2001). Thus, production of ginseng cells under growth regulator-free medium is optimistic. A brief high temperature treatment of in vitro-grown ginseng multiple shoots resulted in the formation of embryogenic callus on hormonefree medium (Asaka et al. 1993b), which was produced in large-scale bioreactor culture (Asaka et al. 1993a). Choi and Jeong (2003) induced hormoneindependent embryogenic callus from cotyledon explants of P. ginseng after treatment with a high concentration of NH4 NO3 . This callus was subsequently mass-produced in a bioreactor. The saponin yield in the embryonic tissue was found to be slightly lower than or the same as that in root callus or cell cultures. Methyl jasmonate elicitation enhanced the synthesis of ginsenoside by cell suspension cultures of P. ginseng in 5-I balloon-type bubble bioreactors (Thanh et al. 2005). 2.4 Commercialization of Cell Culture Nitto Denko Corporation (Ibaraki, Osaka, Japan) produced ginseng cells, using a 20-t tank culture (Furuya and Ushiyama 1994). After these ginseng materials were tested for food safety, the powder and extracts from the cell culture were used for health foods, drinks and cosmetics (Furuya and Ushiyama 1994). This was one of the first successful commercial applications for the production of plant materials using plant tissue culture.

3 Hairy Root Culture of P. ginseng 3.1 Induction of Hairy Roots Hairy roots are formed as a result of the transfer and integration of the genes in the T-DNA of the Ri plasmid of Agrobacterium rhizogenes (Chilton et al. 1982). Hairy roots can offer a valuable source of root-derived secondary metabolites that are useful as pharmaceuticals, cosmetics and food additives. Transformed roots of many plant species have been widely studied for the in vitro production of secondary metabolites (Shanks and Morgan 1999; Giri and Narasu 2000). Fine roots of native and field-cultivated ginseng plants contain 2-fold higher saponin than the tap roots (Kim et al. 1987; Yang and Yang 2000). A. rhizogenestransformed hairy roots synthesize the same component as do the roots of intact plants and root growth is fast in hormone-free medium. It is reported that

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hairy roots produce higher saponin than callus and cultured cells (Furuya et al. 1984; Yoshikawa and Furuya 1987). In view of this, the culture of hairy roots may be a better alternative than callus and cells for ginseng saponin production. The first attempt for transgenic P. ginseng hairy roots was reported by Yoshikawa and Furuya (1987). Mallol et al. (2001) observed that the ginsenoside pattern of transformed ginseng roots varied with their morphology. Woo et al. (2004) selected the hairy root lines producing 4- to 5-fold higher ginsenoside than other hairy roots. It was noted that some of the hairy root lines produced only a single ginsenoside in relatively high amounts. Enhanced production of ginsenosides in ginseng hairy roots was obtained by elicitation, using several elicitors such as salicylic acid, acetylsalicylic acid, yeast elicitor and bacterial elicitor (Jeong et al. 2005) and jasmonic acid (Yu et al. 2002). 3.2 Bioreactor Culture of Hairy Roots Various types of bioreactor systems were designed to enhance biosynthesis and the productivity. The stirred tank bioreactor was found to be unsuitable for the mass production of hairy roots because of strong shear stress. Instead, airlift, bubble column and liquid-dispersed bioreactors were adopted for hairy root culture because of the low shear stress and the simplicity of their design and construction. The growth of hairy roots in both air-bubble column and stirred bioreactor cultures was about 3-fold as high as in flask culture (Jeong et al. 2002), and growth was increased by 55-fold after 39 days in a 5-l bioreactor (Jeong et al. 2003). Palazon et al. (2003) reported that the wave bioreactor appeared to be the most efficient in promoting the growth of ginseng hairy roots. It is clear that hairy roots provide an efficient form of biomass production, owing to their fast growth. Yu et al. (2005) investigated the impact of temperature and light quality on biomass accumulation and ginsenoside production by hairy roots cultivated in large-scale bioreactors. Biomass accumulation and ginsenoside production were optimal under a temperature cycle of 20 ◦ C in the light (12 h) and 13 ◦ C in the dark (8 h). The biomass of hairy roots was highest in the cultures grown in the dark or under red light, while ginsenoside accumulation was optimum under fluorescent light. Hairy roots displayed high biosynthetic capabilities that were comparable with those of natural roots (Giri and Narasu 2000). However, hairy roots have not been exploited for health foods as they need further analysis for the food safety of proteins and compounds expressed by genes introduced on the T-DNA.

4 Adventitious Root Culture in P. ginseng 4.1 Induction of Adventitious Roots Recently, hairy-like adventitious roots were cultured without transformation by A. rhizogenes (Kevers et al. 1999; Choi et al. 2000). Induction and growth

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of adventitious roots were achieved from initial root explants by exogenous application of auxin. To induce adventitious roots, callus was firstly proliferated on MS medium containing auxin (2,4-D). During the subculture of callus on SH medium (Schenk and Hildebrandt 1972) containing indole-3butyric acid (IBA), adventitious roots were induced (Choi et al. 2000). Adventitious roots were then segmented and transferred to medium with IBA or α-naphthaleneacetic acid (NAA) to induce lateral root formation (Fig. 2A; Kim et al. 2004). It was noted that increase in adventitious root fresh weight was affected by the type and concentration of auxin. Amongst the auxins tested [2,4-D, NAA, indole-3-acetic acid (IAA), IBA], IBA was most effective for adventitious root induction from root segments (Choi et al. 2000). Low concentrations of NH+4 ion in the medium fostered the formation of adventitious lateral root-like structures (Kim et al. 2004). The accumulation of secondary metabolites in plants can also be triggered and activated in response to elicitors, the signal compounds of plant defense responses. This was demonstrated by treatment with jasmonic acid (10 mg l−1 ) or methyl jasmonate (100 μM), which resulted in a >5-fold increase in the total ginsenoside content without severe growth retardation of roots (Yu et al. 2002; Kim et al. 2004). 4.2 Commercialization of Adventitious Root Production Son et al. (1999) designed a balloon-type bubble bioreactor (BTBB), which is superior for biomass growth than the bubble column and stirred tank biore-

Fig. 2. Adventitous root culture of P. ginseng. A New lateral root induction from the segments of adventitious roots on half-strength MS medium with 3 mg l−1 IBA after 1 month of culture. Bar 1 cm. B Large-scale culture of adventitious roots in glass balloon-type bubble bioreactors. Bar 15 cm

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actors (Fig. 2B). The fresh weight of ginseng hairy-like adventitious roots cultured in a 20-l bioreactor was 3-fold higher than that in a stirred tank bioreactor. In a 20-l bioreactor, about 2.2 kg fresh weight was obtained after inoculation of 240 g of roots for 42 days. The pilot-scale stainless bioreactor (500 l) was designed according to the BTBB type and growth of ginseng roots was increased by 150-fold after 56 days of culture (Choi et al. 2000). Thus, the biomass increase in the adventitious root culture of P. ginseng appears to be comparable with that of hairy root culture. Based on the pilot-scale balloontype bioreactor, the production of ginseng roots via a 20,000-l bioreactor was attempted for commercial production. In Korea, several companies have been producing ginseng roots commercially using pilot-scale bioreactors (10,000 l to 20,000 l) and the basic design is similar to the balloon-type bubble bioreactor. The root materials produced using these methods are being processed into various health foods and food ingredients.

5 Plant Regeneration of P. ginseng via Organogenesis and Somatic Embryogenesis 5.1 Somatic Embryogenesis Plant regeneration from cultured cells and tissues is a valuable tool not only for the conservation of germplasm, but also for the rapid propagation of ginseng. Moreover, it is important for the production of transgenic plants using genetic engineering. Early attempts to induce organogenesis and somatic embryogenesis in P. ginseng were reported by Butenko et al. (1968) and Jhang et al. (1974). Somatic embryogenesis could be induced from callus derived from various explants such as roots (Butenko et al. 1968; Chang and Hsing 1980), leaves (Butenko et al. 1968), stems (Butenko et al. 1968), flower buds (Shoyama et al. 1988) and zygotic embryos or seedlings (Choi et al. 1998b). Lim et al. (1997) found that petioles from in vitro grown plantlets were best material for callus induction compared with other explants (leaf, flower stalk, root). However, zygotic embryos or young plants were better explants for the rapid production of embryogenic callus than old roots because the latter produced embryogenic callus at a very low frequency after several months (Chang and Hsing 1980). Somatic embryogenesis was obtained mainly from callus cultures (Chang and Hsing 1980; Shoyama et al. 1988; Arya et al. 1991; Tang 2000; Teng et al. 2002). Direct somatic embryogenesis occurred when zygotic embryos were used as explants (Choi and Soh 1994; Ahan et al. 1996). To induce embryogenic callus, MS medium with 2,4-D (1−2 mg l−1 ) alone or in combination with cytokinin (0.1−0.5 mg l−1 ) was employed (Chang and Hsing 1980; Lee et al. 1995; Ahan et al. 1996; Teng et al. 2002). The auxin requirement for somatic embryo induction from P. quinquefolius and P. notoginseng (Shaoyama et al. 1997) is similar

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to P. ginseng. Tang (2000) reported that the carbon source in the medium could also affect somatic embryogenesis in P. ginseng. 5.2 Induction of Somatic Embryos on Hormone-Free Medium Interestingly, excised segments of zygotic embryos of P. ginseng produce somatic embryos near to the excised portion of cotyledons or embryo axis on hormone-free MS-based medium (Choi and Soh 1994). On this medium, somatic embryogenesis and the origin and developmental pattern of somatic embryos can be influenced by the physiological stage of zygotic embryos. The production of somatic embryos is related to wound treatment (Choi and Soh 1996a). Plumules and radicles of zygotic embryos can contain a water-diffusible inhibitor for somatic embryo development. Thus, detachment of cotyledon explants from the embryo axis is more important for somatic embryo induction than wound and excision treatments (Choi and Soh 1996b). Somatic embryo development has been shown to be closely related with the polarity of explants, because embryos form only near the basal portion of excised or wounded explants (Choi and Soh 1996a). This has been demonstrated by treatment with 2,3,5-triiodobenzoic acid (TIBA), an auxin polar transport inhibitor that suppresses somatic embryo formation on the surface of cotyledons (Choi et al. 1997). The presence of macro-elements and high sucrose concentration in the medium also stimulate somatic embryo formation on hormone-free medium, where there is a positive relationship between somatic embryo induction from zygotic embryos and stress-induced loss of germination. This indicates that stress treatment is related to the loss of endogenous control of zygotic embryos (Choi et al. 1998a). The results of other studies show that temporary plasmolyzing pretreatment by 1.0 M sucrose or 1.0 M mannitol greatly enhances the frequency of single cell-derived somatic embryos and the number of embryos per explant (Choi and Soh 1997; Choi et al. 1999a). 5.3 Origin of Somatic Embryos Somatic embryos of ginseng originate from cell masses or single epidermal cells, which are dependent on the maturity of zygotic embryos (Choi and Soh 1994). In immature P. ginseng zygotic embryos, somatic embryos develop from multiple cells in epidermal and sub-epidermal areas. However, in fully matured zygotic embryos with differentiated epidermis and sub-epidermis, somatic embryos develop from single epidermal cells (Fig. 3; Choi and Soh 1994). A plasmolyzing pretreatment of zygotic embryos could greatly induce the formation of single cell-derived somatic embryos, regardless of the developmental stage of the embryo (Choi and Soh 1997; Choi et al. 1999a). It was assumed that factors influencing the origin of somatic embryos coincided with the coordinated behavior of cells participating in embryogenic development (William and Maheswaran 1986). Choi and Soh (1997) reasoned that enhanced single em-

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Fig. 3. Somatic embryo development from epidermal single cells of P. ginseng cotyledons grown on hormone-free medium. A Epidermal cells with dense cytoplasm. Bar 120 μm. B Cell division for somatic embryo development after 1 week of culture. Bar 120 μm. C Early globular embryos after 2 weeks of culture. Bar 150 μm. D Globular embryo after 3 weeks. Bar 120 μm. E Heart-shaped embryo after 1 month of culture. Bar 200 μm

bryo formation over the entire surface of cotyledon explants might be the result of an interruption of cell–cell interaction by plasmolysis pretreatment. As cell–cell interaction is controlled by the intercellular trafficking of signal molecules through plasmodesmata (Roberts and Oparka 2003), plasmolysis treatment might induce the deposition of callose, blocking the plasmodesmatal connections between cells (Radford et al. 1998; Hirano et al. 2004). 5.4 Plant Conversion Results from several lines of study show that plant regeneration is problematic from cultured cells and tissues of P. ginseng. Evidence for this is provided by the conversion of ginseng somatic embryos into multiple shoots without a root system (Butenko et al. 1968; Chang and Hsing 1980; Langhansová et al. 2004), or with inadequate or poor root formation (Kevers et al. 2002). A similar problem has been encountered in American ginseng (Wang 1990). Choi et al. (1998b) suggested that poor root formation might be associated with embryonic structural abnormalities, as observed in morphologically abnormal somatic embryos (multicotyledonary and multiple embryos; Butenko et al. 1968; Chang and Hsing 1980). Langhansová et al. (2004) described the improved regeneration of ginseng somatic embryos by treatments with abscisic acid (ABA) and polyethylene glycol (PEG). Choi et al. (1998b) reported that single cell-derived somatic embryos from cotyledons of fully mature zygotic embryos germinated into plantlets with both shoots and roots. In contrast, somatic embryos formed from cotyledons of immature zygotic embryos fused together with adjacent embryos. Thus these embryos were not converted into whole plantlets because

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they lacked root formation. Therefore, a single cell origin of somatic embryos is important for the production functional plantlets. During germination and plant conversion, full-strength MS medium was found to be unsuitable because root development was not equal to shoot growth (Choi et al. 1998b). This was overcome by lowering or omitting NH4 NO3 that stimulated the root growth of plantlets. Ginseng somatic embryos possessed physiological dormancy after maturing to the cotyledonary stage. GA3 treatment is therefore needed to stimulate germination and plant growth (Choi et al. 1999a). Choi et al. (1998b) attempted field transfer of plantlets. Acclimatization of ginseng plantlets is not difficult, but the plantlets are sensitive to fungus contamination from the air (Choi et al. 1998b). Thus, special management of somatic embryo-derived plantlets to protect against fungus attack is required for successful field transfer. 5.5 Plant Regeneration via Adventitious Shoot Formation In addition to somatic embryogenesis, adventitious buds can be induced by the treatment of cotyledons with cytokinin, which was shown to suppress somatic embryo formation but stimulated direct formation of adventitious buds (Choi et al. 1998c). The presence of IBA in combination with cytokinin enhanced adventitious bud formation, with the highest frequency (40%) at 0.05 mg l−1 IBA and 5.0 mg l−1 BA. Adventitious buds were formed mainly near the distal portion of the cotyledons, while somatic embryos formed near the proximal excised margins. In germinating zygotic embryos after 1 week of culture, excised cotyledons on medium with 0.2 mg l−1 IBA and 2.0 mg l−1 BA formed embryo axis-like shoot and roots, whose morphological and developmental characters were different from those of somatic embryos and adventitious bud development (Choi et al. 1999b). It was suggested that the embryo axis-like structure might represent a regeneration of new an embryo axis (Choi et al. 1999b).

6 Genetic Transformation and Metabolic Engineering 6.1 Genetic Transformation The first attempt at P. ginseng transformation was reported using A. rhizogenes to induce hairy roots (Yoshikawa and Furuya 1987). Several years later, Lee et al. (1995) reported the first successful production of transgenic ginseng plants expressing the β-glucuronidase (GUS) gene by cocultivation of cotyledon explants with A. tumefaciens LBA4404. Transgenic plants were obtained by somatic embryo-derived plant regeneration. Yang and Choi (2000) reported the production of transgenic ginseng plants from hairy roots via somatic embryogenesis using A. rhizogenes-mediated transformation. Transgenic plants possessed actively growing root systems with characteristics similar to those of hairy roots.

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Fig. 4. Enhancement of transient expression of GUS gene by plasmolyzing pretreatment of cotyledon explants. A Unplasmolyzed control. Bar 700 μm. B Plasmolyzed cotyledon pretreated with 0.5 M sucrose for 24 h. Bar 700 μm. C: Plasmolyzed cotyledon pretreated with 0.5 M sucrose for 24 h. Bar 700 μm

A rapid and efficient genetic transformation of P. ginseng was reported by plasmolyzing pretreatment of cotyledon explants (Choi et al. 2001). When cotyledon explants of P. ginseng were pretreated with 1.0 M sucrose, transient expression of the β-glucuronidase (GUS) gene was strongly enhanced following cocultivation with A. tumefaciens harboring the GUS gene (Fig. 4). This enhanced expression coincided with a high frequency of stable transformation (three times greater than non-treatment). The transgenic somatic embryos formed directly on the surface of cotyledons and developed into plantlets (Choi et al. 2001). Approximately 3 months were required to obtain small transgenic ginseng plantlets. Chen and Punza (2002) produced transgenic American ginseng (P. quinquefolius) with A. tumefaciens LBA4404 carrying a rice chitinase gene, under the control of the maize ubiquitin1 promoter, and the phosphinothricin acetyltransferase (bar) gene. In this study, epicotyl explants were pre-cultured for 5–7 days on MS-based medium with NAA and 2,4-D before Agrobacterium infection. A rice chitinase gene was previously shown to enhance resistance to sheath blight (Lin et al. 1995). Herbicide-resistant transgenic P. ginseng plants were also produced by introducing the phosphinothricin acetyl transferase (PAT) gene that confers resistance to the herbicide Basta (bialaphos) through A. tumefaciens-mediated transformation (Choi et al. 2003). Temporary pretreatment of embryogenic callus with 0.5 M sucrose or 0.05 M MgSO4 markedly enhanced transient expression of the β-glucuronidase (gus) gene. Transgenic plants growing in soil were observed to be strongly resistant to Basta application. 6.2 Metabolism and Ginsenoside Biosynthesis The representative secondary compound accumulated in roots of ginseng species is ginsenoside, a triterpenoid saponin. Ginsenosides are considered to be the main bioactive compounds derived from the roots and rhizomes of different Panax species (Araliaceae). Triterpenoids exhibit a wide range of struc-

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tural diversity and biological activity, and these saponins are of economical importance as drugs, detergents, sweeteners and cosmetics (Hostettmann and Marston 1995). In Panax, more than 30 different ginsenosides have been identified (Shibata 2001). The content of each ginsenoside differs with the species. Roots of P. ginseng contain at least seven different triterpenoid saponins (about 5%, namely ginsenosides of dry weight; Shibata 2001) and various phytosterols (Matsumoto et al. 1986). The main active components of P. ginseng are a mixture of triterpenoid saponins referred to as ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf and Rg1. Each ginsenoside has been shown to possess different pharmacological effects, including immune system modulation, antistress activities, and antihyperglycemic activities, anti-inflammatory, antioxidant and anticancer effects (Shibata 2001; Kiefer and Pantuso 2003). Both tetracyclic dammareneand pentacyclic olanane-type triterpene saponins are produced in P. ginseng roots and most triterpenes are of the dammarene-type (Kushiro et al. 1997). The occurrence of a dammarene-type triterpene as a major compound is confined to only a few species, such as P. ginseng (Kushiro et al. 1997) and Gynostemma pentaphyllum (Cui et al. 1999). Triterpene and sterol biosynthesis has a common pathway with C5 isoprenoids. Mevalonate is the preferential precursor for sterol and triterpene biosynthesis (Kuzuyama 2002). The enzyme squalene synthase catalyzes the first step from the central isoprenoid pathway towards sterol and triterpenoid biosynthesis (Abe et al. 1993). Both phytosterols and triterpenes in plants are synthesized from the product of cyclization of 2,3-oxidosqualene, as shown in Fig. 5. The step in ginsenoside synthesis involves cyclization of 2,3-oxidosqualene to oleanane and a dammarene-type triterpene skeleton. Kushiro et al. (1998) isolated several genes encoding oxidosqualene cylase and these genes were shown to be highly homologous with each other. Enzymes at the later step of ginsenoside biosynthesis are cytochrome P450s and glycosyltransferases. These enzymes exist as supergene families in the plant genome. In the ginsenoside biosynthetic pathway, a cytochrome P450 member is involved in hydroxylation of the C-6 position of protopanaxadiol, resulting in protopanaxatriol, with both the latter compounds being used as backbones for ginsenosides. Biosynthesis of ginsenosides from triterpene aglycone involves glycosylation at the C-3 and C-20 hydroxyl positions on the skeleton for the protopanaxadiol type and at the C-6 and C-20 positions for the protopanaxatriol type ginsenoside. Using keyword and domain searches, Choi et al. (2005) identified many candidates for cytochrome P450 and glycosyltransferase candidate genes. 6.3 Metabolic Engineering The genes for biochemical pathways involved in saponin biosynthesis are of considerable interest in the area of ginseng biotechnology. A more detailed understanding of genes involved in saponin biosynthesis would facilitate the

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Fig. 5. The biosynthetic pathway of triterpenes and phytosterols in P. ginseng

development of plants with altered or novel saponin content by genetic engineering. Lee et al. (2004) investigated the roles of squalene synthase (PgSS1) protein on the biosynthesis of phytosterols and triterpenoids. Over-expression of the PgSS1 gene in adventitious roots of transgenic P. ginseng resulted in the up-regulation of the downstream genes, such as squalene epoxidase, β-amyrin synthase and cycloartenol synthase. Transgenic P. ginseng also exhibited a remarkable increase in the production of phytosterol (β-sitosterol, stigmasterol, campesterol) and triterpene saponins (ginsenosides).

7 Genomics in P. ginseng 7.1 BAC Library The chromosome number of P. ginseng (2n = 4x = 48) was reported by Han and Whang (1963). The 1C nuclear DNA content of P. ginseng was estimated to be 3.33 pg (3.12 × 103 Mbp) using flow cytometry. The haploid DNA contents of the two model plant species, Arabidopsis thaliana and rice, are 145 Mb and 420 Mb, respectively (Arumuganathan and Earle 1991). Therefore, the genome size of P. ginseng is 21.5 times that of Arabidopsis and 7.43 times that of rice. Hong et al. (2004) reported that the BAC library consisted of 106,368 clones with an average size of 98.61 kb, amounting to 3.34 genome equivalents.

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Sequencing of 2,167 BAC clones generated 2,492 BAC-end sequences with an average length of 400 bp. Analysis using BLAST and motif searches revealed that 10.2, 20.9 and 3.8% of the BAC-end sequences contained protein-coding regions, transposable elements and microsatellites, respectively. The BAC-end sequences have been submitted to the dbGSS of GenBank (Accession Nos. BZ956677–BZ959168). 7.2 Gene Discovery Involved in Ginsenoside Biosynthesis Recently, large-scale gene isolation from P. ginseng has been carried out in Korea. Expressed sequence tags (ESTs) provide a valuable tool to identify the genes in P. ginseng. Approximately 26,000 ESTs of P. ginseng sequences are available at http://plant.pdrc.re.kr:7777/index.html. Eight cDNA libraries for EST sequencing were constructed from different organs, including the taproot, rhizome, developing seed, in vitro-grown seedlings and soil-grown seedling shoots. Jung et al. (2003) sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides. Only 59% of the ginseng ESTs exhibited significant homology to previously known polypeptide sequences. Stress- and pathogen-response proteins were most abundant in 4-year-old ginseng roots. They identified four oxidosqualene cyclase candidates involved in the cyclization reaction of 2,3-oxidosqualene, nine cytochrome P450 and 12 glycosyltransferse candidates, which may be involved in modification of the triterpene backbone. Methyl jasmonate (MeJA) treatment can increase the concentrations of plant secondary metabolites, including ginsenosides. To create a ginseng gene resource that contains the genes for the biosynthesis of secondary metabolites, including ginsenosides, Choi et al. (2005) generated 3,134 ESTs from MeJAtreated ginseng hairy roots. All ESTs have been submitted to the dbEST and GenBank databases (Accession Nos. CN845540–CN848674). These ESTs were assembled into 370 clusters and 1,680 singletons. Genes that showed high expression were those encoding proteins involved in fatty acid desaturation, the defense response and secondary metabolite biosynthesis. Further analysis revealed that a number of genes, including those encoding oxidosqualene cyclase (OSC), cytochrome P450 and glycosyltransferase, may be involved in ginsenoside biosynthesis. Xu et al. (2005) reported that fungal elicitor induced singlet oxygen generation, subsequently leading to ethylene release and increased saponin synthesis. This was shown by increased mRNA expression of squalene synthase and squalene epoxidase and by accumulation of β-amyrin synthase in cultured cells of P. ginseng C.A. Meyer. The first committed step in ginsenoside synthesis is the cyclization of 2,3-oxidosqualene to dammarenediol II by the oxidosqualene cyclase (dammarenediol synthase). The gene encoding dammarenediol synthase was characterized by Han et al. (2006), who reported that ectopic expression of the dammarenediol synthase gene (DDS) in a yeast mutant (erg7) lacking lanosterol synthase resulted in the pro-

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duction of dammarenediol and hydroxydammarenone. This was confirmed by LC/APCIMS. RNA interference (RNAi) of DDS in transgenic P. ginseng resulted in silencing of DDS expression, which lead to a reduction of ginsenosides production of up to 84.5% in roots. 7.3 Proteomic Studies Involved in the Photoinhibition of P. ginseng The ability of P. ginseng to adapt to light intensity is generally restricted to a 5–20% range of full sunlight. Exposure to excess light greater than 500 μmol photons m−2 s−2 resulted in photoinhibitory symptoms, including decreased photosynthesis, increased photorespiration and chlorosis. Miskell et al. (2002) suggested that ginseng thylakoid membranes possessed relatively more inactive photosynthesis system II (PSII) centers than thylakoids of pea and spinach when grown under similar conditions. It was thought that the susceptibility of P. quinquefolius to photoinhibition might arise as a consequence of a reduced fraction of active PSII centers. This might result in the normal dissipative mechanisms in these plants becoming saturated at elevated, but moderate, light intensities. Nam et al. (2003) performed comparative proteomic analyses in order to understand the physiological responses of P. ginseng to high light. They analyzed the proteins expressed in ginseng leaves. Proteins extracted from leaves after 0–4 h of light exposure were separated by two-dimensional polyacrylamide gel electrophoresis and six light-responsive proteins were identified. These proteins included three cytosolic small heatshock proteins, cytosolic ascorbate peroxidase and putative major latex-like protein that were up-regulated by light. The other three (Rieske Fe/S protein, putative 3-beta hydroxysteroid dehydrogenase/isomerase-like protein, oxygen-evolving enhancer-like protein) were down-regulated. 7.4 Whole Genome Sequence of Chloroplast DNA Kim and Lee (2004) completed the sequencing of the whole chloroplast genome from P. ginseng. The circular double-stranded DNA consists of 156,318 bp, with the genome content and the relative positions of 114 genes (75 peptideencoding genes, 30 tRNA genes, four rRNA genes, five conserved open reading frames). A total of 18 simple sequence repeats have been identified from the chloroplast genome of P. ginseng.

8 Concluding Remarks To date, cells and adventitious roots of P. ginseng have been produced in a pilot-scale bioreactor and this technology has been applied to the commercial production of plant materials using plant tissue culture. Recent advances in

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plant transformation, large-scale EST analysis and the proteomics of ginseng will provide the information necessary to establish a better understanding of the physiological and genetic characters of the plants in this genus. The saponin-regulating metabolic engineering and the production of transgenic ginseng plants resistant to high light and fungus might be promising areas of ginseng biotechnology. Acknowledgements. This work was supported by a grant from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.

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Choi YE, Soh WY (1996b) Effect of plumule and radicle on somatic embryogenesis in the cultures of ginseng zygotic embryos. Plant Cell Tissue Organ Cult 45:137–143 Choi YE, Soh WY (1997) Enhanced somatic single embryo formation by plasmolyzing pretreatment from cultured ginseng cotyledons. Plant Sci 130:197–206 Choi YE, Kim HS, Yang DC, Soh WY (1997) Developmental and structural aspects of somatic embryos formed on medium containing 2,3,5-triiodobenzoic acid. Plant Cell Rep 16:738–744 Choi YE, Yang DC, Choi KT (1998a) Induction of somatic embryos by macrosalt stress from mature zygotic embryos of Panax ginseng. Plant Cell Tissue Organ Cult 52:177–182 Choi YE, Yang DC, Park JC, Soh WY, Choi KT (1998b) Regenerative ability of somatic single and multiple embryos from cotyledons of Korean ginseng on hormone-free medium. Plant Cell Rep 17:544–551 Choi YE, Yang DC, Yoon ES, Choi KT (1998c) Plant regeneration via adventitious bud formation from cotyledon explants of Panax ginseng C.A. Meyer. Plant Cell Rep 17:731–736 Choi YE, Yang DC, Yoon ES, Choi KT (1999a) High efficiency plant production via direct somatic single embryogenesis from pre-plasmolysed cotyledons of Panax ginseng and possible dormancy of somatic embryos. Plant Cell Rep 18:493–499 Choi YE, Yang DC, Yoon ES, Choi KT (1999b) Plant regeneration via direct embryo axis-like shoot and root formation from excised cotyledon explants of ginseng seedlings. In Vitro Cell Dev Biol Plant 35:210–213 Choi YE, Yang DC, Kusano T, Sano H (2001) Rapid and efficient Agrobacterium-mediated genetic transformation by plasmolyzing pretreatment of cotyledons in Panax ginseng. Plant Cell Rep 20:616–621 Choi YE, Jeong JH, In JK, Yang DC (2003) Production of herbicide-resistant transgenic Panax ginseng through the introduction of phosphinotricin acetyl transferase gene and successful soil transfer. Plant Cell Rep 21:563–568 Coleman CI, Hebert JH, Reddy P (2003) The effects of Panax ginseng on quality of life. J Clin Pharm Ther 28:5–15 Cui JF, Eneroth P, Bruhn JC (1999) Gynostemma pentaphyllum: identification of major sapogenins and differentiation from Panax species. Eur J Pharm Sci 8:187–191 Dey L, Xie JT, Wang A, Wu J, Maleckar SA, Yuan CS (2003) Anti-hyperglycemic effects of ginseng: comparison between root and berry. Phytomedicine 10:600–605 Ellis JM, Reddy P (2002) Effects of Panax ginseng on quality of life. Ann Pharmacother 36:375–379 Furuya T, Ushiyama K (1994) Ginseng production in cultures of Panax ginseng cells. In: Shargool P, Ngo TT (eds) Biotechnological application of plant cultures. CRC, Boca Raton, pp 1–22 Furuya T, Yoshikawa T, Orihara Y, Oda H (1983a) Saponin production in cell suspension cultures of Panax ginseng. Planta Med 48:83–87 Furuya T, Yoshikawa T, Ishii T, Kajii K (1983b) Regulation of saponin production in callus cultures of Panax ginseng. Plant Med 47:200–204 Furuya T, Yoshikawa T, Orihara Y, Oda H (1984) Studies of the culture conditions for Panax ginseng cells in jar fermentors. J Nat Prod 47:70–75 Giri A, Narasu ML (2000) Transgenic hairy roots: recent trends and applications. Biotechnol Adv 18:1–22 Han C, Whang J (1963) Development of female gametophyte of Panax ginseng. Kor J Bot 6:3–6 Han JY, Kwon YS, Yang DC, Jung YR, Choi YE (2006) Expression and RNA interference-induced silencing of the dammarenediol synthase gene in Panax ginseng. Plant Cell Physiol 47:1653– 1662 Hirano Y, Pannatier EG, Zimmermann S, Brunner I (2004) Induction of callose in roots of Norway spruce seedlings after short-term exposure to aluminum. Tree Physiol 24:1279–1283 Hong CP, Lee SJ, Park JY, Plaha P, Park YS, Lee YK, Choi JE, Kim KY, Lee JH, Lee J, Jin H, Choi SR, Lim YP (2004) Construction of a BAC library of Korean ginseng and initial analysis of BAC-end sequences. Mol Genet Genomics 271:709–716 Hostettmann K, Marston A (1995) Saponins. Cambridge University Press, Cambridge Jeong GT, Park DH, Hwang B, Park K, Kim SW, Woo JC (2002) Studies on mass production of transformed Panax ginseng hairy roots in bioreactor. Appl Biochem Biotechnol 98:1115–1127

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Jeong GT, Park DH, Hwang B, Woo JC (2003) Comparison of growth characteristics of Panax ginseng hairy roots in various bioreactors. Appl Biochem Biotechnol 107:493–503 Jeong GT, Park DH, Ryu HW, Hwang B, Woo JC, Doman KF, Kim SW (2005) Production of antioxidant compounds by culture of Panax ginseng CA Meyer hairy roots I. Enhanced production of secondary metabolite in hairy root cultures by elicitation. Appl Biochem Biotechnol 121:1147–1157 Jhang JJ, Staba EJ, Kim JU (1974) American and Korean ginseng tissue cultures: growth, chemical analysis and plant production. In Vitro 9:253–259 Jung JD, Park HW, Hahn Y, Hur CG, In DS, Chung HJ, Liu JR, Choi DW (2003) Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags. Plant Cell Rep 22:224–230 Kaioumova D, Kaioumov F, Opelz G, Susal C (2001) Toxic effects of the herbicied 2,4dichlorophenoxyacetic acid on lymphoid organs of the rat. Chemosphere 43:801–805 Kevers C, Jacques P, Thonart P, Gaspar T (1999) In vitro root cultures of Panax ginseng and P. quinquefolium. Plant Growth Regul 27:173–178 Kevers C, Gaspar T, Doommes J (2002) The beneficial role of different auxins and polyamines at successive stages of somatic embryo formation and development of Panax ginseng in vitro. Plant Cell Tissue Organ Cult 70:181–188 Kiefer D, Pantuso T (2003) Panax ginseng. Am Fam Physician 68:1539–1542 Kim KJ, Lee HL (2004) Complete chloroplast genome sequences from Korean ginseng (Panax schinseng Nees) and comparative analysis of sequence evolution among 17 vascular plants. DNA Res 11:247–261 Kim MW, Ko SR, Choi KJ, Kim SC (1987) Distribution of saponin in various sections of Panax ginseng root and changes of its contents according to root age. Kor J Ginseng Sci 11:10–16 Kim YS, Hahn EJ, Murthy HN, Paek KY (2004) Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate. Biotechnol Lett 26:1619–1622 Kushiro T, Ohno Y, Shibuya M, Ebizuka Y (1997) In vitro conversion of 2.3-oxidosqualene into mammarenediol by Panax ginseng microsome. Biol Pharm Bull 20:292–294 Kushiro T, Shibuya M, Ebizuka Y (1998) β-Amyrin synthase: cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants. Eur J Biochem 256:238–244 Kuzuyama T (2002) Mevalonate and nonmevalonate pathways for the biosynthesis of isoprene units. Biosci Biotechnol Biochem 66:1619–1627 Kwon WS, Lee MK, Choi KT (2000) Breeding process and characteristics of Yunpoong, a new variety of Panax ginseng C.A. Meyer. J Ginseng Res 24:1226–8453 Langhansová L, Konrádová H, Vanìk T (2004) Polyethylene glycol and abscisic acid improve maturation and regeneration of Panax ginseng somatic embryos. Plant Cell Rep 22:725–730 Lee HS, Kim SW, Lee KW, Eriksson T, Liu JR (1995) Agrobacterium-mediated transformation of ginseng (Panax ginseng) and mitotic stability of the inserted beta-glucuronidase gene in regenerants from isolated protoplasts. Plant Cell Rep 14:545–549 Lee MH, Jeong JH, Seo JW, Shin CG, Kim YS, In JG, Yang DC, Yi JS, Choi YE (2004) Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene. Plant Cell Physiol 45:976–984 Lim HT, Lee HS, Eriksson T (1997) Regeneration of Panax ginseng C.A. Meyer by organogenesis and nuclear DNA analysis of regenerants. Plant Cell Tissue Organ Cult 49:179–187 Lin W, Anuratha CS, Datta K, Potrykus I, Muthukrishnan S, Datta SK (1995) Genetic engineering of rice for resistance to sheath blight. Bio/Technology 13:686–691 Lu MB, Wong HL, Teng WL (2001) Effects of elicitation on the production of saponin in cell culture of Panax ginseng. Plant Cell Rep 20:674–677 Mallol A, Cusido RM, Palazon J, Bonfill M, Morales C, Pinol MT (2001) Ginsenoside production in different phenotypes of Panax ginseng transformed roots. Phytochemistry 57:365–371

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Matsumoto T, Akihisa T, Soma S, Takido M, Takahashi S (1986) Composition of unsaponifiable lipid from seed oils of Panax ginseng and Panax quinquefolium. J Am Oil Chem Soc 63:544– 546 Miskell JA, Parmenter G, Eaton-Rye JJ (2002) Decreased Hill reaction rates and slow turnover of transitory starch in the obligate shade plant Panax quinquefolius L. (American ginseng). Planta 215:969–979 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue. Physiol Plant 15:473–497 Nam MH, Heo EJ, Kim JY, Kim IIS, Kwon KH, Seo JB, Kwon O, Yoo JS, Park YM (2003) Proteome analysis of the responses of Panax ginseng C.A. Meyer leaves to high light: use of electrospray ionization quadrupole–time of flight mass spectrometry and expressed sequence tag data. Proteomics 3:2351–2367 Palazon J, Mallol A, Eibl R, Lettenbauer C, Cusido RM, Pinol MT (2003) Growth and ginsenoside production in hairy root cultures of Panax ginseng using a novel bioreactor. Planta Med 69:344–349 Radford JE, Vesk M, Overall RL (1998) Callose deposition at plasmodesmata. Protoplasma 201:30–37 Roberts AG, Oparka K (2003) Plasmodesmata and the control of symplastic transfort. Plant Cell Environ 26:103–124 Schenk RU, Hildebrandt AC (1972) Medium and techniques for induction of growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199–204 Shanks JV, Morgan J (1999) Plant ‘hairy root’ culture. Curr Opin Biotechnol 10:151–155 Shibata S (2001) Chemistry and cancer preventing activities of ginseng saponins and some related triterpenoid compounds. J Kor Med Sci 16[Suppl]:S28–S37 Shoyama Y, Kamura K, Nishioka I (1988) Somatic embryogenesis and clonal multiplication of Panax ginseng. Planta Med 54:155–156 Shoyama Y, Zhu XX, Nakai R, Shiraishi S, Kohda H (1997) Micropropagation of Panax notoginseng by somatic embryogenesis and RAPD analysis of regenerated plantlets. Plant Cell Rep 16:450– 453 Son SH, Choi SM, Soo JH, Yun SR, Choi MS, Shin EM, Hong YP (1999) Induction and cultures of mountain ginseng adventitious roots and AFLP analysis for identifying mountain ginseng. Biotechnol Bioprocess Eng 4:119–123 Tang W (2000) High-frequency plant regeneration via somatic embryogenesis and organogenesis and in vitro flowering of regenerated plantlets in Panax ginseng. Plant Cell Rep 19:727–732 Teng WL, Sin T, Teng MC (2002) Explant preparation affects culture initiation and regeneration of Panax ginseng and Panax quinquefolius. Plant Cell Tissue Organ Cult 68:233–239 Thanh NT, Murthy HN, Yu KW, Hahn EJ, Paek KY (2005) Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of Panax ginseng in 5-I balloon type bubble bioreactors. Appl Microbiol Biotechnol 67:197–201 Vogler BK, Pittler MH, Ernst E (1999) The efficacy of ginseng. A systematic review of randomized clinical trials. Eur J Clin Pharmacol 55:567–575 Wang AS (1990) Callus induction and plant regeneration of American ginseng. HortScience 25:571–572 Wen J, Zimmer EA (1996) Phylogeny and biogeography of Panax L. (the ginseng genus, Araliaceae): inferences from ITS sequences of nuclear ribosomal DNA. Mol Phylogenet Evol 6:167–177 William EG, Meheswaran G (1986) Somatic embryogenesis: factors influencing coordinated behavior of cells as an embryogenic group. Ann Bot 57:443–462 Woo SS, Song JS, Lee JY, In DS, Chung HJ, Liu JR, Choi DW (2004) Selection of high ginsenoside producing ginseng hairy root lines using targeted metabolic analysis. Phytochemistry 65:2751–2761 Xu X, Hu X, Neill SJ, Fang J, Cai W (2005) Fungal elicitor induces singlet oxygen generation, ethylene release and saponin synthesis in cultured cells of Panax ginseng C.A. Meyer. Plant Cell Physiol 46:947–954

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Yang DC, Choi YE (2000) Production of transgenic plants via Agrobacterium rhizogenes-mediated transformation of Panax ginseng. Plant Cell Rep 19:491–496 Yang DC, Yang KJ (2000) Pattern and contents of ginsenoside in normal root parts and hairy root lines of Panax ginseng C.A. Meyer. Kor J Plant Tissue Cult 27:485–489 Yoshikawa T, Furuya T (1987) Saponin production by cultures of Panax ginseng transformed with Agrobacterium rhizogens. Plant Cell Rep 6:449–453 Yu KW, Gao W, Hahn EJ, Paek KY (2002) Jasmonic acid improves ginsenoside accumulation in adventitious root culture of Panax ginseng C.A. Meyer. Biochem Eng J 3596:1–5 Yu KW, Murthy HN, Hahn EJ, Paek KY (2005) Ginsenoside production by hairy root cultures of Panax ginseng: influence of temperature and light quality. Biochem Eng J 23:53–56 Yun TK (2001) Panax ginseng – a non-organ-specific cancer preventive? Lancet Oncol 2:49–55 Zhong JJ, Bai Y, Wang SJ (1996) Effects of plant growth regulators on cell growth and ginsenoside saponin production by suspension cultures of Panax quinquefolium. J Biotechnol 45:227–234 Zhu S, Zou K, Fushimi H, Cai S, Komatsu K (2004) Comparative study on triterpene saponins of ginseng drugs. Planta Med 70:666–677

II.2 Opium Poppy J.M. Hagel, B.P. Macleod, and P.J. Facchini1

1 Introduction The beginning of humankind’s relationship with opium poppy (Papaver somniferum L.) predates recorded history. Archeological evidence suggests that opium poppy was amongst the first domesticated plant species and one of the first sources of medicine. Today, opium poppy remains the only source for the analgesic and antitussive drugs morphine and codeine, in addition to a number of other benzylisoquinoline alkaloids of pharmaceutical significance, such as the muscle relaxants papaverine and noscapine. Thebaine, another natural product of opium poppy, is used as a starting material for the production of oxycodone and other semi-synthetic opiates. Naturally occurring alkaloids are found in the latex of the plant, and extraction of this material involves the lancing of unripe seed capsules to allow the collection of exuded sap as raw opium. Although this traditional method of collection is still used, opium alkaloids are now primarily extracted from dried poppy straw. The process of harvesting the straw is highly mechanized, relatively inexpensive on a long-term basis, and improves the regulation of opium poppy agriculture. The latter benefit is significant since illicit opium poppy cultivation for the manufacture of heroin is a global problem. The early domestication and extensive breeding of opium poppy over thousands of years have no doubt guided the evolution of certain agro-morphological traits. Although classic breeding programs are still in place, the available gene pool for the generation of novel, commercial varieties is relatively small. A current goal is the development of hybrid species exhibiting heterosis in terms of vigor and alkaloid yield. While the industrial breeding of opium poppy presents numerous challenges, our understanding of the plant at the molecular level has accelerated in the past decade. The enzymatic synthesis of morphine has been almost completely elucidated, many of the encoding genes have been cloned, and the corresponding recombinant enzymes have been characterized. Benzylisoquinoline alkaloid biosynthetic enzymes have been localized at the cellular and subcellular levels. Recently, advanced technologies, including genomics, DNA microarrays, and proteomics, have been applied to opium poppy. Future prospects include the development of new 1 Department

of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada, e-mail: [email protected]

Biotechnology in Agriculture and Forestry, Vol. 61 Transgenic Crops VI (ed. by E.C. Pua and M.R. Davey) © Springer-Verlag Berlin Heidelberg 2007

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opium poppy varieties exhibiting altered metabolic profiles and/or desirable agronomic characteristics. Potentially, such technologies could facilitate the control of illicit opium poppy cultivation.

2 Origins and History Papaver somniferum is one of the few plant species that was cultivated in prehistoric times. As a result, its origins remain uncertain, although various theories have been proposed. Until the 1930s, Papaver setigerum D.C. was accepted as the immediate predecessor of opium poppy, and based on the native distribution of this species, the Mediterranean region was proposed as its original geographical location. However, this idea was disputed by cytological comparisons of P. setigerum and cultivated opium poppy (Hrishi 1959). The findings of various botanical expeditions suggested the existence of distinguishable gene centers for the two species. It is now generally agreed that the gene center for opium poppy lies in the Middle Asian territories of modern-day India, Iran, and Afghanistan (Simmonds 1976). Preserved seeds and capsules of opium poppy have been found at Neolithic sites in Spain, France, Germany, and Hungary. Early-Neolithic lakeside dwellings in Switzerland also provide a wealth of archeological data, showing a knowledge and cultivation of opium poppy in Europe at least 6,000–7,000 years ago. These ancient farmers also grew linseed and it is possible that both crops were utilized for their oil (Booth 1996). Around 5,400 years ago, opium poppy was being cultivated in the Tigris– Euphrates river systems of lower Mesopotamia. The Sumerians referred to the plant as hul gil – literally “joy plant” – and produced the first written records of opium poppy. The earliest evidence of opium itself comes from ancient Egypt (ca. 3,500 years ago) where a sample was discovered in the tomb of Cha. During this period, the Egyptian city of Thebes was so famous for its poppy fields that Egyptian opium was known as Thebic opium, from which the alkaloid thebaine derives its name. Opium was also common in Greek civilization. In the third century before the common era (BCE), Theophrastus referred to the sap of the capsule as opion, from the Greek opos, meaning juice, and poppy juice as meconion; and in the Corinth region a city was named Mekone, or “Poppy Town”. In the Odyssey, Homer writes of nepenthe, the opium-based drug of forgetfulness, which was employed by Helen, the daughter of Zeus, to soothe mourning warriors. Hippocrates (460–377 BCE) was an early advocate of the medicinal properties of opium poppy, and prescribed preparations of the plant as a hypnotic, narcotic, styptic, and cathartic. He also acknowledged the nutritive properties of the seeds. By the end of the second millennium BCE, knowledge of opium was widespread throughout Europe, North Africa, and the Middle East. References to opium poppy and its preparations appear in the Talmud and the Bible. Roman culture incorporated Greek knowledge of opium poppy along with its

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associated folklore. The routine supplying of opium to Roman soldiers and the extensive growth of the Roman Empire facilitated the widespread cultivation of the plant for food, medicine, and oil. Historically, the direction of opium poppy agriculture varied between regions, with latex-derived, sun-baked opium as the most valued product in Asia, and seeds and seed oil the major commodities in Europe. The history of the introduction of opium poppy to India is not clear, although it is argued that cultivation of the plant began soon after the invasion of Alexander the Great in the fourth century BCE. In the Golden Triangle of Southeast Asia, the Golden Crescent of western Asia, and throughout India opium production continues to depend on methods that have been used for many thousands of years. Today, about 45% of the world’s morphine production depends on traditional methods of opium harvesting (Bryant 1988). For example, 800−1,000 t of licit opium are produced annually in India. However, this figure represents only 5% of the estimated total “black-market” opium largely destined for the illicit production of heroin. Opium production in all parts of the world is tied to local consumption, market considerations, and political climates. The influence of politics was made clear during the first (1838–1842) and second (1856–1860) “opium wars” between Great Britain and China. Currently, international organizations such as the WHO, FAO, and UNODC are mandated to gain control of opium production to reduce illicit trade and consumption. In Europe, the end of the eighteenth century and the first half of the nineteenth century saw the production of large amounts of opium poppy seed oil for food and industrial applications, especially in the regions of Provence, Alsace, and certain German states. However, after this period the use of opium poppy oil was largely replaced by a variety of other plant-derived oils. The purification and isolation of morphine began at small pharmaceutical companies in Europe during the nineteenth century, using opium imported from Turkey and Persia. In the United Kingdom, Macfarlan and Smith (est. 1837) was one of the first companies to specialize in opium processing. As a result of the medical and economical importance of opium products, several other companies soon followed, such as Francopia (est. 1847) in France and Mallinckrodt (est. 1898) in the United States.

3 Modern Cultivation In general, opium poppy straw cultivation is carried out in countries with temperate climates, whereas countries in tropical zones harvest opium. Two main approaches to poppy cultivation are used in temperate zones, which are referred to as “winter” (using autumn-sown seed) and “spring” (using springsown seed) methods. Since the over-wintering of plants cannot be ensured in all regions, spring poppy cultivation is more common in temperate zone

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areas, such as central Europe (Németh 1998). In general, poppies require fertile, free-draining soil which is not overly acidic. Agents such as nitrofene and chlortoluron are frequently used to control weeds, while aminophos-methyl, furathiocarb, dimethoate, diazinon or malathion treatments can provide protection against insects during stem emergence periods. Future prospects for weed control include the use of transgenic poppies rendered resistant to herbicides such as glufosinate ammonium (Chitty et al. 2003). Of particular danger to opium poppy are weevils (Ceutorrhynchus macula-alba, C. denticulatus), poppy flies (Dasyneura papaveris), and poppy gnats (Perrisia papaveris). Weevils chew the leaves and lay eggs in the seed capsule, generating larvae that feed their way through the capsule wall. Causing further damage, flies and gnats can then use the holes made by emerging weevils for laying eggs. Pathogens include powdery mildew (Erysiphe polygoni), root rot (Rhizoctina bataticola), and a variety of viruses. Protection against harmful fungi may be achieved by seed-treatment prior to sowing, application of sulfur- or copper-containing preparations, and optimal harvesting times. Plants infected with a virus are usually burnt to restrict the spread of the infection. In addition to pests, soil quality, topography and availability of irrigation sources are concerns. A common and effective strategy to maintain soil quality is agricultural rotation, which usually involves the cultivation of two or three crop species before poppies are grown again (Laughlin et al. 1998; Chitty et al. 2003). Traditionally, opium poppy cultivation and opium harvesting involve the laborious processes of manually lancing the unripe seed capsule and collecting the latex. In 1928, the Hungarian pharmacist János Kabay developed a method to extract morphine and related compounds from opium poppy straw, which previously was separated as waste from the seeds in the final step of commercial poppy cultivation. This approach circumvented the arduous techniques associated with the harvesting of opium and made it possible to obtain highquality seeds and pharmaceutically valuable raw materials simultaneously. The harvesting of straw has several other advantages over the traditional method of collecting opium. The harvesting and processing of straw can be highly mechanized, thus reducing labor costs. For example, China, a legitimate producer of latex-derived opium, began to produce opium poppy straw in 1998 and gradually increased straw production at the expense of latex-derived opium in subsequent years to reduce the high costs of lancing (United Nations 2001). Moreover, the monitoring and control of straw cultivation is easier than regulating opium production. For these reasons, the ratio of straw to latex-derived opium is steadily increasing. Licit production of opiate raw materials, both latex-derived and poppyderived, is restricted to assigned countries under the Single Convention on Narcotic Drugs 1961 and relevant resolutions of the United Nations Economic and Social Council (United Nations 1961). In compliance with these resolutions, the International Narcotics Control Board (INCB) is responsible for monitoring the licit supply of, and demand for, opiates in addition to maintaining an acceptable global “balance”. For example, if supply dramatically

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exceeds demand by 1 year, INCB will limit opiate production the following year. In addition to regulating the total area of licit opium poppy cultivation, INCB closely follows trends in opiate processing, drug manufacture, trade, and consumption. Outside of licit poppy agriculture, the United Nations Office on Drugs and Crime (UNODC) compiles annual reports documenting the illicit cultivation of opium poppy, although the statistics obtained for such reports are inherently less reliable. Figure 1 shows the major countries engaged in licit and illicit opium poppy agriculture. European countries such as the Netherlands, Germany, Austria, and Poland cultivate opium poppy primarily for seed production, whereas Australia, Spain, France, and Turkey produce poppy straw for the extraction of alkaloids. Seeds are valued directly as food and for their oil, which has both alimentary and industrial applications. Currently, only India exports raw opium, although other Asian countries are entitled to its production. Australia, specifically the island of Tasmania, supplies a large proportion of the world’s opiate material, particularly for the extraction of thebaine. Although not itself used for medicinal purposes, the morphinan alkaloid thebaine is a starting material in the manufacture of several semi-synthetic opiates, including oxycodone, oxymorphone, etorphine, and buprenorphine. Additionally, thebaine is the starting material for the synthesis of naloxone, naltrexone, nalorphine, and nalbuphine, some of which are used to treat opiate poisoning and opium addiction. Until 1998, thebaine was mainly obtained as a byproduct from opium, but since the development of high-thebaine, low-morphine varieties, the alkaloid is now recovered from opium poppy straw. As shown in Table 1, thebaine-accumulating opium poppy has been cultivated in Australia since 1998 and in France since 1999. In 2002, the cultivation of thebaine-rich opium poppy varieties surpassed that of morphine-rich varieties in Australia. Global production of thebaine has increased sharply since 1998. The United States, a major manufacturer, increased thebaine production from 4.6 t in 1996 to 40.3 t in 2000 (United Nations 2001). The increased manufacture of thebaine reflects a rising demand for oxycodone, which is used to treat moderate to severe pain. Oxycodone is marketed as Oxycontin or Percocet (acetaminophen with oxycodone). Abuse of Oxycontin, which produces euphoric “highs” similar to those induced by morphine, prompted the United States Drug Enforcement Administration (DEA) to list this pharmaceutical as a Schedule II drug. Most of the world’s illicit opium and heroin comes from only a few countries. Mexico, Columbia, Afghanistan, Myanmar, and the People’s Democratic Republic of Lao (Laos) cultivate large areas of opium poppy for the illicit drug trade (Fig. 1). Afghanistan currently ranks first among illicit opium poppy growing nations, accounting for about three-quarters of global production (United Nations 2003). From 2000 to 2001, global production of black-market opium decreased by about 65%, mainly due to a ban on opium poppy culti-

174 Table 1. Production of opiate raw materials (opium or poppy straw produced from morphine-rich varieties of Papaver somniferum), consumption of opiates, and the balance between the two, 1988–2002. Area harvested values are in hectares, whereas production, consumption, and balance values are in tonnes of morphine equivalent. Source: United Nations (2001) Country

1988

1989

1990

1991

1992

1993

1994

1995

1996

1997

1998

1999

2000

2001

10,087 68.3 10,753

6,442 47.2 1,707

13,950 56.6 3,448 16.5 40,698 54.3

J.M. Hagel, B.P. Macleod, and P.J. Facchini

Australia Area harvested 3,462 5,011 5,581 7,155 8,030 6,026 6,735 8,139 8,360 9,520 10,682 11,555 15,166 11,275 Production 38.5 38.8 43.0 67.5 89.8 66.9 66.0 55.6 69.0 64.1 79.5 84.0 112.0 52.5 Area harvested – – – – – – – – – – – 809 1,978 5,479 9,300 thebaine-rich varietyb France Area harvested 3,113 2,644 2,656 3,598 3,648 4,158 4,431 4,918 5,677 6,881 7,884 6,091 5,914 5,361 Production 21.4 13.4 19.5 30.2 21.8 28.8 32.9 48.9 47.3 52.0 64.8 59.0 40.0 30.0 – – – – – – – – – – 1,822 1,883 2,297 Area harvested – – thebaine-rich varietyb India Area harvested 19,858 15,019 14,253 14,145 14,361 11,907 12,694 22,798 22,596 24,591 10,098 29,163 32,085 18,086 Production 70.2 59.3 52.8 47.4 59.7 41.9 51.5 88.8 92.1 110.3 29.3 118.3 146.2 79.9 Spain Area harvested 2,935 2,151 1,464 4,200 3,084 3,930 2,539 3,622 1,180 1,002 1,640 3,913 5,698 5,536 Production 10.8 5.7 8.0 24.2 12.8 9.0 5.2 4.2 4.4 1.9 7.5 18.0 34.8 36.4 Turkey Area harvested 18,260 8,378 9,025 27,030 16,393 6,930 25,321 60,051 11,942 29,681 49,207 87,193 27,554 45,836 Production 24.7 7.2 13.3 57.9 18.7 7.8 41.1 75.2 16.1 38.3 86.7 97.1 35.8 62.0

2002a

Country

1988

1989

1990

1991

1992

1993

1994

1995

1996

1997

1998

1999

2000

2001

2002

Opium Poppy

Table 1. continued

Other countries Area harvested – – – – – – – – – – – – – – – Production 36.9 18.4 38.0 31.2 14.9 13.2 21.5 25.5 16.9 6.1 7.3 10.3 15.5 11.0 11.0 Total area harvested Morphine-rich 47,628 33,203 32,979 56,128 45,516 32,951 51,720 99,528 49,755 71,675 79,511 137,915 86,417 86,094 74,625 variety – – – – – – – – – – 809 3,800 7,362 11,597 12,460 Thebaine-rich varietyb Total 202.5 142.8 174.6 258.4 217.7 167.6 218.2 298.2 245.8 272.7 275.1 386.7 384.3 271.8 253.9 production (1) Total 200.9 204.3 196.1 217.8 212.4 236.6 225.7 237.8 245.1 240.0 247.7 244.0 232.0 240.0 240.0 consumption (2) Balance 1.6 –61.5 –21.5 40.6 5.3 –69.0 –7.5 60.3 0.7 32.7 27.3 142.7 152.3 31.8 13.9 [(1) minus (2)] a b

Figures for 2002 are International Narcotics Control Board projections. A new variety of P. somniferum with a high thebaine content.

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vation by the Taliban regime in Afghanistan. Under this regime, opium poppy production in Afghanistan was reduced by 94%. However, the end of Taliban rule saw a return to large-scale opium poppy cultivation, bringing the total area used for illicit opium poppy agriculture back to 180,000 ha worldwide in 2002, which is comparable with the 222,000 ha grown in 2000. Although Myanmar is the second-largest producer of illicit opium, Afghanistan’s irrigated fields typically produce about four-fold the amount of opium per hectare compared with rain-fed, mountainous Myanmar terrain. From 1999 to 2002, opium production in Southeast Asia declined by about 40% as a result of strict government enforced anti-drug policies, a trend observed much earlier in neighboring Thailand. Prior to 1977, opium, the oxidized, resinous latex obtained by lancing the unripe seed capsules, was the main source for the extraction of morphine. In traditional, morphine-rich opium poppy varieties, raw opium contains 4–21% morphine, depending on moisture level and quality. Codeine is usually present at 0.7–2.5% and thebaine is generally present at even lower levels. Most licit opium is used for the extraction of alkaloids, whereas about 5% is processed directly into medicinal preparations in some countries. China, North Korea, India, and Japan are the only countries permitted by international law to cultivate opium poppy for the production of raw opium. However, only India produces substantial quantities of the product. As shown in Table 1, nearly onethird of the 2001 worldwide opiate supply was obtained from opium produced in India. In some cases, illicit opium is seized from drug traffickers and released for medicinal use. For example, in 1999 the Islamic Republic of Iran added 218 t of opium to its total licit quantity of about 1,000 t.

Fig. 1. Major producers of licit (gray) and illicit (black) opiate raw materials and/or poppy seeds. Northeast (NE) Europe includes The Netherlands, Germany, Poland, Austria, Czech Republic, Slovakia, and Hungary. The Netherlands, Germany, Poland and Austria cultivate opium poppy for seed production only

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4 Classic Breeding Opium poppy is a herbaceous annual with a distinctive vegetative phase, characterized by several, horizontally spread large pinnatisect leaves, and a reproductive stage during which flowering stems and drooping buds are formed. Maturation of the capsule occurs about 110–150 days after sowing. The long history of domestication and breeding led to the development of many different opium poppy land races, which are chemotype varieties and cultivars adapted to particular uses and climatic conditions. As a result, cultivation of the plant extends over a wide area, from Mexico to Russia to Tasmania (Krikorian and Ledbetter 1975). P. somniferum (2n = 22) is considered as a predominantly self-pollinating species, although out-crossing occurs at various rates depending on variety and environmental factors. Large, often colorful flowers with numerous stamens and large quantities of pollen attract insects, especially bees. However, pollination also occurs by wind (Patra et al. 1992). The size and shape of the seed capsule vary widely, depending on the cultivar and origin. Five different varieties have been identified based on the respective width-tolength ratios of the capsule, namely oval, broad oval, orbicular, flat, and conical. The former two shapes are common to Indian varieties, while the latter three shapes are typical of European varieties. Capsule shape is also related to seed yield, with orbicular and conical capsules found in European varieties yielding greater quantities of seed than flat or oval capsules. This feature is likely the result of traditional breeding practices, as East- and Central European nations have long valued opium poppy for its seeds and seed oil. The success of any breeding program necessitates the availability of a highly varied gene pool. Evaluations of the genetic variation in cultivated germplasms of P. somniferum have shown that only limited variation occurs in Indian (Singh and Khanna 1991) and European genetic stocks (Dubedout 1993) for most agronomic and chemical traits, a feature related to the narrow genetic base of genotypes with common ancestry. In Europe during the early 1960s, the genetic and breeding aspects of opium poppy were investigated with the aim of increasing yields for straw, seeds, and seed oil. India, in contrast, historically aimed at increasing latex yield and morphine content. Also, the different climates of Europe and India directed the breeding of diverse cultivars with variations in height, susceptibility to lodging, disease resistance, photoperiod requirements, latex yield, and morphine content. Opium poppy breeders use a variety of selection techniques in the development of improved cultivars (Levy and Milo 1998). However, the most successful breeding method, which has generated several commercial cultivars, is the pedigree selection process whereby desired traits are combined through the hybridization of parents with a variety of different characteristics. The pedigree selection approach has been used successfully to increase capsule numbers (Tarahich 1974), seed and opium yield (Khanna and Shukla 1989), morphine content, and lodging resistance (Lörincz 1978). A disadvantage of this approach is that it markedly reduces genetic variability and contributes

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to the genetic narrowing of the cultivated germplasm. Nonetheless, through the use of genetic, and to a smaller extent agro-technological improvements, France has increased its morphine yield from 4.5 kg ha−1 in 1961 to 10.5 kg ha−1 in 1991 without significantly altering the yield of dry matter (Dubedout 1993). However, the continued selection of new opium poppy lines is an essential and ongoing part of successful breeding programs to ensure the renewal of a large base of genetic variation. Although male sterility, either genic or genic-cytoplasmic, is widely employed in the commercial production of hybrid lines for most crops, the natural occurrence of male sterility have not been reported for opium poppy. However, irradiation of opium poppy seeds with gamma rays allows isolation of male-sterile plants in the M1 generation (Singh and Khanna 1970). Male-sterile plants have also been observed in the F2 generation of an inter-specific hybrid between P. somniferum and P. setigerum (Hrishi and Hrishi 1960). In many crop species, difficulties in promoting cross-pollination are circumvented by the use of male-sterile varieties. The development of male-sterile lines of opium poppy could increase hybrid vigor and heterosis in terms of morphine yield and/or seed content. Self-incompatibility might also facilitate the production of hybrid seeds, although little work has been done in this area. Most breeding programs use a selection index to maximize the inheritance of desirable traits. A multi-character index involves criteria such as days to flowering, plant height, capsule and leaf number, and capsule husk weight. Positive correlations have been drawn between capsule size and opium yield, although Dubedout (1993) found no relationship between the agro-morphological characteristics and the content of morphinan alkaloids of poppy capsules. In a direct approach, Kaicker et al. (1975) used an index based on a single criterion – opium yield – for selection purposes. Dubedout (1993) described a selection index based on a study of 24 European opium poppy varieties and their hybrids, which took into account the heritability and correlation coefficients of different components governing morphine yield. The total yield of morphine equivalents, defined as 100% of the morphine content, 96.9% of the codeine content, plus 91.6% of the thebaine content, was considered the most important criterion.

5 Biochemistry and Molecular Biology Research in the field of plant alkaloid biochemistry began with the isolation of morphine in 1806. Over the past half-century, several key technologies, including radioactive tracing of metabolites, plant cell culture methods, and molecular biology, have contributed to a dramatic improvement in our understanding of alkaloid metabolism. Benzylisoquinoline alkaloids, such as morphine, are a large and diverse group of about 2500 defined structures, many of which are used as pharmaceuticals (Facchini 2001, 2006). The structural complexity

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of these compounds generally precludes chemical synthesis as an alternative to plant cultivation for commercial purposes. Benzylisoquinoline alkaloid biosynthesis begins with a lattice of decarboxylations, ortho-hydroxylations, and deaminations to generate both dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) from tyrosine. Genes encoding the aromatic l-amino acid decarboxylase (TYDC) converting tyrosine and dopa to tyramine and dopamine, respectively, have been isolated from opium poppy (Facchini and De Luca 1994). The condensation of dopamine and 4-HPAA is catalyzed by norcoclaurine synthase (NCS) to yield (S)-norcoclaurine, the central precursor to all benzylisoquinoline alkaloids in plants (Fig. 2). The isolation of a cDNA for NCS from the meadow rue Thalictrum flavum (Samanani et al. 2004) facilitated the cloning of the corresponding gene from opium poppy (Liscombe et al. 2005). A 6-O-methyltransferase (6OMT), an N-methyltransferase (CNMT), a P450 hydroxylase (CYP80B1), and a 4 -O-methyltransferase (4 OMT) are responsible for the conversion of (S)-norcoclaurine to (S)-reticuline (Fig. 2). Molecular clones encoding each enzyme have all been isolated from opium poppy (Huang and Kutchan 2000; Facchini and Park 2003). (S)-Reticuline is a key branchpoint intermediate in benzylisoquinoline alkaloid biosynthesis and a variety of subsequent enzymatic reactions determine the structural type of alkaloid produced. (S)-Reticuline can be converted to laudanine by (R,S)-reticuline 7-O-methyltransferase (Ounaroon et al. 2003), to (S)-scoulerine by the berberine bridge enzyme (BBE), or to 1,2-dehydroreticuline (Hirata et al. 2004). The reaction catalyzed by BBE represents the first committed step in the branch pathway leading to the benzophenanthridine alkaloid sanguinarine, and the opium poppy genes encoding BBE have been characterized (Facchini et al. 1996a). Two P450-dependent oxidases convert (S)-scoulerine to (S)-stylopine, which subsequently undergoes N-methylation by a specific N-methyltransferase. A cDNA encoding tetrahydroprotoberberine cis-N-methyltransferase (TNMT) has been isolated from opium poppy (Liscombe and Facchini 2007). Molecular clones for (S)-stylopine synthase (CYP719A2, CYP719A3) were recently identified from Eschscholzia californica (Ikezawa et al. 2007), but the opium poppy orthologs have not yet been reported. Two additional P450-dependent enzymes convert (S)-cis-N-methylstylopine to dihydrosanguinarine, which is oxidized to yield sanguinarine. Opium poppy roots generally accumulate the highest concentrations of sanguinarine, which exhibits antimicrobial activity. The oxidation and subsequent reduction of (S)-reticuline to (R)-reticuline, via 1,2-dehydroreticuline, are the first committed steps in morphinan alkaloid biosynthesis (De-Eknamkul and Zenk 1992; Hirata et al. 2004). (R)-Reticuline is converted in two enzymatic steps to salutaridinol by a P450-dependent enzyme and a short-chain dehydrogenase/reductase, respectively. A cDNA encoding the latter enzyme, salutaridine reductase (SAR), was identified recently in opium poppy using comparative transcript and alkaloid profiling of different Papaver species (Ziegler et al. 2006). Acetyl coenzyme A:salutaridinol-7O-acetyltransferase (SAT) catalyzes the conversion of salutaridinol to saluta-

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Fig. 2. Schematic representation of the biosynthetic pathway from l-dopa to laudanine, (S)-scoulerine, and morphine in opium poppy. Molecular clones have been obtained for the enzymes indicated. TYDC Tyrosine decarboxylase, NCS norcoclaurine synthase, 6OMT (S)-norcoclaurine-6-O-methyltransferase, CNMT (S)-coclaurine-Nmethyltransferase, CYP80B1 (S)-N-methylcoclaurine-3 -hydroxylase, 4 OMT (S)-3 -hydroxy-Nmethylcoclaurine-4 -O-methyltransferase, 7OMT (R,S)-reticuline-7-O-methyltransferase, BBE berberine bridge enzyme, CYP719A (S)-stylopine synthase, TNMT tetrahydroprotoberberine cis-N-methyltransferase, SAR salutaridine reductase, SAT salutaridinol-7-O-acetyltransferase, COR codeinone reductase. Reactions blocked in top1 mutants are also shown

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ridinol-7-O-acetate, which spontaneously produces thebaine. The subsequent methylation of thebaine results in the formation of either neopinone or oripavine. Neopinone spontaneously forms codeinone, which is reduced by the NADPH-dependent enzyme codeinone reductase (COR) to form codeine. In turn, codeine is demethylated, yielding morphine. An alternate route for morphine biosynthesis involves the production of morphinone from oripavine, followed by COR-catalyzed reduction. Although molecular clones encoding SAT and COR have been isolated from opium poppy, most other morphinanspecific biosynthetic enzymes remain poorly characterized. The cell type-specific localization of benzylisoquinoline alkaloid biosynthesis in opium poppy was recently determined (Bird et al. 2003). In opium poppy, gene transcripts encoding CYP80B1, BBE, and COR are restricted to companion cells, whereas the corresponding enzymes are localized to adjacent sieve elements of the phloem in the vascular system of the plant. The biosynthesis of morphine and related alkaloids in the phloem breaks the long-standing paradigm that sieve element functions are limited to the translocation of solutes and information macromolecules in plants. Laticifers, the specialized cells in the phloem that contain the latex, are now known to serve as the site of alkaloid accumulation, not biosynthesis, in opium poppy. Overall, the process from gene expression through alkaloid accumulation requires three specialized cell types and necessitates the intercellular translocation of biosynthetic enzymes and products. Due to the toxicity of pathway intermediates and products, benzylisoquinoline alkaloid biosynthetic enzymes are also compartmentalized at the subcellular level (Bird and Facchini 2001). The non-cytosolic enzymes involved in the benzophenanthridine and morphinan branch pathways are localized to the endoplasmic reticulum (ER), or ER-derived endomembranes (Facchini 2001).

6 Biotechnology Cell cultures of opium poppy were first established in the late 1950s through in vitro studies on the growth of immature ovulary tissue. Work with callus and cell cultures of opium poppy is now common, although the regeneration of intact plants proves more arduous. Somatic embryogenesis and organogenesis of roots or shoots appear to be generally inducible by transferring cultured tissues to medium containing low quantities of exogenous hormones (Facchini and Bird 1998). However, tissue and culture browning is a consistent problem, in addition to the low frequencies of shoot formation and difficulties transferring rooted plants to soil. In contrast to the differentiated tissues and organs of opium poppy plants, where morphine, sanguinarine, and/or several other alkaloids accumulate to high concentrations, dedifferentiated opium poppy cell cultures accumulate only sanguinarine, and only under certain conditions such as treatment with fungal-derived elicitors (Eilert et al. 1985;

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Facchini et al. 1996b). Reports of the accumulation of morphine and codeine are rare, and usually involve newly established callus cultures (Facchini and Bird 1998). Despite the general inability of cultured opium poppy cells to generate morphinan alkaloids, the known morphinan-specific biosynthetic enzymes are active in dedifferentiated cell cultures. This phenomenon has long been interpreted to suggest that the biosynthesis and accumulation of benzylisoquinoline alkaloids require a network of factors found only within an integrated assembly of differentiated cell types in opium poppy. The localization of biosynthetic enzymes to sieve elements of the phloem demonstrates the cell type-specific context of benzylisoquinoline alkaloid pathways (Bird et al. 2003). Despite difficulties in regenerating intact plants from cultured tissue, there are several reports on the genetic transformation of opium poppy organs and plants. Park and Facchini (2000b) used Agrobacterium rhizogenes to produce transgenic hairy roots from wounded opium poppy seedlings. Similarly, Le Flem-Bonhomme et al. (2004) infected wounded hypocotyls using a sonication-assisted Agrobacterium-mediated transformation (SAAT) protocol and induced hairy root formation. Sonicated hypocotyls were found to be highly susceptible to transformation with A. rhizogenes strain LBA 9402. Although Park and Facchini (2000b) showed that agar-solidified media could be used to induce transgenic roots, Le Flem-Bonhomme et al. (2004) suggested that opium poppy hairy roots could be obtained only in liquid medium without growth regulators. Nessler (1998) documented the production of transgenic opium poppy plants by either microparticle bombardment or Agrobacterium tumefaciens, although the work was preliminary and did not define the genotype. Park and Facchini (2000a) described the A. tumefaciens-mediated transformation of opium poppy, using seedling hypocotyls as explants and regeneration by shoot organogenesis. Recently, Chitty et al. (2003) applied the A. tumefaciensmediated infection of hypocotyl explants to produce antibiotic- and herbicideresistant embryonic callus. Following induction of somatic embryos, full plant development was achieved. The T0 plants exhibited normal morphology and were self-fertile, permitting stable transgenic inheritance in both the T1 and T2 generations. The technique was reportedly efficient and effective across a range of opium poppy varieties. The continued refinement of reliable transformation protocols is a key component in the establishment of biotechnology as a driving force in the opium poppy industry. With the ability to genetically transform opium poppy comes the unprecedented capacity to engineer new plant varieties with commercially desirable traits. Although conventional plant breeding produced a doubling in morphine levels over the past two decades, continued progress using traditional approaches was limited. Metabolic engineering using genetically transformed plants, a growing arsenal of cloned genes, and a steadily improving understanding of the regulation of alkaloid biosynthetic pathways, should provide new strategies to further expand the frontiers of opium poppy breeding.

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Indeed, genetic modification with the goal of developing new opium poppy varieties has been underway for decades. A traditional method of altering metabolic profiles that remains popular today for plants, animals, and microorganisms is the mutagenesis of a population using chemicals or ionizing radiation. Chemical mutagens, such as ethylmethanesulfonate (EMS), primarily cause point mutations and are favored because they generate a relatively high density of irreversible genetic lesions. Furthermore, the production of commercially valuable, non-transgenic plant varieties has particular appeal within the industry. In a recent effort, the seeds of a commercial opium poppy cultivar were mutagenized using EMS and the plants were screened for altered latex metabolite profiles (Millgate et al. 2004). The mutant top1 was found to accumulate thebaine and oripavine at the expense of morphine, conferring a pigmented color to the latex, rather than the normal white color. Further investigation of the top1 mutant confirmed a block at thebaine and oripavine, which occur in two branches of the bifurcated pathway leading to morphine (Fig. 2). The mutation is possibly due to a defect in a single enzyme responsible for the demethylation of thebaine and oripavine. The top1 variety is now extensively cultivated in Tasmania (Table 1), demonstrating the potentially powerful impact of mutagenesis on the opium poppy industry. Interestingly, a variety of opium poppy exhibiting a high-thebaine, low-morphine phenotype was also isolated from a natural (i.e., chemically untreated) population (Nyman 1980). Opium poppy lines with altered alkaloid profiles have also been produced by genetic transformation. Frick et al. (2004) reported altered alkaloid ratios in the latex, but not roots, in opium poppy lines transformed with an antisenseBBE construct. In California poppy (Eschscholzia californica Cham.), which is also a member of the Papaveraceae, benzophenanthradine alkaloid accumulation was elevated in root cultures expressing BBE from opium poppy, whereas transgenic roots harboring an antisense-BBE construct displayed reduced levels of sanguinarine and related benzophenanthridine alkaloids (Park et al. 2003). The suppression of benzophenanthradine alkaloid biosynthesis was also shown to occur in California poppy cell cultures transformed with antisense-BBE and antisense-CYP80B1 constructs (Park et al. 2002). RNA interference (RNAi) was recently used to silence COR expression in opium poppy with the surprising outcome that only (S)-reticuline, but no morphinan branch pathway intermediates, accumulated to high levels (Allen et al. 2004). Transcript levels for seven other benzylisoquinoline alkaloid biosynthetic enzymes in the pathway, both before and after (S)-reticuline, were unchanged (Fig. 2). Thus, the absence of a single enzyme can prevent (S)reticuline from entering the morphine-specific biosynthesis, possibly due to the existence of a requisite metabolic complex of morphinan branch pathway enzymes. The regulation of benzylisoquinoline alkaloid metabolism is complex and our understanding of opium poppy biochemistry at the molecular level can be advanced with genetic transformation and metabolic engineering biotechnology.

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Genomic and proteomic strategies, such as DNA microarrays and random sequencing of proteins separated by two-dimensional gel electrophoresis, respectively, are beginning to offer much-needed, comprehensive perspectives on alkaloid metabolism in opium poppy. The development of DNA microarrays using organ- and tissue-specific expressed sequence tag collections provides a powerful platform for the discovery of new genes. As part of their analysis of the top1 mutant, Millgate et al. (2004) developed a 17,000-gene microarray and used it to explore global changes in gene expression patterns. In another study, an opium poppy latex cDNA library was used to identify cell wall-degrading enzyme homologs, whose expression levels were examined by RNA gel blot hybridization analysis (PilatzkeWunderlich and Nessler 2001). Poppy laticifers are classified as articulated and anastomosing due to their compound origin and the perforations that develop between the lateral walls of adjacent latex vessels. At maturity, the continuous laticifer networks allow the exudation of large volumes of latex, which is under positive pressure, by lancing the unripe seed capsules. Understanding the development of laticifers and the mechanisms responsible for the degradation of adjoining cell walls, in particular, it might be possible to engineer opium poppy varieties lacking continuous laticifer networks to prevent the illicit collection of opium using the lancing technique. At the post-trancriptional level, two-dimensional gel electrophoresis followed by direct peptide sequencing was used to develop a database of both cytosolic and vesicular latex proteins (Decker et al. 2000). Similar proteomic analysis was used also to clone and characterize the two alkaloid biosynthetic enzymes (R,S)-reticuline 7-O-methyltransferase and (R,S)-norcoclaurine 6-Omethyltransferase (Ounaroon et al. 2003). Other genomic techniques, such as amplified restriction fragment length polymorphic (AFLP) analysis, have been used to evaluate the genetic diversity of breeding populations to provide information on those lines with desired genetic heterogeneity (Saunders et al. 2001).

7 Future Prospects Our understanding of the biological processes underlying the biosynthesis and accumulation of benzylisoquinoline alkaloids in opium poppy has advanced considerably over the past decade. This rapid progress is largely due to the availability of reliable molecular tools. These tools, combined with recent advancements in plant genomics, will undoubtedly expedite our knowledge of opium poppy biology at the biosynthetic and regulatory levels. Coupling molecular biology with agronomy is key to the generation of novel, commercially valuable lines of opium poppy. In the future, new varieties exhibiting desirable metabolite profiles, such as increased morphine or codeine content, might be derived through a combination of classic breeding,

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mutagenesis, and genetic transformation. The development of reliable, robust transformation protocols remains an important objective to realize these goals, although mutagenesis combined with targeting induced local lesions in genomes (TILLING) technology provides an alternative means to obtain genespecific knock-out varieties. Beyond commercial interests, the global problems surrounding the trade of “black-market” opium for the production of heroin might be addressed by the development of opium poppy varieties that accumulate alkaloids other than morphine, or that prevent the easy extraction of opium. The value of opium poppy as a source for both food and medicine will encourage continued interest in the development and application of new biotechnologies.

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Park S-U, Yu M, Facchini PJ (2002) Antisense RNA-mediated suppression of benzophenanthradine alkaloid biosynthesis in transgenic cell cultures of California poppy. Plant Physiol 128:696– 706 Park S-U, Yu M, Facchini PJ (2003) Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids. Plant Mol Biol 51:153–164 Patra NK, Ram RS, Chauhan SP, Singh AK (1992) Quantitative studies on the mating system of opium poppy (Papaver somniferum L.). Theor Appl Genet 84:299–302 Pilatzke-Wunderlich I, Nessler CL (2001) Expression and activity of cell-wall-degrading enzymes in the latex of opium poppy, Papaver somniferum L. Plant Mol Biol 45:567–576 Samanani N, Liscombe DK, Facchini PJ (2004) Molecular cloning and characterization of norcoclauarine synthase, an enzyme catalyzing the first committed step in benzylisoquinoline alkaloid biosynthesis. Plant J 40:302–313 Saunders JA, Pedroni MJ, Penrose LDJ, Fist AJ (2001) AFLP analysis of opium poppy. Crop Sci 41:1596–1601 Simmonds NW (1976) Evolution of crop plants. Longman, London Singh SP, Khanna KR (1970) Male sterility in opium poppy. Sci Cult 36:554–556 Singh SP, Khanna KR (1991) Genetic variability for some traits in opium poppy (Papaver somniverum L.). Narendra Deva J Agric Res 6:88–92 Tarahich AP (1974) Intervarietal hybridization in breeding poppy. Plant Breed Abstr 45:3829 United Nations (1961) Single convention on narcotic drugs 1961. Economic and Social Council/Commission on Narcotic Drugs, United Nations, New York United Nations (2001) Narcotic drugs: estimated world requirements for 2002 – statistics for 2000. International Narcotics Control Board, United Nations, New York United Nations (2003) Global illicit drug trends 2003. Office on Drugs and Crime, United Nations, New York Ziegler J, Voigtländer S, Schmidt J, Kramell R, Miersch O, Ammer C, Gesell A, Kutchan TM (2006) Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis. Plant J 48:177–192

II.3 Henbane, Belladonna, Datura and Duboisia R. Arroo1 , J. Woolley1 , and K.-M. Oksman-Caldentey2

1 Introduction Alkaloids are a structurally diverse class of plant-derived compounds, which often possess a strong physiological activity and, over the centuries, have found many clinical applications. More than 12,000 alkaloids have been identified in the plant kingdom (Kutchan 1995). Although the pharmacological effects of alkaloids have been well studied, the pathways by which these compounds are synthesised in plants are still obscure. Alkaloids may be classified based on the amino acids from which they are derived (Dewick 2002). The best studied groups, for which the enzymes and genes involved in alkaloid biosynthesis have been partially characterised, are the tyrosine-derived isoquinoline alkaloids, the tryptophan-derived indole alkaloids and the ornithine-derived nicotine and tropane alkaloids (Suzuki et al. 1999).

2 Tropane Alkaloids, Uses and Outlook Atropine and hyoscyamine were among the first alkaloids to be isolated and structurally identified (Geiger and Hesse 1833; Mein 1833; Fig. 1), while scopolamine was identified a few decades later (Schmidt 1892; Fig. 2). However, the use of herbs that possessed tropane alkaloids as their main active ingredient dates back to ancient times. The principal alkaloids of medicinal interest are the tropane ester (–)-hyoscyamine, its more stable racemate atropine and scopolamine (hyoscine). These three specific alkaloids are confined to plants in the Solanaceae (nightshade family). The species that are used for commercial production of tropane alkaloids are deadly nightshade (Atropa belladonna), thornapple (Datura stramonium), henbane (Hyoscyamus niger) H. muticus and Duboisia (Duboisia myoporoides, D. leichhardtii and their hybrids). Hyoscyamine and scopolamine are also found in two other solanaceous plants, namely scopolia (Scopolia carniolica) and mandrake (Mandragora officinarum), but these plants find little current commercial use (Dewick 2002). 1 Leicester School of Pharmacy, Natural Products Research, De Montfort University, The Gateway,

Leicester LE1 9BH, United Kingdom Technical Research Centre of Finland, Plant Biotechnology, P.O. Box 1000, FIN-02044 Espoo, Finland, e-mail: Kirsi-Marja.Oksman@vtt.fi

2 VTT

Biotechnology in Agriculture and Forestry, Vol. 61 Transgenic Crops VI (ed. by E.C. Pua and M.R. Davey) © Springer-Verlag Berlin Heidelberg 2007

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Fig. 1. Atropine

Fig. 2. Scopolamine

The Solanaceae is a large plant family (94 genera, 2,950 species), including a number of food crops, e.g. potato (Solanum tuberosum), egg plant (S. melongena) and cape gooseberry (Physalis peruviana). Although edible parts of these food crops are non-toxic, they do contain tropane alkaloid-like compounds. The occurrence of calystegines, polyhydroxy nor-tropanes that are structurally and biosynthetically closely related to the tropane esters, has been reported in most Solanaceae tested so far, including food crops (Dräger 2004; Fig. 3). Analysis of the polar calystegines demands special techniques for extraction and separation, which are time-consuming. Thus, the inventory of the Solanaceae is far from comprehensive. Another well known tropane base is cocaine (Fig. 4), extracted from Erythroxylum spp (Erythroxylaceae). This compound is used medicinally as a powerful local anaesthetic, but is not reviewed here. Atropine and scopolamine are anticholinergic and amongst the oldest known muscarinic antagonists. Muscarinic receptors in smooth muscle regulate cardiac contractions, gut motility and bronchial constriction. Muscarinic receptors in exocrine glands stimulate gastric acid secretion, salivation and lacrimation. In addition, muscarinic receptors are found throughout the brain. Anticholinergics are generally used to control the secretion of saliva and gastric acid, slow gut motility and prevent vomiting. Atropine and scopolamine salts are

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Fig. 3. Calystegine A3

Fig. 4. Cocaine

used in eye drops to dilate the pupil and relax the lens so that eye examinations can be carried out in ophthalmology. Tropane alkaloid injections may be given as a pre-anaesthetic medication to inhibit excessive salivary and bronchial secretions and to diminish the risk of vagal inhibition of the heart. Injections are also used to reverse muscarinic effects associated with toxic exposure to anticholinesterase compounds (e.g. organophosphate pesticides, or some nerve gases). Scopolamine hydrobromide tablets are commonly used to prevent motion sickness. Tropane alkaloids also have a limited therapeutic use for the treatment of Parkinson’s disease. In large doses, however, the muscarinic antagonists with tertiary amines have profound central effects, including hallucinations and memory disturbances. The latter effect is more pronounced with scopolamine than it is with hyoscyamine/atropine, but much less with Tiglissin (tigloidine). Muscarinic antagonists, such as scopolamine or atropine, readily cross the blood–brain barrier, but their quaternary N-methylated salts carry a permanent positive charge and do not cross as easily. Such considerations are useful for predicting the site of action of a drug, the central nervous system (CNS) or parasympathetic nervous system (PNS), and for restricting the action of a class of drugs to the PNS. The semi-synthetic quaternary muscarinic antagonists ipratroprium (Fig. 5) and, more recently, tiotropium (Fig. 6), are two effective bronchodilators that have been developed and are used extensively in the treatment of chronically obstructed pulmonary disorders.

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Fig. 5. Ipatropium

Fig. 6. Tiotropium

3 Economic Importance of Tropane Alkaloid-Containing Crops The economic importance of solanaceous medicinal crops lies mainly in their accumulation of hyoscyamine, or in the more valuable scopolamine. The roots are the principal site of alkaloid biosynthesis and generally have a higher alkaloid content and a wider variety of alkaloids than either parts of the plants. However, secondary modifications of tropane alkaloids, e.g. epoxidation of hyoscyamine to give scopolamine, occur in the aerial parts. As a consequence, leaves are often the preferred source for alkaloid production. Datura stramonium is a bushy annual attaining a height of about 1.5 m that is cultivated in central Europe (e.g. Germany, France, Hungary). The plant is widespread in both the Old and New Worlds. Dried leaves and flowering tops are traded as Stramonium Leaf, and usually contain 0.2–0.45% (on a dry weight basis) of alkaloids, the chief of which are hyoscyamine and scopolamine. Young plants predominantly contain scopolamine, but the ratio changes with age. Henbane (Hyoscyamus niger) is a biannual or annual, depending on the variety. Dried leaves and flowering tops typically contain 0.05–0.14% of alkaloids, of which hyoscyamine and scopolamine are the principal ones. Henbane is cul-

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tivated in Central Europe and the United States. Egyptian henbane (H. muticus) is a perennial stout fleshy plant, about 30−60 cm in height. It is indigenous to desert regions in the Middle East and is cultivated in Southern California. The leaves contain up to 1.7% of alkaloids, mainly hyoscyamine. Deadly nightshade, Atropa belladonna, is a perennial shrub which can grow up to 1.5 m and is cultivated in Europe and the United States. Plants about 3 years old are sufficiently large to harvest the leaves. Belladonna herb, the dried leaves and flowering tops, contains 0.3–0.6% of alkaloids, almost entirely hyoscyamine. Belladonna root, also collected from plants at least 3 years old, contains about 0.4–0.8% of alkaloids, mainly hyoscyamine and atropine. Two species of Duboisia, D. myoporoides and D. leichhardtii, are indigenous to Australia. They and their hybrid are medium-sized trees, which can be trimmed twice a year. Leaves of Duboisia typically contain 2–4% of total alkaloids, with more than 60% of scopolamine and 30% of hyoscyamine and, globally, are the main source of tropane alkaloids. Conventional breeding has resulted in varieties that can accumulate up to 6% of scopolamine on a dry weight basis. Plantations have been established in Australia, Ecuador and Brazil and annually yield 1 t/ha of scopolamine. Most of the crop is exported to Europe and Japan for processing (Boehringer–Ingelheim 2005).

4 Tropane Alkaloid Biosynthetic Pathway Although a considerable amount of information is available on the pharmacological effects of tropane alkaloids, surprisingly little is known about how the plants synthesise these compounds and almost nothing is known about how synthesis is regulated. Most biosynthetic elucidation was based on classic biochemical studies, such as feeding labelled precursors. After an extensive series of degradation studies on tropine and ecgonine (the tropane base of cocaine), Richard Wilstätter (1896) was the first to chemically synthesise tropinone. Later, Robert Robinson presented a much simpler synthetic route to tropinone by condensing together succindialdehyde, N-methylamine and acetone in the same vessel at physiological pH in water (Robinson 1917). This was the first biomimetic synthesis of a natural product. Subsequently, Robinson proposed that ornithine was the biological precursor of succindialdehyde (Robinson 1928). In the same paper, he suggested that tropic acid was derived from the condensation of three glyceraldehyde residues, a prescient adumbration of the shikimic pathway. Later, Robinson proposed that the biosynthesis of the tropane ring might occur via an analogous route, i.e. the condensation of an amino acid-derived N-methylpyrrolidine with an “acetone equivalent” (Robinson 1955). Leete et al. (1954) showed that, after feeding [2-14C]ornithine to Datura stramonium plants, the label was specifically incorporated into hyoscyamine. Subsequent feeding experiments, with D. stramonium, Hyoscyamus albus and Ery-

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throxylum coca, confirmed the general biosynthetic pathway from ornithine, via putrescine, methylputrescine, to an N-methylpyrrolinium intermediate (Leete et al. 1954; Fig. 7). This part of the biosynthetic pathway is now generally accepted. More recent feeding experiments with D. stramonium indicated the existence of an alternative biosynthetic pathway starting from the amino acid arginine (Walton et al. 1990). Biosynthetically, ornithine is derived from arginine. Therefore, it is plausible that arginine incorporation proceeded via ornithine. However, when [U-14C]agmatine was fed to D. stramonium root cultures, there was more incorporation than with [5-14C]ornithine. Thus, since agmatine could not have been incorporated via ornithine, the existence of an alternative pathway was confirmed (Walton et al. 1990; Fig. 8). The next steps in the biosynthesis of the tropane ring system require the condensation of an appropriate acetate-derived intermediate with N-methylpyrrolinium. However, the details of this condensation reaction have not been resolved and some intriguing stereochemical questions remain unanswered (Robins et al. 1997; Hemscheidt 2000; Humphrey and O’Hagan 2001). The condensation product of N-methylpyrrolinium salt and the as yet unspecified acetate-derived intermediate, is tropinone, which is considered a key intermediate in the biosynthesis of tropane esters in the Solanaceae. Reduction of tropinone results in the formation of either tropine or its stereoisomer pseudotropine (Portsteffen et al. 1992). Whereas the former is the precursor of most clinically used tropane esters, the latter is an intermediate in the formation of some rarer esters (e.g. tigloidine, which was used to control Parkinson’s

Fig. 7. Ornithine as a precursor in tropane alkaloid biosynthesis

Fig. 8. Arginine as a precursor in tropane alkaloid biosynthesis

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Fig. 9. Stereospecific reduction of tropinone to pseudotropine and tropine

Fig. 10. Biosynthetic steps leading to hyoscyamine and scopolamine

symptoms, but is now obsolete). In addition, pseudotropine is considered to be the precursor of the calystegines (Fig. 9). A wide range of acids are found esterified with tropane bases. The structures of esterifying acids found in the tropane alkaloids have been thoroughly reviewed (Lounasmaa 1988; Lounasmaa and Tamminen 1993). The dominant metabolic reaction in most commercially grown species is the formation of hyoscyamine, the ester of tropine with (S)-tropic acid. The formation of this ester is not as straightforward as one might suspect. Tropine becomes initially esterified with phenyllactic acid to form littorine. The next step is then an intramolecular rearrangement of the phenyllactate moiety of the alkaloid, resulting in the formation of hyoscyamine (Robins et al. 1994). Further modifications to the tropane skeleton, e.g. the formation of a 6, 7β-epoxide as in scopolamine, occur at the ester and not on the aminoalcohol level (Hashimoto et al. 1993; Fig. 10).

5 Current Research and Development in Transgenic Technology The introduction of root cultures made by transforming the appropriate tropane-producers, notably Hyoscyamus, Datura, Atropa and Duboisia, con-

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siderably aided the advance of biosynthetic studies in the 1980s and 1990s (Robins and Walton 1993). The transformation process involves the insertion of a small piece of DNA, carried on a plasmid in Agrobacterium rhizogenes, into the genome of the plant. This transferred DNA (T-DNA) carries a number of genes that stimulate the formation of roots from the point of infection; the aux-genes (tms-genes) direct auxin synthesis in transformed cells, whereas the rol-genes (rooting locus), seem to increase the sensitivity of transformed cells to auxins. These roots may be excised and propagated aseptically in vitro using simple, defined media. The transgenic root cultures with hairy appearance are usually referred to as “hairy root cultures” when grown in vitro. Hairy root cultures grown in vitro generally have the same characteristics as normal plant roots, i.e. they accumulate the same secondary metabolites in roughly the same amounts (Sevón and Oksman-Caldentey 2002). In contrast to root and shoot cultures (Khanam et al. 2001a, b), undifferentiated plant cell suspension cultures often seem to lose their ability to synthesise tropane alkaloids, or will only accumulate low concentrations (Bourgaud et al. 2001). Thus, hairy root cultures are an ideal model system to investigate the regulation of tropane alkaloid biosynthesis (Robins 1998). They have been used extensively to identify enzymes and genes involved in tropane alkaloid biosynthesis, and to study regulation of gene expression. Several enzymes that play a role in tropane alkaloid biosynthesis in plants, and the genes coding for these enzymes, have been identified. The occurrence of ornithine decarboxylase (ODC; EC 4.1.1.17) and arginine decarboxylase (ADC; EC 4.1.1.19) is not limited to plants. The main role of these enzymes is in the biosynthesis of the putrescine and other polyamines, like spermidine and spermine, that are essential in the regulation of cell growth and differentiation in both plants and animals (Fig. 11). Over-expression of ADC and ODC in tobacco (Burtin and Michael 1997; Mayer and Michael 2003) did not result in a comparable increase in the level of free or conjugated polyamines. Plant ODC and ADC activities seem to be highly regulated and it is unlikely that overexpression of a single step can result in an increase in flux and end-product accumulation. An additional complication here is that excess polyamines disrupt normal cellular homeostasis; feedback inhibition can affect the transcriptional, post-transcriptional, translational and post-translational regulation of the polyamine biosynthetic enzymes. Putrescine N-methyl transferase (PMT; EC 2.1.1.53) is the first committed enzyme in the biosynthetic pathway leading from putrescine to tropane and related pyrrolidine alkaloids. Over-expression of the pmt gene, in general, seems to have a moderate effect only on tropane alkaloid accumulation (Moyano et al. 2002; Rothe et al. 2003). However, the effect can be very different between various plant species (Moyano et al. 2003; Lee et al. 2005). No reports have been published on the over-expression of the gene coding for N-methylputrescine oxidase in tropane alkaloid containing plants. As this

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Fig. 11. Overview of the tropane alkaloid biosynthetic pathway and the enzymes catalysing individual bioconversions. ADC Arginine decarboxylase, H6H hyoscyamine 6β-hydroxylase, ODC ornithine decarboxylase, PAT phenylalanine aminotransferase, PMT putrescine N-methyltransferase, TR-I tropinone reductase I, TR-II tropinone reductase II

enzyme is classified as a diamine oxidase (DAO; EC 1.4.3.6) that has an affinity for a wide range of diamines (Boswell et al. 1999), it may play a role in polyamine metabolism. Thus, plant polyamine homeostatic mechanisms are likely to compensate for increased enzyme activity, as mentioned earlier for ODC and ADC. Over-expression of the tr-1 gene, coding for the enzyme tropinone reductase 1 (TR-I; EC 1.1.1.206) that catalyses the reduction of tropinone to form tropine, was accompanied by a three-fold increase in hyoscyamine and fivefold increase in scopolamine in A. belladonna. In contrast, over-expression of tr-2, coding for tropinone reductase 2 (TR-II; EC 1.1.1.236), resulted in more pseudotropine and led to an increased accumulation of calystegines in the roots (Richter et al. 2005).

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Since scopolamine is the most valuable of the tropane alkaloids, there is an increasing interest to obtain plants with enhanced contents of this compound. Yun and co-workers showed that introduction of the h6h gene [encoding hyoscyamine-6β-hydroxylase (H6H; EC 1.14.11.11) previously identified in Hyoscyamus niger L.] into Atropa belladonna L. resulted in the accumulation of scopolamine (Yun et al. 1992). Whereas normally only trace amounts are found in A. belladonna, in transgenic plants virtually all hyoscyamine was converted to scopolamine. This is the first example of the successful engineering of an important medicinal plant in order to produce a valuable end product. An even more drastic effect was obtained when h6h was over-expressed in Hyoscyamus muticus hairy roots. In this case not only were large amounts of scopolamine produced, but also high concentrations of hyoscyamine accumulated in the hairy roots (Jouhikainen et al. 1999). The examples described above elegantly show the successful metabolic engineering experiments when over-expression of only one biosynthetic enzyme at the end of the whole biosynthesis route is needed. However, if the bottleneck is somewhere in the beginning or in the middle of the route, the application of metabolic engineering does not seem possible without knowing each single step of the biosynthesis. An excellent result on scopolamine production was achieved by introducing and over-expressing the pmt and h6h genes simultaneously in hairy roots of H. niger. The best line produced over 400 mg/l scopolamine, concentrations never before reached in a biotechnological process (Zhang et al. 2004).

6 Use of Hairy Root Cultures for Tropane Alkaloid Production Several attempts have been made to use hairy roots, cultured in specially designed bioreactors, as a novel means of alkaloid production (Eibl and Eibl 2002). Hairy root cultures, that can be cultured in artificial media in the complete absence of additional phytohormones, have been shown to be a more efficient system for tropane alkaloid production than adventitious root cultures, i.e. non-transgenic roots that are cultured in the presence of auxin (Sevón and Oksman-Caldentey 2002; Yoshimatsu et al. 2004). In general, exogenously applied auxins seem to decrease the rate of secondary metabolism (Goddijn et al. 1992; Arroo et al. 1995). Expression of T-DNA-derived genes in hairy roots has been shown to affect the rate of tropane alkaloid biosynthesis. Expression of the aux1 gene (tms1) in a Duboisia hybrid and in Datura metel tended to result in callus-like roots which accumulated relatively low concentrations of tropane alkaloids (Moyano et al. 1999). However, transformation of the aux genes is not necessary to induce the hairy root phenotype; transfer of rol genes only is sufficient. Expression of the rolA gene was shown to increase polyamine metabolism in tobacco (Altabella et al. 1995). Expression of the rol genes, notably rolC, in-

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creased accumulation of tropane alkaloids in Atropa belladonna hairy roots 12-fold when compared with untransformed roots (Bonhomme et al. 2000a, b). Careful optimisation of the culture conditions in bioreactors is often needed to maximise the production of medicinal compounds from plant cell cultures. Several elicitors, for instance, have been applied in an attempt to enhance tropane alkaloid production in hairy root cultures of Solanaceae (Table 1). Although plant cell or tissue cultures were discussed recently as possible alternatives to agricultural processes, only a few such systems are used commercially (Kim et al. 2002; Sevón and Oksman-Caldentey 2002). At the moment, maybe the most remarkable example is Phyton GmbH in Germany (currently part of DFB Pharmaceuticals Inc.), which produces paclitaxel in plant cell bioreactors. Compared with agriculture, plant cell fermentation is a relatively expensive production system and has its main commercial application in the production of low-volume/high-price compounds. The current value for paclitaxel is U.S.$ 13,500/g, compared with the value of scopolamine at U.S.$ 12/g. In addition, the world-wide production of tropane alkaloids is well over 1,000 t/year. Thus, it does not seem likely that bioreactors will be used as an alternative production system for tropane alkaloids. Table 1. Overview of efforts that have been made to enhance the accumulation of tropane alkaloids in root cultures of several species of Solanaceae

Cultural condition

Root culture used

Reference

Nutrients Sucrose

Atropa belladonna

Rothe et al. (2001), Rothe and Dräger (2002)

Scopolia parviflora Brugmansia candida A. belladonna Hyoscyamus muticus B. suaveolens A. belladonna S. parviflora A. belladonna B. suaveolens B. suaveolens A. belladonna B. candida S. parviflora B. candida B. candida

Jung et al. (2003) Pitta-Alvarez and Giulietti (1999) Rothe et al. (2001) Sevón et al. (1992) Zayed and Wink (2004) Rothe et al. (2001) Kang et al. (2004) Sasaki et al. (2002) Zayed and Wink (2004) Zayed and Wink (2004) Lee et al. (2001) Pitta-Alvarez et al. (2000) Kang et al. (2004) Pitta-Alvarez et al. (2000) Pitta-Alvarez et al. (2000)

A. belladonna A. belladonna

Rothe et al. (2001) Rothe and Dräger (2002)

Elicitors Bacteria Chitosan

Methyl jasmonate Phytosulfokine-α Quercetin Salicylic acid

Silver nitrate Yeast extract Phytohormones Abscisic acid Auxin

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7 Novel Developments and Future Challenges Although tropane alkaloid-based drugs have been known for a long time, they are still recognised as effective and readily available drugs and in most cases have not been superseded by better or cheaper alternatives. Novel derivatives of naturally occurring tropane bases have been developed and are likely to be used in the foreseeable future. As an example, pervilleine A, a novel tropane alkaloid isolated from Erythoxylum pervillei has shown its potential to act as a multi-drug resistance (MRD)-reversing agent (Mi et al. 2001). To understand the regulation of the tropane alkaloid pathway, with a view to its control, it is necessary to consider not only the genes coding for enzymes catalysing individual steps in the pathway, but also the homeotic genes controlling transcription of numerous genes involved in regulation of the whole pathway and cellular pathways that interconnect with it. The relatively new areas of functional genomics and metabolomics seem to be the most promising way forward. A novel and rapid gene discovery platform based on functional genomics was described recently (Oksman-Caldentey and Inzé 2004). Using the combination of transcript and metabolic profiling it is possible to obtain a large number of genes whose expression correlates with secondary metabolite accumulation (Rischer et al. 2006). The transcript profiling was performed first using tobacco cells as a model system, and over 500 putative biosynthetic and regulatory genes were discovered (Goossens et al. 2003). In a single experiment it was thus possible to find all the genes so far known and additionally a number of putative genes involved in the nicotine alkaloid pathway. The candidate genes, alone and in combination, were then subjected to functional analysis to further characterise their roles. Also recently, suppression of one cytochrome P450 gene by virus-induced gene-silencing resulted in the accumulation of littorine in Hyoscyamus muticus, at the expense of hyoscyamine and scopolamine (Li et al. 2005). Thus, the gene/enzyme involved in littorine–hyoscyamine rearrangement that has been elusive for the past decade (Robins et al. 1995; Patterson and O’Hagan 2002) seems to have been identified. No clinical tests have been published yet on the recently discovered calystegines, although in vitro assays show their original and very promising activities as glycosidase inhibitors. Some glycosidase inhibitors are applied as pharmaceutical compounds for diabetic patients (miglitol, acarbose) and for the treatment of metabolic disorders like Gaucher’s disease (N-butyldeoxynojirimycin), a very severe autoimmune disease. Other glycosidase inhibitors show promising activities as anti-cancer or anti-viral compounds (Asano et al. 2001). However, since calystegine concentrations in plants are generally low (Dräger 2004) and procedures for the chemical synthesis of these compounds are becoming more and more efficient, it is not likely that these compounds will play an important role in the commercial development of medicinal crops.

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Genomics approaches will undoubtedly offer new challenges to discover novel natural compounds in transgenic plants through so-called combinatorial biosynthesis. The concept is based on the fact that different, but either closely or more distinctly related plants synthesise structurally related but nevertheless very different molecules. As such, it is expected that an enzyme with a certain substrate specificity isolated from one plant might encounter new but related substrates when introduced in another plant. Thus, when genes involved in the biosynthesis of a given alkaloid are isolated from one plant and subsequently introduced into another plant that synthesises related molecules, one expects to find novel chemical structures. Several experimental data already support the feasibility of this approach (Oksman-Caldentey and Inzé 2004). Therefore combinatorial biochemistry will offer new structures with potential pharmaceutical interest. This technique has already been successful in micro-organisms for the production of novel pharmaceuticals, such as antibiotics (Menzella et al. 2005).

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Geiger PL, Hesse K (1833) Darstellung des Atropins. Ann Pharm 5:43 Goddijn OJM, Dekam RJ, Zanetti A, Schilperoort RA, Hoge JHC (1992) Auxin rapidly downregulates transcription of the tryptophan decarboxylase gene from Catharanthus roseus. Plant Mol Biol 18:1113–1120 Goossens A, Häkkinen ST, Laakso I, Seppänen-Laakso T, Biondi S, De Sutter V, Lammertyn F, Nuutila AM, Söderlund H, Zabeau M, Inzé D, Oksman-Caldentey KM (2003) A functional genomics approach toward the understanding of secondary metabolism in plant cells. Proc Natl Acad Sci USA 100:8595–8600 Hashimoto T, Matsuda J, Yamada Y (1993) 2-Step epoxidation of hyoscyamine to scopolamine is catalyzed by bifunctional hyoscyamine 6-β-hydroxylase. FEBS Lett 329:35–39 Hemscheidt T (2000) Tropane and related alkaloids. Topics Curr Chem 209:175–206 Humphrey AJ, O’Hagan D (2001) Tropane alkaloid biosynthesis. A century old problem unresolved. Nat Prod Rep 18:494–502 Jouhikainen K, Lindgren L, Jokelainen T, Hiltunen R, Teeri TH, Oksman-Caldentey KM (1999) Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering. Planta 208:545–551 Jung HY, Kang SM, Kang YM, Yun DJ, Bahk JD, Yang DZ, Choi MS (2003) Enhanced production of scopolamine by bacterial elicitors in adventitious root cultures of Scopolia parviflora. Enzyme Microb Technol 33:987–990 Kang SM, Jung HY, Kang YM, Yun DJ, Bahk JD, Yang JK, Choi MS (2004) Effects of methyl jasmonate and salicylic acid on the production of tropane alkaloids and the expression of PMT and H6H in adventitious root cultures of Scopolia parviflora. Plant Sci 166:745–751 Khanam N, Khoo C, Close R, Khan AG (2001a) Tropane alkaloid production by shoot culture of Duboisia myoporoides Rev Br Phytochem 56:59–65 Khanam N, Khoo C, Khan AG (2001b) Effects of cytokinin–auxin combinations on cell arrangement in the basal stems and tropane alkaloid production in cultured non-rooted shoots of Duboisia myoporoides. Aust J Bot 49:443–450 Kim Y, Wyslouzil BE, Weathers PJ (2002) Invited review: secondary metabolism of hairy root cultures in bioreactors. In Vitro Cell Dev Biol Plant 38:1–10 Kutchan T (1995) Alkaloid biosynthesis – the basis for metabolic engineering of medicinal plants. Plant Cell 7:1059–1070 Lee KT, Hirano H, Yamakawa T, Kodama T, Igarashi Y, Shimomura K (2001) Responses of transformed root culture of Atropa belladonna to salicylic acid stress. J Biosci Bioeng 91:586– 589 Lee OS, Kang YM, Jung HY, Min JY, Kang SM, Karigar CS, Prasad DT, Bahk JD, Choi MS (2005) Enhanced production of tropane alkaloids in Scopolia parviflora by introducing the PMT (putrescine N-methyltransferase) gene. In Vitro Cell Dev Biol Plant 41:167–172 Leete E, Marion L, Spenser ID (1954) Biogenesis of hyoscyamine. Nature 174:650–651 Li R, Page J, Reed DW, Liu E, Nowak J, Pelcher L, Covello PS (2005) Carbon skeleton rearrangement in the tropane alkaloid pathway: lessons from functional genomics. Can Chem Conf Nat Prod Chem Biol 88:1102 Lounasmaa M (1988) The tropane alkaloids. In: Brossi A (ed) The alkaloids, vol 33. Academic, New York, pp 2–81 Lounasmaa M, Tamminen T (1993) The tropane alkaloids. In: Cordell GA (ed) The alkaloids: chemistry and pharmacology, vol 44. Academic, San Diego, pp 1–114 Mayer MJ, Michael AJ (2003) Polyamine homeostasis in transgenic plants overexpressing ornithine decarboxylase includes ornithine limitation. J Biochem 134:765–772 Mein (1833) Über die Darstellung des Atropins in weißen Krystallen. Ann Pharm 6:67–72 Menzella HG, Reid R, Carney JR, Chandran SS, Reisinger SJ, Patel KG, Hopwood DA, Santi DV (2005) Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nat Biotechnol 23:1171–1176 Mi Q, Cui B, Silva GL, Lantvit D, Lim E, Chai H, You M, Hollingshead MG, Mayo JG, Kinghorn AD, Pezzuto JM (2001) Pervilleine A, a novel tropane alkaloid that reverses the multidrugresistance phenotype. Cancer Res 61:4030–4037

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Moyano E, Fornale S, Palazon J, Cusido R, Bonfill M, Morales C, Pinol MT (1999) Effect of Agrobacterium rhizogenes T-DNA on alkaloid production in Solanaceae plants. Phytochemistry 52:1287–1292 Moyano E, Fornale S, Palazon J, Cusido RM, Bagni N, Pinol MT (2002) Alkaloid production in Duboisia hybrid hairy root cultures overexpressing the pmt gene. Phytochemistry 59:697–702 Moyano E, Jouhikainen K, Tammela P, Palazon J, Cusido RM, Pinol MT, Teeri TH, OksmanCaldentey KM (2003) Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus. J Exp Bot 54:203–211 Oksman-Caldentey KM, Inzé D (2004) Plant cell factories in the post genomic era: new ways to produce designer secondary metabolites. Trends Plant Sci 9:433–440 Patterson S, O’Hagan D (2002) Biosynthetic studies on the tropane alkaloid hyoscyamine in Datura stramonium; hyoscyamine is stable to in vivo oxidation and is not derived from littorine via a vicinal interchange process. Phytochemistry 61:323–329 Pitta-Alvarez SI, Giulietti AM (1999) Influence of chitosan, acetic acid and citric acid on growth and tropane alkaloid production in transformed roots of Brugmansia candida. Plant Cell Tissue Organ Cult 59:31–38 Pitta-Alvarez SI, Spollansky TC, Giulietti AM (2000) The influence of different biotic and abiotic elicitors on the production and profile of tropane alkaloids in hairy root cultures of Brugmansia candida. Enzyme Microb Technol 26:252–258 Portsteffen A, Dräger B, Nahrstedt A (1992) Two tropinone reducing enzymes from Datura stramonium transformed root cultures. Phytochemistry 31:1135–1138 Richter U, Rothe G, Fabian AK, Rahfeld B, Dräger B (2005) Overexpression of tropinone reductases alters alkaloid composition in Atropa belladonna root cultures. J Exp Bot 56:645–652 Rischer H, Oresic M, Seppänen-Laakso T, Katajamaa M, Lammertyn F, Ardiles-Diaz W, Van Montagu M, Inzé D, Oksman-Caldentey KM, Goossens A (2006) Gene-to-metabolite networks for terpenoid indole alkaloid in Catharanthus roseus cells. Proc Natl Acad Sci USA 100:8595– 8600 Robins RJ (1998) The application of root cultures to problems of biological chemistry. Nat Prod Rep 15:549–570 Robins RJ, Walton NJ (1993) The biosynthesis of tropane alkaloids. In: Cordell GA (ed) The alkaloids: chemistry and pharmacology, vol 44. Academic, San Diego, pp 115–187 Robins RJ, Bachmann P, Woolley JG (1994) Biosynthesis of hyoscyamine involves an intramolecular rearrangement of littorine. J Chem Soc Perkin Trans 1:615–619 Robins RJ, Chesters NCJE, O’Hagan D, Parr AJ, Walton NJ, Woolley JG (1995) The biosynthesis of hyoscyamine: The process by which littorine rearranges to hyoscyamine. J Chem Soc Perkin Trans 1:481–485 Robins RJ, Abraham TW, Parr AJ, Eagles J, Walton NJ (1997) The biosynthesis of tropane alkaloids in Datura stramonium: the identity of the intermediates between N-methylpyrrolinium salt and tropinone. J Am Chem Soc 119:10929–10934 Robinson R (1917) A synthesis of tropinone. J Chem Soc Trans 111:762–768 Robinson R (1928) The relationship of some complex natural products to the simple sugars and amino acids. Proc Univ Durham Philos Soc 8:14–19 Robinson R (1955) The structural relations of natural products. Clarendon, Oxford Rothe G, Dräger B (2002) Tropane alkaloids – metabolic response to carbohydrate signal in root cultures of Atropa belladonna. Plant Sci 163:979–985 Rothe G, Garske U, Dräger B (2001) Calystegines in root cultures of Atropa belladonna respond to sucrose, not to elicitation. Plant Sci 160:1043–1053 Rothe G, Hachiya A, Yamada Y, Hashimoto T, Dräger B (2003) Alkaloids in plants and root cultures of Atropa belladonna overexpressing putrescine N-methyltransferase. J Exp Bot 54:2065–2070 Sasaki K, Ishise T, Shimomura K, Kobayashi T, Matsubayashi Y, Sakagami Y, Umetsu H, Kamada H (2002) Effects of phytosulfokine-alpha on growth and tropane alkaloid production in transformed roots of Atropa belladonna. Plant Growth Regul 36:87–90 Schmidt E (1892) Über Scopolamin. Arch Pharm 230:207

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Sevón N, Hiltunen R, Oksman-Caldentey K-M (1992) Chitosan increases hyoscyamine content in hairy root cultures of Hyoscyamus muticus. Pharm Pharmacol Lett 2:96–99 Sevón N, Oksman-Caldentey KM (2002) Agrobacterium rhizogenes-mediated transformation: root cultures as a source of alkaloids. Planta Med 68:859–868 Suzuki K, Yamada Y, Hashimoto T (1999) Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle. Plant Cell Physiol 40:289–297 Walton NJ, Robins RJ, Peerless ACJ (1990) Enzymes of N-methylputrescine biosynthesis in relation to hyoscyamine formation in transformed root cultures of Datura stramonium and Atropa belladonna. Planta 182:136–141 Willstätter R (1896) Über das Tropinon. Chem Ber 1896:393–403 Yoshimatsu K, Sudo H, Kamada H, Kiuchi F, Kikuchi Y, Sawada J, Shimomura K (2004) Tropane alkaloid production and shoot regeneration in hairy and adventitious root cultures of Duboisia myoporoides x D. leichhardtii hybrid. Biol Pharm Bull 27:1261–1265 Yun DJ, Hashimoto T, Yamada Y (1992) Metabolic engineering of medicinal plants: transgenic Atropa belladonna with an improved alkaloid composition. Proc Natl Acad Sci USA 89:11799– 11803 Zayed R, Wink M (2004) Induction of tropane alkaloid formation in transformed root cultures of Brugmansia suavolens (Solanaceae). Z Naturforsch C 59:863–867 Zhang JH, Ding R, Chai Y, Bonfill M, Moyano E, Oksman-Caldentey KM, Xu T, Pi Y, Wang Z, Zhang H, Kai G, Liao Z, Sun X, Tang K (2004) Engineering tropane alkaloid biosynthetic pathway in Hyoscyamus niger hairy root cultures. Proc Natl Acad Sci USA 101:6786–6791

II.4 Taxus M.T. Piñol, R.M. Cusidó, J. Palazón, and M. Bonfill1

1 Introduction The members of the genus Taxus, which is the only genus in the family Taxaceae, are characterised by leaves shaped like flat needles, male flowers with 6–14 anthers in the form of shields, and a female apparatus with an ovule surrounded by scales. The fecundated ovule is wrapped in a red aril which becomes fleshy when ripe. The species has been known since antiquity for the substantial toxicity of all its parts (except for the arils) to humans and animals. Many Taxus species are distributed throughout the northern hemisphere. The most representative species include T. brevifolia (Pacific yew), T. baccata (European yew), T. canadensis (Canadian yew), T. mairei, T. yunnanensis, and T. chinensis (Chinese yews), T. wallichiana (Himalayan yew), T. cuspidata (Japanese yew), and hybrids derived from the crossing of these species, such as T. x media and T. x hunnewelliana. Distinguishing these entities based on morphological criteria is difficult because there are a large number of ornamental cultivars. Some authors even consider that all yews are just varieties within one species (Appendino 1993). The most interesting constituents obtained from Taxus leaves and stems are diterpenes with a taxane nucleus. Some compounds are strictly diterpenoids (e.g. baccatins), whereas others have an amide function (e.g. taxol), or are esters of 3-dimethylamino-3-phenylpropionic acid (e.g. taxines). In view of these, they are often considered to be pseudoalkaloids. Taxol (paclitaxel; NSC-125973; Fig. 1) has been a highly successful anticancer drug since it was approved initially for the treatment of breast and ovarian cancers (Suffness and Wall 1995). Other molecular targets for taxanes are still being investigated, such as multidrug resistance inhibition, apoptosis inhibitor binding, and treatments for non-small-cell lung cancer and AIDSrelated Kaposi’s sarcoma (Thayer 2000). Thus, demands on the supply of taxol continue to grow as a result of its expanding use in early intervention therapies and its use in combination with other chemotherapeutic agents. Taxol, like some other natural substances, is a mitotic spindle poison, but its mode of action is highly specific. In mitotic cells, it binds tubulin heterodimers, promotes 1 Seccion

de Fisiología Vegetal, Facultad de Farmacia, Universidad de Barcelona, Avd. Diagonal 643, E-08028 Barcelona, Spain, e-mail: [email protected]

Biotechnology in Agriculture and Forestry, Vol. 61 Transgenic Crops VI (ed. by E.C. Pua and M.R. Davey) © Springer-Verlag Berlin Heidelberg 2007

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Fig. 1. General structure of taxol (paclitaxel, 1) and baccatin III (2). Ac Acetyl group, Ph phenyl group

and stabilises microtubule assembly, and disrupts cellular division (Jordon and Wilson 1995). The limited supply of taxol from the original source, the bark of the Pacific yew (T. brevifolia), prompted intense efforts to develop alternate sources and means of production. To date, methods have been developed for the semisynthesis of taxol (and its analog docetaxel, or Taxotere) from related taxanes, such as 10-deacetilbaccatin III and baccatin III (Guénard et al. 1993), present in substantial quantities (from 0.2 g kg−1 to 1 g kg−1 ) in the leaves of the European yew, T. baccata, and other yews (e.g. Himalayan yew, T. wallichiana), which is renewable source material. However, this methodology requires the continued use of precursors from natural sources and significant amounts of solvents, both of which can cause environmental problems. Another alternative for obtaining taxol and its synthetically useful progenitors is plant cell suspension culture. Using plant cell culture has several advantages, which are: (1) it is similar to well established procedures that have been used successfully in bacterial fermentation, (2) it facilitates basic studies on taxol biosynthesis (Bonfill et al. 2003; Ketchum et al. 2003; Palazón et al. 2003a; Cusidó et al. 2007), and (3) its process can provide an environmentally friendly path to develop methods to produce taxol and enhance its productivity through cell suspension systems (Ketchum et al. 1999; Cusidó et al. 2002; Navia-Osorio et al. 2002a, b; Kim et al. 2004), or cell immobilised systems (Seki et al. 1997; Bentebibel et al. 2005). Some of the results are promising, and commercial production of taxol from plant cell culture is now a reality (Sohn and Okos 1998). In this chapter, we discuss the production of taxol and related taxanes by cultured cells of Taxus species, mainly from our own research.

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2 Biosynthesis of Taxol Taxol is a highly substituted, polyoxygenated cyclic diterpenoid characterised by the taxane ring system. It differs from other known taxanes either in the substitution pattern, the nature of the ester side-chains, or in the presence of the oxetane ring (D-ring) system (Fig. 1). Potent anti-mitotic activity seems to be restricted to taxanes such as taxol, which possesses a Nbenzoyl-3-phenylisoserine side-chain at C-13, the oxetane ring function, and a benzoil group at C-2. The complex chemical structure of taxol means that total chemical synthesis is not commercially viable at present, and an efficient and economical supply of the drug must rely on biological production systems for the foreseeable future (Cragg et al. 1993). Up-regulation of the taxol biosynthetic pathway by over-expression of selected genes in Taxus cells can potentially address the supply issue. In all cases, improving the biological production yields of taxol depends critically upon a detailed understanding of the biosynthetic pathway, the enzymes catalysing this sequence of reactions, especially the slow steps, and the genes encoding these proteins. The first committed step in taxol biosynthesis (Fig. 2) involves the cyclisation of the universal precursor of plant diterpenoids, geranylgeranyl diphosphate (GGPP), to taxa-4(5),11(12)-diene by taxadiene synthase to establish the taxane core structure. This parent olefin is then hydroxylated at the C5 position by a cytochrome P450 enzyme, representing the first of eight oxygenation steps (of the core) en route to taxol. The resulting intermediate, taxa-4(20),11(12)dien-5-ol, can be acetylated by a well defined acetyltransferase (Jennewein and Croteau 2001). Studies using microsomal preparations from Taxus cells induced for taxol production showed that taxa-4(20),11(12)-dien-5-yl acetate is converted by cytochrome P450-dependent reactions to polyoxygenated taxanes by way of taxa-4(20),11(12)-dien-5α-acetoxy-10β-ol (Jennewein et al. 2003). This same set of experiments also demonstrated the microsomal cytochrome P450-dependent conversion of taxa-4(20),11(12)-dien-5α-ol itself to polyoxygenated derivatives by means of taxa-4(20),11(12)-dien-5α,13α-diol, thereby creating some uncertainty as to the precise order of the early pathway hydroxylation and acylation steps that diverge from the confirmed intermediate taxadienol. Genes encoding several of these taxol biosynthetic enzymes have been isolated, including those for geranylgeranyl diphosphate synthase, taxadiene synthase (TXS), and several acyltransferases (Schoendorf et al. 2001). The biosynthetic pathway of taxol is now quite well known, although some doubts remain about the exact order of oxygenations and acylations on the taxadiene core stucture. DeJong et al. (2006) have been able to successfully reconstruct the early steps of taxol biosynthesis in Saccharomyces cerevisiae, using the genes isolated and cloned by Croteau’s group. Regarding the GGPP substrate used by TXS, it appears to be derived mostly from the mevalonate-independent (methylerytritol 4-phosphate) pathway (Eisenreich et al. 1996; Palazón et al. 2003a; Cusidó et al. 2007), which operates in

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Fig. 2. Outline of taxol biosynthetic pathway. The enzymes involved are: a taxadiene synthase, b cytochrome P450 taxadiene 5α-hydroxylase (involving allylic rearrangement), c taxa-4(20),11(12)dien-5α-o-acetyl transferase, d cytochrome P450 taxane-13α-hydroxylase, and e cytochrome P450 taxane-10β-hydroxylase. The broken arrows signify undefined steps. Modified from Jennewein and Croteau (2001)

parallel with the classic cytosolic acetate/mevalonate pathway for the biosynthesis in the plastids of the universal terpenoid precursor isopentenyl diphosphate. In addition to the formation of the oxetane D-ring, an additional esterification reaction, which is also essential for biological activity, is represented by the attachment of the C-13 side chain to the taxane core. As shown in Fig. 3, it was suggested that this process involved the initial esterification of the C-13 hydroxyl with phenylisoserine followed by N-benzoylation, the preformed phenylisoserine being generated by aminomutase reaction of phenylalaline to β-phenylalanine, followed by α-hydroxylation (Walker and Floss 1998). An alternative mechanism can be envisaged, whereby the C-13 hydroxyl group is first esterified with β-phenylalanine, which is then converted to phenylisoserine via the α-hydroxylation reaction, followed by N-benzoylation of the side-chain (Jennewein and Croteau 2001). As with the order of the oxygenation reactions of taxol biosynthesis, the precise sequence of the acylation reactions and the timing of the epoxidation and ring expansion steps are not yet fully defined, and several inosculating routes to taxol may be possible.

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Fig. 3. Proposed biosynthetic scheme for the origin of the taxol C-13 side-chain by addition of phenylisoserine to baccatin III, followed by N-benzoylation (from Jennewein and Croteau 2001)

3 In Vitro Culture In order to increase the taxol and related taxane productivity in cell and tissue cultures, various strategies have been examined, including optimisation of culture conditions, selection of high taxol-producing cell lines, the use of elicitors, the addition of precursors, and other approaches (Srinivasan et al. 1996; Yukimune et al. 1996; Ketchum et al. 1999; Cusidó et al. 2002; Kim et al. 2004; Bentebibel et al. 2005). Although large-scale production of taxol using Taxus cell cultures is now commercially feasible, and indeed such commercial production is carried out by Phyton Catalytic and ESCA Genetics in the United States, no details of the production processes are available. Generally, when plant cells perceive environmental changes via specific receptors or perception mechanisms, they generate biological responses through specific signal transduction. The accumulation of taxol and related taxanes in Taxus plants is thought to be a biological response to specific external stimuli (Yukimune et al. 1996). Jasmonates are reported to play an important role in a signal transduction process that regulates defence genes in plants (Farmer and Ryan 1990). Exogenously applied methyl jasmonate enhances production of secondary metabolites in a variety of plant species and, particularly, it is the most effective chemical for eliciting taxane production in various Taxus suspension cultures (Cusidó et al. 2002; Bonfill et al. 2003; Ketchum et al. 2003). Moreover, the development of Taxus cell cultures capable of producing significant amounts of taxol and related taxanes, and inducible by elicitation, provides an excellent tool to improve our understanding of how the biosynthesis of these compounds is regulated in vitro. As far as a specific culture system and elicitor is concerned, exposure time and dosage are the two main variables that affect cell growth and yield of secondary metabolites. It appears that plant cells respond differently to exposure time and dosage (Yukimune et al. 1996; Ketchum et al. 1999), which may be attributed to differences not only between plant species, but also between the cell lines within a given species.

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Table 1. Significant recent studies on the capacity of Taxus spp cell cultures to produce taxol and related taxanes

Research summary

Reference

A novel type of bioreactor was successfully developed for the production of taxol and its precursors by culturing cells of T. cuspidata on a pilot scale. Approx. 3 mg l−1 of taxol and 74 mg l−1 total taxanes were obtained after 27 days of culture. All stereoisomers of methyl jasmonate were prepared, and their effects on cell yield and the promotion of taxol and baccatin III production were investigated in cell suspension cultures of T. media. As described in Section 3.2.1, a two-stage culture for T. media cell suspension was carried out using a 5-l stirred bioreactor. A content of 21.12 mg l−1 of taxol and 56.03 mg l−1 of baccatin III was obtained after 20 days of culture. The effects of inoculum size and age on biomass growth and taxol production of elicitor-treated T. yunnanensis cell cultures were studied. Growth rate and capacity to accumulate taxol and baccatin III were measured. Suspension cultures of T. baccata and T. wallichiana were grown in a 20-l bioreactor, and their growth rate and capacity to accumulate taxol and baccatin III were measured. The kinetics of growth and production of taxol and baccatin III by a cell suspension of T. wallichiana were compared in shake flasks and in a 20-l airlift bioreactor. The synergistic effects of various elicitors were investigated in suspension cultures of T. chinensis. The results showed that mixtures of elicitors with different acting paths had a synergistic effect on taxol production, while those with the same acting paths did not. Taxol transport in T. baccata suspension cultures was studied using [14 C]-taxol as a tracer. The absorbed molecule was localised both in walls (20%) and in protoplasts (80%), suggesting an accumulation within vacuoles. Taxol release into the culture medium was demonstrated not to depend on cell lysis. As described in Section 3.1.1, the inhibition of taxol and baccatin III accumulation by mevinolin and fosmidomycin in suspension cultures of T. baccata was studied. The principal taxanes produced by T. media cell suspension cultures during normal growth and upon elicitation with methyl jasmonate were described. The influence of elicitors on taxane production and 3-hydroxy-3-methylglutaril coenzyme A reductase activity in T. media cells was studied. The effects of low-energy ultrasound on oxidative burst, jasmonic acid biosynthesis, and taxol production in T. chinensis suspension cultures were investigated.

Son et al. (2000)

Yukimune et al. (2000)

Cusidó et al. (2002)

Zang et al. (2002)

Navia-Osorio et al. (2002a) Navia-Osorio et al. (2002b) Yuan et al. (2002)

Fornalè et al. (2002)

Palazón et al. (2003a) Ketchum et al. (2003) Bonfill et al. (2003) Wu and Ge (2004)

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Table 1. continued

Research summary

Reference

Two groups of replicate T. cuspidata cell cultures were generated either with mixing flasks or a segregation of parental flasks at each subculture. The results showed that the production level of taxol in subcultures resulting from mixing inocula was sustained at a higher level than from segregated subcultures, which is consistent with the possibility of taxol production being induced by cell signaling within the population. The induction by methyl jasmonate and salicylic acid (SA) of taxol and relevant taxane biosynthesis in suspension cultures of T. chinensis was studied. The results indicated that SA might increase taxol production by blocking the biosynthesis pathway from baccatin III to cephalomannine. As described in Section 3.2.2, the production of taxol and baccatin III using free and immobilised cells of T. baccata was investigated in three different bioreactor types (stirred, airlift, wave). The stirred bioreactor was the most efficient in promoting immobilised cell production of taxol, giving a content of 43.43 mg l−1 after 16 days of culture. As described in Section 3.1.1, the source of isopentenyl diphosphate for the formation of the taxane ring system of taxol and baccatin III in T. baccata cell cultures was studied.

Kim et al. (2004)

Wang et al. (2004)

Bentebibel et al. (2005)

Cusidó et al. (2007)

There are excellent reviews of the early research on establishing Taxus cell cultures, the taxane content achieved, and the medium conditions and elicitors for optimising taxane production (Fett-Neto and DiCosmo 1997; Roberts and Shuler 1998; Takeya 2003; Tabata 2004). Therefore, this chapter discusses the more recent work reported on the capacity of Taxus cell cultures to produce taxol and related taxanes (see Table 1). The main focus of research was to establish the capacity of cell cultures to produce taxol and the precursors used for its semisynthesis, and to establish the suitable conditions for scale-up to commercial production. Since its discovery, the supply of taxol has been limited and, with increasing applications in chemotherapy, the availability and cost of the drug will remain important issues. 3.1 Shake Flask Cultures 3.1.1 Cell Suspensions Secondary metabolite production in plant cell cultures is a process that is not usually dependent on growth. This is the case of taxol in Taxus cell cultures, where the production of this secondary product mainly takes place when the lineal growth phase is completed and the culture is in its stationary growth

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phase (Srinivasan et al. 1996; Fett-Neto and DiCosmo 1997; Cusidó et al. 1999). In such cases, a two-stage culture system seems to be the most adequate in order to stimulate secondary metabolite production. First, plant cells are cultured in a medium that is optimised for their growth, and second, after the optimised culture medium is removed, the cell biomass continues its growth in a production medium that mainly stimulates the biosynthesis of secondary metabolites. At the same time, this system has the advantage of permitting the addition of biosynthetic precursors and elicitors when the secondary metabolite production is at its highest, that is, during the second stage of the culture. The combined effects of the above-mentioned two-stage culture on taxol and baccatin III yields from a T. media cell suspension, together with elicitation and the feeding of putative precursors, were studied by Cusidó et al. (2002). The elicitors assayed were arachidonic acid (AA; 5 g g−1 fresh weight; FW), methyl jasmonate (MJ; 220 g g−1 FW) and vanadyl sulphate (VS; 81.5 g g−1 FW). With regard to biosynthetic precursors, different concentrations of mevalonate (0.19, 0.38, 0.76 mM) and N-benzoylglycine (0.1, 0.2, 0.5 mM) were tested. For taxol and baccatin III measurements, taxanes were extracted from lyophilised cells and the culture medium, as previously described by Cusidó et al. (1999), and quantified using an indirect competitive enzyme immunoassay (CIEIA). Both growth and production media were selected after assaying 24 different culture media. Our results revealed that McCown’s woody plant medium (Lloyd and McCown 1980) with 0.5% sucrose + 0.5% fructose, 2 mg l−1 of Picloram, and 0.1 mg l−1 of kinetin was optimum for cell growth (growth medium), while Gamborg’s B5 medium (Gamborg and Miller 1968) with 3% sucrose, 2 mg l−1 of 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 mg l−1 of 6-benzylaminopurine (BAP; production medium) was optimum for the yield of both taxol and baccatin III. Under the conditions assayed, the addition of MJ to the production medium was the best single strategy for increasing the total (cell-associated + extracellular) taxol and baccatin III content (13.76 mg l−1 on day 18 and 7.10 mg l−1 on day 16, respectively) (data not shown). However, the fact that the greatest concentrations of both compounds were achieved in the latter phase of the culture suggested that the stimulatory action of this elicitor on the biosynthesis of the specific taxanes could not take place until the cells had formed sufficient precursors in response to its presence in the culture medium. For this reason, we tested the combined effect of adding MJ to the production medium together with the most effective concentrations of assayed biosynthetic precursors for increasing taxol yield, i.e. mevalonate and N-benzoylglycine at 0.38 mM and 0.20 mM, respectively. As shown in Fig. 4, in the trials where MJ was combined with the indicated concentrations of mevalonate and N-benzoylglycine, the maximum total taxol and baccatin III content (achieved for both taxanes on day 12) was 15.72 mg l−1 and 10.38 mg l−1 , respectively. Although these increases were not very important with respect to trials with the elicitor alone (factor of 1.2 and 1.5, respectively), these contents of taxol and baccatin III were achieved in a much shorter period of time (6 and 4 days earlier, respectively).

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Regarding basic studies on taxol and related taxane biosynthesis, recent examples of the successful use of Taxus cell cultures include work by Bonfill et al. (2003) and Palazón et al. (2003a). In the former study, we reported how the production of taxol and related taxanes (10-deacetylbaccatin III, baccatin III, 10-deacetyltaxol, cephalomannine) was affected by adding the elicitors AA, MJ and VS to cells of T. media growing in the same production medium and culture conditions described above, but in this case, in order to quantify the taxanes in different samples, a high performance liquid chromatography (HPLC) method was used. From the results obtained, it can be seen that the yields of baccatin III, 10-deacetyltaxol and taxol were significantly (P < 0.001, t-test) increased in the presence of MJ (up to 3-fold, 5-fold, 12-fold, respectively), whereas those of 10-deacetylbaccatin III and cephalomannine were significantly (P < 0.001) increased in the presence of VS (up to 40-fold) and AA (up to 4-fold), respectively. Our results suggest that MJ contributes to taxol production not only by activating the biosynthesis steps from GGPP to baccatin III, but also those from baccatin III to taxol, and possibly even the benzoylation of N-debenzoyltaxol. The fact that taxol increases much more than cephalomannine, when both taxanes have a baccatin III moiety, may be due to the lower activation of tigloyation than benzoylation by the assayed MJ concentration or the lack of a donor for a side-chain, such as tiglic acid. Cephalomannine is very similar to taxol structurally, differing in that it has a tigloyl group in position C-3 of the C-13 chain instead of a benzoyl group. Considering that isopentenyl diphosphate (IPP) is not only an essential precursor but also the first intermediate in taxane biosynthesis, it is important to elucidate its source in this process in order to know to what extent cytosolic mevalonate and plastid non-mevalonate pathways contribute to the formation of the taxane ring system of taxol. In our previous work (Palazón

Fig. 4. Comparison of total (cell-associated plus extracellular) content of taxol and baccatin III in cell suspensions of T. media grown in production medium in shake flasks supplemented with a combination of methyl jasmonate (220 μg g−1 FW), mevalonate (0.38 mM), and N-benzoylglycine (0.20 mM), and with the elicitor alone. In all cases, the inoculum consisted of 100 g l−1 of cells (FW). Data represent average values from three replicates ± SD. Black columns Methyl jasmonate. Grey columns Methyl jasmonate + mevalonate + N-benzoylglycine

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et al. 2003a; Cusidó et al. 2007) using Taxus cell lines, the dependence of methyl jasmonate-induced and non-induced taxol and baccatin III production on IPP derived from mevalonate and non-mevalonate pathways was detected by selectively blocking the IPP biosynthesis with specific inhibitors: fosmidomycin as an inhibitor of the non-mevalonate branch of the pathway and mevilonin as an inhibitor of the mevalonate branch. Fosmidomycin is an inhibitor of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DOXP reductoisomerase) and mevinolin competitively inhibits the binding of the natural substrate hydroxymethylglutaryl-CoA (HMG-CoA) to the active site of the enzyme HMG-CoA reductase. On the basis of these results, it can be inferred that, although the cell line type was an important variable for taxol and baccatin III production, the biosynthesis of both taxanes was reduced by fosmidomycin to a much greater extent than by mevinolin, irrespective of the presence of 100 μM methyl jasmonate. These results clearly suggest that the biosynthesis of IPP responsible for the taxane ring formation takes place mainly via the non-mevalonate pathway, which fosmidomycin blocks. 3.1.2 Immobilised Cells There are various studies on the use of immobilised plant cells for the continuous production of valuable plant metabolites. Plant cells have been immobilised within various kinds of matrices, such as alginate, carrageenan, and different synthetic supports, but there has been little evaluation of the effect of the variation of immobilisation parameters on the production of a secondary metabolite such as taxol. Recently (Bentebibel et al. 2005), we investigated the effect of MJ elicitation and cell immobilisation on cell viability, growth rate, and the production of taxol and its synthetically useful progenitor, baccatin III, in cell cultures of T. baccata growing in a medium that specifically stimulates the biosynthesis of both taxanes. To our knowledge, this is the first study of the combined effect of these particular conditions on the biosynthesis of taxol and related taxanes. Seki et al. (1997) reported an immobilised Taxus cell system, but did not experiment with elicitation. The T. baccata cell suspension was maintained in 175-ml flasks in the dark at 25.0 ± 0.2 ◦ C and 100 ± 1 rpm in a shaker-incubator. Every 10–12 days, 1 ± 0.2 g of cells were used as inoculum in 10 ml of Gamborg’s B5 medium with 0.5% sucrose, 0.5% fructose, 2 mg l−1 of NAA and 0.1 mg l−1 of BAP (growth medium), which was previously demonstrated to be optimum for the cell growth of this species (Palazón et al. 2003a). For the preparation of biocatalyst beads, sodium alginate at concentrations of 1.5%, 2.0%, or 2.5% was used for cell immobilisation. Considering that the taxol production in Taxus cell cultures takes place mainly when the lineal growth phase has ended and the culture is in its stationary phase, 2.0 ± 0.2 g wet weight of free cells grown for 13 days in growth medium (the length of time

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necessary for them to enter the stationary growth phase) were mixed with 10 ml of a sterile solution of sodium alginate (High Viscosity; Sigma) and dropped into 100 ml of a 2.5% sterile calcium chloride solution to form biocatalyst beads by ionotropic gelation of alginate. The beads of calcium alginate with entrapped cells were washed for 10 min with distilled water and hardened in the calcium chloride solution for about 20 min. The beads were transferred to 10 ml of B5 medium with 3% sucrose, 2 mg l−1 of Picloram and 0.1 mg l−1 of kinetin (production medium), which was previously selected as optimum for both taxol and baccatin III yield in T. baccata cultured cells (Palazón et al. 2003a), and then submitted to the same culture conditions as free cells. Control experiments were performed with free cells in their stationary growth phase, cultured in the production medium supplemented with the elicitor MJ (100 μM) and the precursors mevalonate (0.38 mM) and N-benzoylglycine (0.2 mM). The concentrations of elicitor and both precursors were those established previously as optimum for taxol biosynthesis (Cusidó et al. 2002). At the end of culture, the calcium alginate support was dissolved and liberated cells and free cells were lyophilised. Taxane extraction and quantification of taxol and baccatin III from lyophilised cells and culture medium was performed as indicated in Section 3.1.1. The total production values of taxol and baccatin III (cell-associated plus extracellular) in Fig. 5 show that cell immobilisation stimulated the production of both taxanes, although there were differences according to the alginate concentration. The maximum accumulation of taxol (13.20 mg l−1 ) was achieved at day 24 with 1.5% alginate, while the maximum accumulation with alginate concentrations of 2.0% and 2.5% was achieved at the end of the culture (10.85 mg l−1 , 11.90 mg l−1 , respectively), and was less. Although the effect of the alginate concentration on the baccatin III production was evident, it differed clearly from that reported for taxol. Hence, the maximum accumulation (4.62 mg l−1 ) was achieved at day 24 using a concentration of 2.5%, while with concentrations of 2.0% and 1.5%, the maximum accumulation of taxane was obtained at day 8 (3.21 mg l−1 ) and day 24 (2.22 mg l−1 ), respectively. These variations may reflect differences in the enzyme concentrations induced by the three alginate concentrations tested. In this respect, the charged nature and/or calcium binding capacity of alginate may play a role. 3.2 Bioreactor Since taxol was approved for the treatment of ovarian cancer in 1992 by the FDA, the number of publications related to the cell culture of Taxus species aimed at the production of taxol have increased steadily. Typically, such studies are performed in shake flasks, which are inexpensive and facilitate multiple experiments with replicates. Nevertheless, there are relatively few reports on the growth of cell suspensions of Taxus species in bioreactors. Commercial bioreactors that provide more complete control of operating conditions and

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Fig. 5. Comparison of total (cell-associated plus extracellular) concentration of taxol and baccatin III in T. baccata free cells and alginate-entrapped cells using 1%, 1.5% and 2.5% alginate solution, all grown in production medium supplemented with methyl jasmonate (100 μM), mevalonate (0.38 mM), and N-benzoylglycine (0.2 mM). In all cases, the inoculum consisted of 100 g l−1 of cells in stationary growth state. Data represent average values from five separate experiments ± SD. White columns 1.5% alginate, grey columns 2% alginate, black columns 2.5% alginate

facilitate monitoring are expensive. This is particularly true when many units are needed simultaneously, but an increase in volumetric productivity is crucial for potential commercialisation. The scale-up of taxol production using Taxus cell suspensions from shake flask to bioreactor is the first and, in many cases, the most difficult step in scaling-up. This is because production processes using plant cell cultures often result in reduced productivities (Roberts and Shuler 1998). However, the successful cultivation of Taxus cells for taxol production has been carried out by several groups using different types of bioreactors (Srinivasan et al. 1995; Pestchanker et al. 1996; Son et al. 2000; Cusidó et al. 2002; Navia-Osorio et al. 2002a, b; Bentebibel et al. 2005). Here, we describe only the scale-up carried out by our group from shake flask cultures of T. media cell suspension culture and T. baccata immobilised cell culture, both considered previously (Sections 3.1.1 and 3.1.2, respectively). 3.2.1 Scale-Up from Shake Flask Cultures of Taxus Media Cell Suspension The taxane production values obtained during our shake flask experiments (Section 3.1.1) clearly showed that a reliable way to improve the yield of both taxanes was to supplement the production medium with MJ (220 μg g−1 FW) together with mevalonate (0.38 mM) and N-benzoylglycine (0.20 mM). For this reason, a two-stage culture (the first stage being for cell growth, the second for taxol and baccatin III yield) was carried out in a 5-l turbine stirred tank bioreactor (Applikon Dependable Instruments, Schiedam, The Netherlands), aerated with a sintered steel sparger. The flow was set at 0.8 l min−1 at the beginning of the experiment, gradually increased to 1.5 l min−1 , and maintained

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Fig. 6. Time courses of biomass accumulation (A) and total (cell-associated plus extracellular) concentration of taxol (B) and baccatin III (C) by a two-stage culture of T. media cell suspension in a 5-l stirred bioreactor running for 30 days. The first stage was in growth medium for 12 days, and the second stage was either in production medium without supplement (control), or supplemented as indicated in Fig. 4. The cultivation parameters are given in Section 3.2.1. Data represent average values from three replicates ± SD

at this level until the end of the culture period with a mass flow control system. The working volume was kept at 3.5-l culture medium, at a temperature of 25 ◦ C. The inoculum always consisted of 100 g l−1 (FW) of cells. T. media cells were cultured in growth medium for the first 12 days, which was when the stationary growth phase commenced. Subsequently, the medium was removed and the resulting cell biomass continued growing in the production medium for an additional 24 days, either under control conditions, or supplemented with the elicitor and two precursors in the concentrations indicated earlier. As shown in Fig. 6A, under control conditions, the cell biomass increased more than 2.0-fold during the 12 days in the growth medium, while the resulting biomass (19.13 g l−1 dry weight) increased only 1.2-fold during the additional 24 days in the production medium, as could be deduced from the maximum value of biomass yield (24.17 g l−1 dry weight) reached at the end of the 36-day culture period. It seems evident from these results that the growth of cells in the resulting biomass was significantly inhibited (P < 0.001) in the

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production medium, suggesting that their stationary growth phase began almost immediately. Compared with the control, cell growth was barely affected by the addition of MJ, mevalonate, and N-benzoylglycine to the production medium. However, notable differences in total taxol and baccatin III content (cell-associated plus extracellular) were observed (Fig. 6B, C). The greatest total contents of taxol (21.12 mg l−1 ) and baccatin III (55.63 mg l−1 ), both reached on day 20 (after 8 days of culture in the production medium), were 9- and 26fold higher, respectively, in the supplemented trials. The fact that there was a greater accumulation of taxol and baccatin III in the bioreactor culture than in shake flasks (factors of 1.4 and 5.4, respectively; see results in Fig. 4), when both culture types were in the highest productive state, confirms the suitability of the selected growth conditions used in the bioreactor culture. 3.2.2 Scale-Up from Shake Flask Cultures of Taxus baccata Immobilised Cells In contrast to the culture beads prepared with 1.5% alginate (which during shake flask studies gave the greatest production of taxol; see Fig. 5, Section 3.1.2), those prepared with 2% alginate were more resistant to the agitation selected for their culture in the stirred bioreactor, so they were chosen for the subsequent scale-up study. This was performed in three different commercially available types of bioreactor: a 5-l turbine stirred tank reactor (Applikon Dependable Instruments), a 4-l multi-purpose tower airlift reactor (Applikon Dependable Instruments), and a 2-l wave reactor (Wave Biotech, Switzerland). The new wave reactor described by Palazón et al. (2003b) is a mechanically driven submerged bioreactor consisting of a measuring and control unit, a cultivation chamber or cellbag, and a pneumatically operated rocking unit. The turbulent, but shear-reduced mixing in the cellbag is caused by a wave motion of the rocking unit. The conditions established for the growth of free and immobilised cells of T. baccata are summarised in Table 2. When enough cellular biomass in a stationary growth phase was available from the respective small scale cultures, it was transferred to an inoculation flask with the corresponding volume of production medium supplemented with 100 μM MJ, 0.38 mM mevalonate, and 0.2 mM N-benzoylglycine (see Section 3.1.2), and then to the vessel of the stirred and airlift bioreactors or the cellbag of the wave bioreactor. When comparing the total taxol and baccatin III production (cell-associated plus extracellular) during the culture of free and immobilised cells in the bioreactors (Fig. 7A–C), it is evident that cell entrapment in calcium alginate markedly stimulated the yield of both taxanes, although there were considerable differences according to the type of reactor used. In the stirred bioreactor, at day 16, when free and immobilised cells reached their maximum taxol production, the content of this taxane in the alginate-entrapped cells was 43.43 mg l−1 , significantly higher (P < 0.001) than that of free growing cells (more than 5-fold). It should be emphasised that this taxol production

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Table 2. Cultivation parameters for free and immobilised cells of Taxus baccata cultured in a 5-l stirred reactor, a 4-l airlift reactor, and a 2-l wave reactor running for 24 days in batch mode. Standard conditions for all cultivations: inoculum/culture medium ratio = 1/10; temperature = 25 ◦ C

Bioreactor type

Stirred

Airlift

Wave

Culture type

Parameters Shaker (rpm)

Rocking Wave frequency angle (waves min−1 ) (2−10◦ )

Air Working flux volume (l) (l min−1 )

100 100

– –

– –

1.0 1.0

1.5 1.5

– –

– –

– –

2.0 2.0

1.5 1.5

–

6◦

0.3

0.4

Immobilised – cells

6◦

Days: 0 (20), 1 (24), 10 (34), 14 (40) Days: 0 (20), 1 (24), 10 (34), 14 (40)

0.3

0.4

Free cells Immobilised cells Free cells Immobilised cells Free cells

Fig. 7. Comparison of growth index (final fresh weight/inoculum fresh weight) and total concentracion (cell-associated + extracellular) of taxol and baccatin III in free and immobilised cells of T. baccata cultures in 5-l stirred (A), 4-l airlift (B) and 2-l wave (C) bioreactors running for 24 days in batch mode under the cultivation parameters given in Table 2. In all cases, the studies were performed using the inoculum and supplemented production medium described in Fig. 5. Data represent average values from three separate experiments ± SD

was 7.2-fold greater than the taxane content that Seki et al. (1997) achieved after 20 days of bioreactor culture of a cell suspension of T. cuspidata, also immobilised within calcium alginate beads.

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As can be deduced from the values in Fig. 7A–C, the best strategy for increasing the yield of taxol is to combine the effects of cell immobilisation, a culture medium that stimulates the product biosynthesis to the detriment of the growth of productive cells, and the stirred reactor system. This is also applicable to baccatin III using the wave reactor system, in which the content of 7.78 mg l−1 was 2.4-fold higher than in the shake flask culture. The taxol production of 43.43 mg l−1 obtained at 16 days of culture in the stirred bioreactor, equivalent to a rate of 2.71 mg l−1 day−1 , exceeded that reported by academic laboratories, including ours, for other Taxus spp cultures in bioreactors, where the optimum values were 18.7 mg l−1 by day 27 (Srinivasan et al. 1995), 22 mg l−1 by day 20 (Pestchanker et al. 1996), 3.0 mg l−1 by day 27 (Son et al. 2000), 21.04 mg l−1 by day 24 (Navia-Osorio et al. 2002b), and 21.12 mg l−1 by day 20 (Cusidó et al. 2002). Considerably greater taxol concentrations (the maximum level being 295 mg l−1 reached at day 14; Tabata 2004), have been reported by industrial groups.

4 Conclusions and Prospects Taxol constitutes an important anti-cancer drug, but despite intense efforts in total chemical synthesis, the semisynthetic method and production by Taxus cell cultures are presently the only commercially viable routes to this important chemotherapeutic agent. However, the semisynthesis of taxol relies on the extraction of advanced taxanes (e.g. 10-deacetylbaccatin III, baccatin III) from the plant material, which is subject to significant variation in taxane content due to epigenetic and environmental factors, and necessarily involves costly purification of the target metabolite from co-occurring taxanes. Therefore, the best alternative for obtaining taxol and its synthetically useful progenitors are Taxus cell culture systems. It is evident that the combined effects of various enhancement strategies can stimulate taxol and related taxane production much more than any individual approach and this can be particularly valuable in large-scale systems. However, sustaining such high production of secondary metabolites is a well documented problem in plant cell culture systems. With a full understanding of the taxol biosynthetic pathway and the availability of the genes responsible, it may be possible to bioengineer Taxus cell cultures for commercially sustainable production rates of this important chemotherapeutic agent and its synthetically useful progenitors. Thus, it should be possible to increase the production of desirable taxanes by over-expressing genes that control the slow biosynthetic steps, while at the same time suppressing the production of undesired taxanes by anti-sense technology. Both approaches would improve flux to the desired end-product and simplify downstream purification processes.

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References Appendino G (1993) Taxol (paclitaxel): historical and ecological aspects. Fitoterapia 64[Suppl 1]:5–25 Bentebibel S, Moyano E, Palazón J, Cusidó RM, Bonfill M, Eibl R, Piñol MT (2005) Effects of immobilization by entrapment in alginate and scale-up on paclitaxel and baccatin III in cell suspension cultures of Taxus baccata. Biotechnol Bioeng 89:647–655 Bonfill M, Palazón J, Cusidó RM, Joly S, Morales C, Piñol MT (2003) Influence on taxane production and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in Taxus media cells. Plant Physiol Biochem 41:91–96 Cragg GM, Schepartz SA, Suffmess M, Grever MR (1993) The taxol supply crisis. New NCI policies for handling the large-scale production of novel natural product anticancer and anti-HIV agents. J Nat Prod 56:1657–1668 Cusidó RM, Palazón J, Navia-Osorio A, Mallol A, Bonfill M, Morales C, Piñol MT (1999) Production of taxol and baccatin III by a selected Taxus baccata callus line and its derived cell suspension culture. Plant Sci 146:101–107 Cusidó RM, Palazón J, Bonfill M, Navia-Osorio A, Morales C, Piñol MT (2002) Improved paclitaxel and baccatin III production in suspension cultures of Taxus media. Biotechnol Prog 18:418– 423 Cusidó RM, Palazón J, Bonfill M, Expósito O, Moyano E, Piñol MT (2007) Source of isopentenyl diphosphate for taxol and baccatin III biosynthesis in cell cultures of Taxus baccta. Biochem Eng J 33:159–167 DeJong JH, Liu Y, Bollon AP, Long RM, Jennewein S, Williams D, Croteau RB (2006) Genetic engineering of taxol biosynthetic genes in Saccharomyces cerevisiae. Biotechnol Bioeng 93:212–224 Eisenreich W, Menhard B, Hylands PJ, Zenk MH, Bacher A (1996) Studies on the biosynthesis of taxol: taxane carbon skeleton is not of mevalonoid origin. Proc Natl Acad Sci USA 93:6431– 6436 Farmer EE, Ryan CA (1990) Interplant communication: airborne methyl jasmonates induces synthesis of proteinase inhibitors in plant leaves. Proc Natl Acad Sci USA 87:7713–7716 Fett-Neto AG, DiCosmo F (1997) Taxol and taxane production by cell culture. In: Meyers RA (ed) Encyclopedia of molecular biology and molecular medicine, vol 6. Wiley, Weinheim, pp 10–17 Fornalè S, Esposti DD, Navia-Osorio A, Cusidó RM, Palazón J, Piñol MT, Bagni N (2002) Taxol transport in Taxus baccata cell suspension cultures. Plant Physiol Biochem 40:81–88 Gamborg OL, Miller RA (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158 Guénard D, Guéritte-Voegelein F, Potier P (1993) Taxol and taxotere: discovery, chemistry, and structure–activity relationships. Acc Chem Res 26:160–167 Jennewein S, Croteau R (2001) Taxol biosynthesis, molecular genetics, and biotechnological applications. Appl Microbiol Biotechnol 57:13–19 Jennewein S, Rithner CD, Williams RM, Croteau R (2003) Taxoid metabolism: taxoid 14βhydroxylase is a cytochrome P450-dependent monooxigenase. Arch Biochem Biophys 413:262–270 Jordon MA, Wilson L (1995) Microtubele polymerisation dynamics, mitotic block, and cell death by paclitaxel at low concetrations. In: Georg GI, Chen TT, Ojima I, Vyas DM (eds) Taxane anticancer agents. (ACS symposium series 583) American Chemical Society, Washington, D.C., pp 138–153 Ketchum REB, Gibson D, Croteau R, Shuler, ML (1999) The kinetics of taxoidaccumulation in cell suspension cultures of Taxus following elicitation with methyljasmonate. Biotechnol Bioeng 62:97–105 Ketchum REB, Rithner CD, Qiu D, Kim YS, Williams RM, Croteau RB (2003) Taxus metabolomics: methyl jasmonate preferentially induces production of taxoids oxygenated at C-13 in Taxus media cell cultures. Phytochemistry 62:901–909

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Kim BJ, Gibson DM, Shuler ML (2004) Effect of subculture and elicitation on instability of taxol production in Taxus sp. suspension cultures. Biotechnol Prog 20:1666–1673 Lloyd G, McCown B (1980) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Int Plant Soc Proc 30:421 Navia-Osorio A, Garden H, Cusidó RM, Palazón J, Alfermann AW, Piñol MT (2002a) Taxol and baccatin III production in suspension cultures of Taxus baccata and Taxus wallichiana in an airlift bioreactor. J Plant Physiol 159:97–102 Navia-Osorio A, Garden H, Cusidó RM, Palazón J, Alfermann AW, Piñol MT (2002b) Production of paclitaxel and baccatin III in a 20-L airlift bioreactor by a cell suspension of Taxus wallichiana. Planta Med 68:336–340 Palazón J, Cusidó RM, Bonfill M, Morales C, Piñol MT (2003a) Inhibition of paclitaxel and baccatin III accumulation by mevinolin and fosmidomycin in suspension cultures of Taxus baccata. J Biotechnol 191:157–163 Palazón J, Mallol A, Eibl R, Lettenbauer C, Cusidó RM, Piñol MT (2003b) Growth and ginsenoside production in hairy root cultures of Panax ginseng using a novel bioreactor. Planta Med 69:344–349 Pestchanker LJ, Roberts SC, Shuler ML (1996) Kinetics of taxol production and nutrient use in suspension cultures of Taxus cuspidata in shake flasks and Wilson-type bioreactor. Enzyme Microb Technol 19:256–260 Roberts S, Shuler ML (1998) Strategies for bioproduct optimisation in plant cell tissue cultures. In: Zaborsky OR (ed) BioHydrogen. (Proceedings of an international conference on biological hydrogen production) Plenum, New York, pp 483–491 Schoendorf A, Rithner CD, Williams RM, Croteau R (2001) Molecular cloning of a cytochrome P450 taxane 10 β-hydroxylase cDNA from Taxus and functional expression in yeast. Proc Natl Acad Sci USA 98:1501–1506 Seki M, Ohzora C, Takeda M, Furusaki S (1997) Taxol (paclitaxel) production using free and immobilized cells of Taxus cuspidate. Biotechnol Bioeng 53:214–219 Sohn H, Okos MR (1998) Paclitaxel (taxol): From nut to drug. J Microbiol Biotechnol 8:427–440 Son SH, Choi SM, Lee YH, Choi KB, Yun SR, Kim JK, Park HJ, Kwon OW, Noh EW, Seon JH, Park YG (2000) Large-scale growth and taxane production in cell cultures of Taxus cuspidata (Japanese yew) using a novel bioreactor. Plant Cell Rep 19:628–633 Srinivasan V, Pestchanker L, Moser S, Hirasuna T, Taticek RA, Shuler ML (1995) Taxol production in bioreactors: kinetics of biomass accumulation, nutrient uptake, and taxol production by cell suspensions of Taxus baccata. Biotechnol Bioeng 47:666–676 Srinivasan V, Ciddi V, Bring V, Shuler ML (1996) Metabolic inhibitors, elicitors and precursors as tools for probing yield limitation in taxane production by Taxus chinensis cell cultures. Biotechnol Prog 12:457–465 Suffness M, Wall ME (1995) Discovery and development of taxol. In: Suffness M (ed) Taxol: sciences and applications. CRC, Boca Raton, pp 3–25 Tabata H (2004) Paclitaxel production by plant-cell-culture technology. Adv Biochem Eng Biotechnol 87:1–23 Takeya K (2003) Plant tissue culture of taxoids. In: Itokawa H, Lee K-H (eds) Taxus. The genus Taxus. Taylor and Francis, London, New York, pp 134-150 Thayer AM (2000) Busting down a blockbuster drug. Chem Eng News 78:20–21 Walker KD, Floss HG (1998) Detection of phenylalanine amino-mutase in cell free extracts of Taxus brevifolia and preliminary characterization of its reaction. J Am Chem Soc 120:5333– 5334 Wang YD, Yuan YJ, Wu JC (2004) Induction studies of methyl jasmonate and salicylic acid on taxane production in suspension cultures of Taxus chinensis var. mairei. Biochem Eng J 19:259–265 Wu J, Ge X (2004) Oxidative burts, jasmonic acid biosynthesis, and taxol production induced by low-energy ultrasound in Taxus chinensis cell suspension cultures. Biotechnol Bioeng 85:714–721

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Section III Ornamental Crops

III.1 Roses S.S. Korban1

1 Introduction Roses (Rosa) have been cultivated as ornamental plants for more than 2,000 years, and they are amongst the most economically important ornamental crops grown world-wide (Phillips and Rix 1988). The genus Rosa, belonging to the family Rosaceae, includes more than 150 species and thousands of cultivars (Gudin 2000). Most modern roses do not belong to a single rose species, but are complex hybrids derived from multiple species (Gudin 2000). Wild species are often diploids (2x = 14), while almost all cultivated roses are tetraploids (4x = 28). Rose chromosomes are small, with an average DNA content of 1.1 pg/2C for diploid roses (Yokoya et al. 2000; Rajapakse et al. 2001). As most rose cultivars are tetraploids, it is difficult to conduct genetic analysis with this group of plants. Thus, diploid species and varieties of rose have been used instead for genetic studies (Debener 1999; Debener and Mattiesch 1999; Gudin 2000). Despite the low chromosome number and small genome size, little is known on the genetics of rose (De Vries and Dubois 1996; Gudin 2000). This is attributed to heterozygosity, varying ploidy amongst species, difficulties in sexual hybridization and reproduction, low seed set, and poor seed germination. Nevertheless, many breeding efforts are underway, resulting in the ongoing development and release of new cultivars. However, it is with availability of molecular markers, genetic linkage maps, genomic approaches, and genetic engineering that advances will be made in the genetic improvement of roses. This review focuses on advances in these areas of rose research.

2 Advances in Molecular Markers for Genetic Studies and Breeding 2.1 Genetic Diversity and Identification In recent years, various molecular markers, including random amplified polymorphic DNAs (RAPDs), restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs), and simple sequence re1 Department

of Natural Resources & Environmental Sciences, 310 ERML, University of Illinois, 1201 W. Gregory, Urbana, IL 61801, USA, e-mail: [email protected]

Biotechnology in Agriculture and Forestry, Vol. 61 Transgenic Crops VI (ed. by E.C. Pua and M.R. Davey) © Springer-Verlag Berlin Heidelberg 2007

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peats (SSRs), have been used to distinguish rose cultivars and to determine genetic relationships (Ballard et al. 1996; Debener et al. 1996; Matsumoto and Fukuri 1996; Millan et al. 1996; Reynders-Aloisi and Bollerau 1996; Xu et al. 2004). These various marker systems have been highly valuable in genotyping most rose varieties (Ballard et al. 1996; Debener et al. 1996). More recently, Esselink et al. (2003) identified 24 polymorphic sequenced-tagged microsatellite site (STMS) markers with easily scorable allele profiles, from six different linkage groups, to characterize 46 hybrid tea cultivars and 30 rootstock varieties belonging to different species. Another important source of new rose cultivars is the selection of sport mutants derived via spontaneous mutations (Schum and Preil 1998). These mutants most commonly involve variations in flower color, flower shape, or other ornamental characters. Several studies have been conducted to discern and identify genetic differences between sports and original parental cultivars, using various molecular markers. However, differences were not detected between sport mutants and the original cultivars from which they were derived (Weising et al. 1995; Esselink et al. 2003). Recently, Debener et al. (2000) used RAPDs as well as AFLPs to characterize sports of two cut rose varieties, as well as a garden rose variety. Although polymorphisms were not detected between sports and the original variety of cut rose, five polymorphisms were detected between the garden rose variety and its sports. In contrast, large numbers of polymorphisms were detected between parents and offspring derived from sexual hybridizations. 2.2 Mapping and Linkage of Traits of Economic Importance Advances in molecular genetic mapping studies have contributed to better understanding of rose genetics, as well as those genes controlling various important horticultural traits (Malek et al. 2000; Rajapakse et al. 2001; Crespel et al. 2002; Debener 2003; Kaufmann et al. 2003). Debener (1999) investigated the presence/absence of prickles and recurrent flowering in segregating populations of diploid R. multiflora hybrids, deemed valuable for investigating important horticultural traits. He reported that traits, including pink flower color, double flowers, and prickles, were inherited as single dominant genes or pairs of dominant complementary genes in these populations. In contrast, recurrent flowering was confirmed to be inherited as a single recessive gene. Using a diploid population of R. multiflora hybrids, Debener and Mattiesch (1999) constructed the first molecular genetic linkage map for rose covering over 300 RAPD and AFLP markers. Seven pairs of homologous linkage groups were identified. Genes were mapped controlling pink flower color (Blfa) and double flowers (Blfo). Later, Debener et al. (2001) saturated this map with additional AFLPs, SSRs, RFLPs, and sequence-specific amplified regions (SCARs), and were able to map yet another gene, a resistance gene (Rdr1) to blackspot (Diplocarpon rosae) disease. Rajapakse et al. (2001) developed two genetic

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maps using a tetraploid population and identified genes for prickles and the enzyme malate dehydrogenase (Mdh). Crespel et al. (2002) published a map of a dihaploid population using AFLP markers and mapped genes controlling such traits as the number of prickles, double corolla, and recurrent blooming. All these maps consisted of medium marker densities and provided initial tools for genetic research, as well as contributing to efficient introgression of useful traits via marker-assisted breeding of roses (Rajapakse 2003). Debener et al. (2003) used AFLP markers to determine whether a selfincompatible diploid rose genotype was capable of self-fertilization under field conditions. These authors concluded that all plants were outcrossed events, thus demonstrating that isolation distances of 250 m from large rose plantations were insufficient to prevent gene flow between populations. Recent work on rose genetics and molecular biology concentrated on ornamental characters, including flower structure, recurrent flowering, plant morphology, scent (Debener 1999; Channeliere et al. 2002), and resistance to the most important pathogens (Malek and Debener 1998; Hattendorf et al. 2004). Kaufmann et al. (2003) constructed a bacterial artificial chromosome (BAC) library for R. rugosa consisting of 27,264 clones, 0.5 pg or 500 Mb, and corresponding to 5.2× haploid genome equivalents. This BAC library provides opportunities for physical mapping as well as positional cloning of genes of interest. In fact, this BAC library was used to assemble a BAC contig spanning the Rdr1 locus conferring resistance to blackspot. Using bulked segregant analysis and 816 AFLP primer combinations, a fine-scale map of the Rdr1 locus was constructed and used to assemble the BAC contig (400 kb), consisting of a minimal tiling path of six BAC clones covering the Rdr1 locus. A cluster of at least five resistance gene analogs belonging to the TIR-NBS-LRR family was identified within this contig, suggesting the presence of a cluster of resistance genes around Rdr1. Linde et al. (2004) demonstrated that in the diploid rose population 97/9, resistance to powdery mildew race 9 was controlled by a major dominant resistance gene, Rpp1. Several AFLP molecular markers were identified closely linked to Rpp1 at intervals of 5 cM between two adjacent markers, via bulked segregant analysis, and these were readily converted into reliable and robust SCARs. No linkage was found between the two R genes Rpp1 and Rdr1. Furthermore, the genetic model based on a single dominant resistance gene was supported by the marker data. Yan et al. (2005) established a high-density genetic map with a number of anchor markers to dissect genetic variation in rose. Linkage maps for the diploid 94/1 population, consisting of 88 individuals, were constructed using a total of 520 molecular markers, including AFLP, SSR, PK, RGA, RFLP, SCAR, and morphological markers. Seven linkage groups, putatively corresponding to the seven haploid rose chromosomes, were identified for each parent, spanning 487 cM and 490 cM, respectively. The average length of 70 cM may cover more than 90% of the rose genome. An integrated map was constructed by incorporating the homologous parental linkage groups, resulting in seven linkage groups with a total length of 545 cM. This linkage map is currently the most

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advanced map in rose. The SSR markers, together with RFLP markers, provide anchor points for future map alignment studies in rose and related species. The codominant AFLP markers were used to integrate the parental maps.

3 Cloning and Characterization of Genes of Economic Value 3.1 Ethylene Receptors Genes involved in ethylene perception are of particular interest as the role of ethylene is central in flower initiation and senescence, amongst other physiological and developmental processes. Molecular studies have been conducted to understand ethylene perception and signal transduction pathways by taking advantage of knowledge obtained from alterations in the triple response in ethylene insensitive mutants in Arabidopsis. Müller et al. (2000) reported that differences in flower longevity in R. hybrida were due partly to differences in ethylene receptor levels, and they were able to isolate and partially sequence four genes, RhETR1, RhETR2, RhETR3, and RhETR4, belonging to different subgroups of ethylene receptor genes. Expression analysis of these various genes suggest they are differentially expressed throughout flower development and that differences in flower longevity amongst rose cultivars are likely attributed to differences in levels of expression of these various genes. For example, while RhETR2 is expressed constitutively throughout flower development, RhETR3 expression increases in senescing flowers of a cultivar having a short floral life, but remains low in flowers of a cultivar having long-lasting flowers (Müller et al. 2000). Moreover, ethylene and abscisic acid (ABA) treatments may modulate flower sensitivity to ethylene by regulating the transcript levels of some of these four RhETR genes. Wang et al. (2004) isolated seven putative 1-aminocyclopropane-1carboxylate (ACC) synthase clones from a cDNA library of aging rose petals of R. hybrida cv. Kardinal. A full-length ACC synthase cDNA was cloned and sequenced, designated PKacc7, deemed a member of a multigene family and expressed specifically in rose petals, ovaries, and sepals. Accumulation of ACC synthase transcripts correlated with ethylene concentrations during flower development and senescence. 3.2 Genes Associated with Floral Scent Over the years, rose fragrance seems to have been lost during the later stages of breeding, particularly in Hybrid Teas destined for cut-flower production which are essentially selected primarily for flower form and vase life. In recent years, efforts have focused on identifying, cloning, and characterizing several genes involved in scent formation in roses. Floral scent is one of the most important valuable traits in roses, and more than 400 volatile compounds, including

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hydrocarbons, alcohols, esters, aromatic ethers, aldehydes, oxides, and norisoprenes, amongst others, have been identified in the floral scent of various rose cultivars (Lavid et al. 2002). Also, Channeliere et al. (2002) constructed a cDNA library from petal tissue harvested from the diploid R. chinensis variety Old Blush, and 1,794 rose petal cDNA clones were sequenced. Cluster analysis identified 242 groups of sequences and 635 singletons, indicating that the database represents a total of 877 genes. Expression analysis indicated that transcripts of several of the genes identified accumulated specifically in petals and stamens, and are involved in the biosynthesis of terpenoid and methyl-ether volatile components of floral scent. Guterman et al. (2002) also constructed cDNA libraries from petals of the two tetraploid R. hybrida cultivars, ‘Fragrant Cloud’ and ‘Golden Gate’, and sequenced over 3,000 cDNA clones from these libraries. An annotated petal expressed sequence tag (EST) database of ∼2,100 unique genes was generated and used to create a microarray. Expression profiling, focusing on secondary metabolism-related genes associated with scent production, combined with chemical analysis of volatile composition, led to the discovery of novel flower fragrance-related genes. Functional analysis of these ESTs identified two closely related cDNAs present in both rose cultivars, with homology to known O-methyltransferases (OMTs; Guterman et al. 2002). Both OOMT1 and OOMT2 encode a protein capable of methylating orcinol and orcinol monomethyl ether, respectively, and optimum OOMT1 and OOMT2 transcripts are associated with emission of an orcinol dimethyl ether product (3,5-dimethoxytoluene; DMT), the major scent compound of R. hybrida cultivars (Lavid et al. 2002). Scalliet et al. (2006) explored the evolutionary pathway of scent production in a number of European (R. gallica officinalis, R. damascena) and Chinese rose (R. chinensis, R. gigantea) species, both progenitors of modern hybrid roses, R. hybrida. They reported that in DMT-producing roses, OOMTs were localized specifically in the petals and were more abundant in the adaxial epidermal cells, and thus likely to be associated with membranes of the scent secretory machinery of plant cells. Although OOMT gene sequences were detected in the two non-DMT producing rose species, gene products were not detected, thus suggesting that up-regulation of OOMT gene expression was critical in the evolution of scent production (Scalliet et al. 2006). Shalit et al. (2003) isolated and characterized a gene, designated RhAAT1, expressed exclusively in developing rose petals of ‘Fragrant Cloud’ that encodes a protein that catalyzes the formation codes for alcohol acetyltransferase (AAT) activity. High levels of expression of RhAAT1 coincide with peak scent emission in floral petal tissues. Recently, Guterman et al. (2006) introduced RhAAT into a plant cloning vector, driven by the constitutive promoter cauliflower mosaic virus (CaMV) 35S promoter, and used the construct to transform petunia plants using Agrobacterium-mediated gene transfer. Flowers of transgenic petunia expressing RhAAT emitted the acetate esters, benzyl acetate and phenylethyl acetate, the concentrations of which were 3-fold higher than in flowers of control Petunia plants.

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3.3 Other Genes With available genomic tools, interest has expanded into cloning and characterization of various disease resistance genes, such as Rdr1 and Rdr2, as well as resistance gene analogs (RGAs; Hattendorf et al. 2004). In a recent report, Hattendorf and Debner (2006) identified 40 different RGA families in an RGA library of rose of variable size and studied their phylogenetic relationships among each other and in relation to other RGA and R genes from various plant species.

4 Advances in Genetic Transformation and Recovery of Transgenic Plants 4.1 Regeneration Systems For many years now, efforts have been underway to utilize the tools of tissue culture and genetic engineering to either manipulate in vitro cell differentiation and regeneration and/or to develop gene transfer protocols for rose. A prerequisite for any successful genetic engineering effort is the availability of a reliable and efficient regeneration system for the recovery of transgenic plants. Regeneration via both shoot organogenesis and somatic embryogenesis from various tissues and organs is reported for many rose cultivars and a few species of rose, although emphasis focuses primarily on somatic embryogenesis. Embryogenic callus was initiated from in vitro-derived leaf or stem segments of R. hybrida cv. Carl Red and R. canina (Visessuwan et al. 1997), R. hybrida cv. Carefree Beauty, and R. chinensis minima cv. Baby Katie (Hsia and Korban 1996). Embryogenic callus was also induced in leaves of R. hybrida cvs. Domingo and Vicky Brown (De Wit et al. 1990), petioles and roots of R. hybrida cvs. Trumpeter and Glad Tidings (Marchant et al. 1996), root explants of both R. hybrida cv. Moneyway (Van der Salm et al. 1996) and R. Heritage x Alista Stella Gray (Sarasan et al. 2001), petals of R. hybrida cv. Arizona (Murali 1996), and immature seeds of R. rugosa (Kunitake et al. 1993). This was also achieved using immature leaf or stem segments of R. hybrida cv. Landora (Rout et al. 1991), in vivo mature leaves of R. hybrida cv. Soraya (Kintzios et al. 1999), anther filaments of R. hybrida cv. Royalty (Noriega and Söndahl 1991), as well as anthers, petals, receptacles, and leaves of R. hybrida cv. Meirutal (Arene et al. 1993). Kim et al. (2003) induced somatic embryos from immature zygotic embryos of R. hybrida cv. Sumpath, while Kamo et al. (2004) were able to increase plant regeneration from somatic embryos of R. hybrida cvs. Kardinal and Classy by dispersing embryogenic callus in liquid medium for 3 h, followed by fractionation to isolate proembryogenic masses that were smaller than 530 μm in size.

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Organogenesis was also reported from various tissues, including callus cultures of R. hybrida cv. Bridal Pink (Burger et al. 1990), R. damascena (Ishioka and Tanimoto 1990), R. hybrida cv. Landora (Rout et al. 1992), R. multiflora cv. Thornless (Rosu et al. 1995), R. hybrida cvs. Madelon, Only Love, Presto, Sonia, and Tineke (Dubois and de Vries 1995), R. hybrida cv. Carefree Beauty and R. chinensis minima cv. Baby Katie (Hsia and Korban 1996), and R. hybrida cv. Moneyway (Van der Salm et al. 1996). Interestingly, protoplasts were isolated from different rose genotypes (Mathews et al. 1991), and both somatic embryos and plantlets were obtained, primarily from protoplasts isolated from embryogenic material (Mathews et al. 1991; Schum and Hofmann 2001). Using polyethylene glycol (PEG)-mediated protoplast fusion, somatic hybrid calli, confirmed by AFLP and flow cytometric analyses, were obtained from fusions of cv. Heckenzauber with R. wichuraiana and cv. Praiser Charme with R. wichuraiana. However, no plant regeneration from these somatic hybrids was reported (Schum and Hofmann 2001). The wide range of explants and experimental approaches that have been employed with different rose species and cultivars strongly suggest that it is difficult to develop a universal genotype-independent method for the production of embryogenic callus in rose (Marchant et al. 1996). Progress on rose regeneration was reviewed thoroughly by Rout et al. (1999). More recently, Korban (2006) reported on the latest developments in somatic embryogenesis of rose. Induction of secondary embryogenesis is highly desirable for both micropropagation and genetic transformation (Raemakers et al. 1995). Li et al. (2002a) reported that inducing secondary embryogenesis from primary somatic embryos can be accomplished by transferring primary embryogenic callus onto Petri plates with half-strength MS basal salts (Murashige and Skoog 1962), fullstrength vitamins, and solidified with 2.5 g Gelrite gellan gum for a period of 1 month, followed by transfer to a plant growth regulator (PGR)-free medium with monthly subculture. Proliferation of these somatic embryos can be maintained for at least 1 year. 4.2 Maturation and Germination of Somatic Embryos Maturation and germination of somatic embryos is achieved by transferring individual clumps of somatic embryos onto a similar medium as described above, but with slight modification. Essentially, the medium consists of halfstrength MS basal salts, full-strength MS vitamins, 30 g sucrose, 3.8 μM ABA, and solidified with 2.5 g Gelrite gellan gum. Bipolar plantlets are then excised, and transferred individually to a PGR-free shoot elongation medium consisting of half-strength MS medium, full-strength vitamins, and 30 g sucrose for a period of 1 month. This medium also promotes shoot elongation and root development. Recently, Kamo et al. (2005) reported on the maturation of somatic embryos of R. hybrida cv. Kardinal on MS medium lacking growth regula-

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tors, and on the important role of ABA in plantlet conversion from long-term embryogenic callus. Rooted plantlets are transferred to a soil mix (1:1:1, by volume of soil:peat:perlite) in 4-cm plastic pots for a period of 2 weeks and covered with a clear plastic bag. A clear plastic cover can be used if plantlets are in trays (either plastic or wood). The plastic cover is gradually removed or opened to allow for acclimatization of plantlets. This process can take 2–3 weeks. Acclimatized plants are then transferred to the glasshouse and grown at 23 ◦ C. Plants are watered daily using a drip-irrigation system and fertilized once every 2 weeks with 250 ppm of a 20-20-20 NPK fertilizer solution. Once plants are well established they can be transferred to the field. 4.3 Genetic Transformation Systems As with all plant species, rose transformation is achieved either via Agrobacterium-mediated transformation or via microprojectile bombardment. However, a limited number of target cells typically receive the foreign DNA during these transformation events, and even fewer of these cells survive selection and subsequent regeneration of stable transformants. Therefore, the transformation efficiency is quite low and, in rose, it is highly genotype-dependent. The first published report on rose transformation was by Firoozabady et al. (1991), using Agrobacterium-mediated transformation of embryogenic tissues induced from filament cultures of R. hybrida cv. Royalty. Later, transgenic rose plants were obtained by gene insertion into friable embryogenic tissues of rose, also derived from filament cultures, with either Agrobacterium tumefaciens or A. rhizogenes (Firoozabady et al. 1994). Mathews et al. (1991, 1994) established protocols for isolating protoplasts from root and shoot cultures of various rose genotypes and for regenerating whole plants from protoplasts prepared from embryogenic cell lines of R. persica x R. xanthina. Schum and Hofmann (2001) reported on transient gene expression of the green fluorescent protein reporter gene from a number of rose genotypes, including R. persica x R. xanthina, R. multiflora, and R. wichuraiana, using PEG-mediated transformation of protoplasts. Although results also indicated the presence of stable transformants, no molecular (i.e., PCR, Southern blot) evidence was provided. Van der Salm et al. (1997) obtained transgenic plants from roots initiated from stem slices of the rootstock R. hybrida cv. Moneyway following co-cultivation with A. tumefaciens strain GV3101 containing an nptII gene and individual rol genes from A. rhizogenes. Grafting the transformed rootstock resulted in stimulation of both root development of the rootstock and axillary-bud break of the untransformed scion (Van der Salm et al. 1998). Marchant et al. (1998a) regenerated transgenic plants from embryogenic callus of R. hybrida following microprojectile bombardment. Subsequently, Marchant et al. (1998b) introduced a chitinase gene into R. hybrida cv. Glad Tidings and found that expression of the chitinase transgene reduced the severity of

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black spot (Diplocarpon rosae Wolf.) development by 13–43%. Dohm et al. (2001) introduced various combinations of antifungal resistance genes, encoding a Class II chitinase, Class I β-1,3-glucanase, and a Type I ribosome inhibiting protein (RIP) from barley, as well as a T4 lysozyme gene into the garden rose cultivars Heckenzauber and Pariser Charme, and obtained ∼80 confirmed transgenic plants. When these transgenic plants were screened for resistance to blackspot, only those carrying the RIP gene exhibited enhanced resistance. Li et al. (2002b) reported an enhanced efficiency of Agrobacterium-mediated transformation of embryogenic cultures of R. hybrida cv. Carefree Beauty by taking advantage of induced secondary somatic embryogenesis (Li et al. 2002a). As transformed embryogenic cells act independently from neighboring cells, these develop into somatic embryos that further undergo secondary embryogenesis. It was observed that transgenic plants with similar Southern hybridization profiles exhibited the same level of transcription as demonstrated by similar band intensities in Northern blots. Therefore, the transformation efficiency was estimated to be at least 9%. As the number of transgenic plants developing from the same transformation event was high (having undergone secondary somatic embryogenesis), this approach avoided the recovery of chimeric transgenic plants. This finding is especially important for plant species that rely on vegetative propagation. In a later study, Li et al. (2003) used the above transformation protocol to introduce an antimicrobial protein encoding gene, Ace-AMP1, into R. hybrida cv. Carefree Beauty. Some of the selected transgenic plants exhibited enhanced resistance to the fungal pathogen powdery mildew [Sphaerotheca pannosa (Wallr.: Fr.) Lev. var. rosae]. This was demonstrated in both a detached leaf assay and an in vivo greenhouse assay of whole plants. These promising findings offer new opportunities for developing roses with resistance to various economically important diseases in addition to other useful and desirable traits, such as flowering and growth habits, flower quality and longevity, and floral scent. Recently, Kim et al. (2004) reported on transformation of embryogenic callus initiated from petioles of R. hybrida cv. Tineke using A. tumefaciens strain LBA4404 harboring the green fluorescent protein 5, gfp5, gene as a visual reporter gene. They also reported that the transformation frequency increased from 6.6% to 12.6%, by including additional copies of virE and virG genes into the bi-functional fusion marker gene cassette. A detailed description has been reported recently of an Agrobacteriummediated transformation protocol for rose, along with recovery of transgenic plants, as used in our own laboratory (Korban et al. 2006).

5 Conclusions Significant progress has been made in the area of rose biotechnology, including the development of molecular markers useful for genotyping and assessing

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genetic diversity, along with the availability of linkage maps for a selected number of traits of economic importance, as well as the availability of genetic maps. The availability of genomic resources, such as BAC libraries and a small number of EST datasets and unigene sets, enables progress to be made in the gene cloning and characterization, as well as analysis of genes of high value, particularly those involved in scent production. The latter is amongst the most valuable traits of rose. These genomic resources, with the availability of genes isolated and cloned from rose, together with various other genes available from other plant species, and combined with the availability of both regeneration and genetic transformation systems, all provide promising tools for the development of transgenic roses with enhanced traits. However, much work is needed to determine the functionality of various groups of genes, as well as to enhance the transformation system for rose so that it can be useful for a range of genotypes.

References Arene L, Pellegrino C, Gudin S (1993) A comparison of the somaclonal variation level of Rosa hybrida L. cv. Meirutral plants regenerated from callus of direct induction from different vegetative and embryonic tissues. Euphytica 71:83–90 Ballard R, Rajapakse S, Abbott A, Byrne D (1996) DNA markers in rose and their use for cultivar identification and genome mapping. Acta Hortic 424:265–268 Burger DW, Liu L, Zary KW, Lee CI (1990) Organogenesis and plant regeneration from immature embryos of Rosa hybrida L. Plant Cell Tissue Organ Cult 21:147–152 Channeliere S, Riviere S, Scalliet G, Jullien F, Szecsi J, Dolle C, Vergne P, Dumas C, Bendahmane M, Hugueney P, Cock JM (2002) Analysis of gene expression in rose petals using expressed sequence tags. FEBS Lett 515:35–38 Crespel L, Chirollet M, Durel E, Zhang D, Meynet J, Gudin S (2002) Mapping of qualitative and quantitative phenotypic traits in Rosa using AFLP markers. Theor Appl Genet 105:1207–1214 Debener T (1999) Genetic analysis of horticulturally important morphological and physiological characters in diploid roses. Gartenbauwissenschaft 64:14–20 Debener T (2003) Inheritance of characters. In: Roberts A, Debener T, Gudin S (eds) Encyclopedia of rose sciences. Elsevier, Oxford, pp 286–329 Debener T, Mattiesch L (1998) Effective pairwise combination of long primers for RAPD analyses in roses. Plant Breed 117:147–151 Debener T, Mattiesch L (1999) Construction of a genetic linkage map for roses using RAPD and AFLP markers. Theor Appl Genet 99:891–899 Debener T, Bartels C, Mattiesch L (1996) RAPD analysis of genetic variation between a group of rose cultivars and selected wild rose species. Mol Breed 2:321–327 Debener T, Janakiram T, Mattiesch L (2000) Sports and seedlings of rose varieties analysed with molecular markers. Plant Breed 119:71–74 Debener T, Mattiesch L, Vosman B (2001) A molecular marker map for roses. Acta Hortic 547:283–287 Debener T, Dohm A, Mattiesch L (2003) Use of diploid self incompatible rose genotypes as a tool for gene flow analyses in roses. Plant Breed 122:285–287 De Vries DP, Dubois LAM (1996) Rose breeding: past, present, prospects. Acta Hortic 424:241–248 De Wit JC, Esendam HF, Honkanen JJ, Tuominen U (1990) Somatic embryogenesis and regeneration of flowering plants in rose. Plant Cell Rep 9:456–458

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Lavid N, Wang J, Shalit M, Guterman I, Bar E, Beurerle T, Menda N, Shafir S, Zamir D, Adam Z, Vainstein A, Weiss D, Pichersky E, Lewinsohn E (2002) O-Methyltransferases involved in the biosynthesis of volatile phenolic derivatives in rose petals. Plant Physiol 129:1899–1907 Li X, Krasnyanski S, Korban SS (2002a) Somatic embryogenesis, secondary somatic embryogenesis, and shoot organogenesis in Rosa. J Plant Physiol 159:313–319 Li X, Krasnyanski S, Korban SS (2002b) Optimization of the uidA gene transfer into somatic embryos of rose via Agrobacterium tumefaciens. Plant Physiol Biochem 40:453–459 Li X, Gasic K, Cammue B, Broekaert W, Korban SS (2003) Transgenic rose lines harboring an antimicrobial protein gene, Ace-AMP1, demonstrate enhanced resistance to powdery mildew (Sphaerotheca pannosa). Planta 218:226–232 Linde M, Mattiesch L, Debener T (2004) Rpp1, a dominant gene providing race-specific resistance to rose powdery mildew (Podosphaera pannosa): molecular mapping, SCAR development and confirmation of disease resistance data. Theor Appl Genet 10:1261–1266 Malek VB, Debener T (1998) Genetic analysis of resistance to blackspot (Diplocarpan rosae) in tetraploid roses. Theor Appl Genet 96:228–231 Malek VB, Weber WE, Debener T (2000) Identification of molecular markers linked to Rdr1, a gene conferring resistance to blackspot in roses. Theor Appl Genet 101:977–983 Marchant R, Davey MR, Lucas JA, Power JB (1996) Somatic embryogenesis and plant regeneration in floribunda rose (Rosa hybrida L. cvs. Trumpeter and Glad Tidings). Plant Sci 120:95–105 Marchant R, Power JB, Lucas JA, Davey MR (1998a) Biolistic transformation of rose (Rosa hybrida L.). Ann Bot 81:109–114 Marchant R, Davey MR, Lucas JA, Lamb CJ, Dixon RA, Power JB (1998b) Expression of a chitinase in rose (Rosa hybrida L.) reduces development of black spot disease (Diplocarpon rosae Wolf). Mol Breed 4:187–194 Mathews D, Mottley J, Horan I, Roberts AV (1991) A protoplast to plant system in roses. Plant Cell Tissue Organ Cult 24:173–180 Mathews D, Mottley J, Yokoya K, Roberts AV (1994) Regeneration of plants from protoplasts of Rosa species (roses). In: Baja YPS (ed) Plant protoplasts and genetics engineering. (Biotechnology in agriculture and forestry, vol 29) Springer, Berlin Heidelberg New York, pp 146–160 Matsumoto S, Fukuri H (1996) Identification of Rosa cultivars and clonal plants by random amplified polymorphic DNA. Sci Hortic 67:49–54 Millan T, Osuna F, Cobos S, Torres AM, Cubero JI (1996) Using RAPDs to study phylogenetic relationships in Rosa. Theor Appl Genet 92:273–277 Müller R, Stummann BM, Serek M (2000) Characterization of an ethylene receptor family with differential expression in rose (Rosa hybrida L.) flowers. Plant Cell Rep 19:1232–1239 Murali S, Sreedhar D, Lokeswari TS (1996) Regeneration through somatic embryogenesis from petal-derived calli of Rosa hybrida L. Arizona (hybrid tea). Euphytica 91:271–275 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–493 Noriega C, Sondahl MR (1991) Somatic embryogenesis in hybrid tea roses. Bio/Technology 9:991–993 Phillips R, Rix M (1988) Roses. Random House, New York Raemakers CJJM, Jacobsen E, Visser RGF (1995) Secondary somatic embryogenesis and applications in plant breeding. Euphytica 81:93–107 Rajapakse S (2003) Gene mapping. In: Roberts A, Debener T, Gudin S (eds) Encyclopedia of rose sciences. Elsevier, Oxford, pp 326–334 Rajapakse S, Byrne DH, Zhang L, Anderson N, Arumuganathan K, Ballard RE (2001) Two genetic linkage maps of tetraploid roses. Theor Appl Genet 103:575–583 Reynders-Aloisi S, Bollerau P (1996) Characterization of genetic diversity in genus Rosa by randomly amplified polymorphic DNA. Acta Hort 424:253–260

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Rosu A, Skirvin RM, Bein A, Norton MA, Kushad M, Otterbacher AG (1995) The development of putative adventitious shoots from a chimeral thornless rose (Rosa multiflora Thurb. Ex J Murr.) in vitro. J Hortic Sci 70:901–907 Rout GR, Debata BK, Das P (1991) Somatic embryogenesis in callus culture of Rosa hybrida L. cv. Landora. Plant Cell Tissue Organ Cult 27:65–69 Rout GR, Debata BK, Das P (1992) In vitro generation of shoots from callus culture of Rosa hybrida L. cv. Landora. Indian J Exp Biol 30:15–18 Rout GR, Samantaray S, Mottey J, Das P (1999) Biotechnology of the rose: a review of recent progress. Sci Hortic 81:201–228 Sarasan V, Roberts AV, Rout GR (2001) Methyl laurate and 6-benzyladenine promote the germination of somatic embryos of a hybrid rose. Plant Cell Rep 20:183–186 Scalliet G, Lionnet C, Le Bechec M, Dutron L, Magnard J-L, Baudino S, Bergougnoux V, Jullien F, Chambrier P, Vergne P, Dumas C, Cock JM, Hugueney P (2006) Role of petal-specific orcinol O-methyltransferases in the evolution of rose scent. Plant Physiol 140:18–29 Schum A, Hofmann K (2001) Use of isolated protoplasts in rose breeding. Acta Hortic 547:35–45 Schum A, Preil W (1998) Induced mutations in ornamental plants. In: Jain SM, Brar DS, Ahloowalia BS (eds) Somaclonal variation and induced mutations in crop improvement. Kluwer, Dordrecht, pp 333–366 Shalit M, Guterman I, Volping H, Bar E, Tamari T, Menda N, Adam Z, Zamir D, Vainstein A, Weiss D, Pichersky E, Lewinsohn E (2003) Volatile ester formation in roses. Identification of an acetyl-coenzyme A geranoil/citronellol acetyltransferase in developing rose petals. Plant Physiol 131:1868–1876 Van der Salm TPM, Van der Toorn CJG, Hanisch ten Cate CH, Dons HJM (1996) Somatic embryogenesis and shoot regeneration from excised adventitious roots of the rootstock Rosa hybrida cv. Money Way. Plant Cell Rep 15:522–526 Van der Salm TPM, Van der Toorn CJG, Bouwer R, Don HJM (1997) Production of rol gene transformed plants Rosa hybrida L. and characterisation of their rooting ability. Mol Breed 3:39–47 Van der Salm TPM, Bouwer R, Van Dijk AJ, Keizer LCP, Hanish ten Cate CH, Van der Plas LHW, Dons JJM (1998) Stimulation of scion bud release by rol gene transformed rootstocks of Rosa hybrida L. J Exp Bot 49:847–852 Visessuwan R, Kawai T, Mii M (1997) Plant regeneration systems from leaf segment culture through embryogenic callus formation of Rosa hybrida and R. canina. Breed Sci 47:217–222 Wang D, Fan J, Ranu RS (2004) Cloning and expression of 1-aminocyclopropane-1-carboxylate synthase cDNA from rose (Rosa x hyrbida). Plant Cell Rep 22:422–429 Weising K, Nybom H, Wolff K, Meyer W (1995) DNA fingerprinting in plants and fungi. CRC, Boca Raton Xu ML, Li X, Korban SS (2004) DNA-methylation alterations and exchanges during in vitro cellular differentiation in rose (Rosa hybrida L.). Theor Appl Genet 109:899–910 Yan Z, Denneboom C, Hattendorf A, Dolstra O, Debener T, Stam P, Visser PB (2005) Construction of an integrated map of rose with AFLP, SSR, PK, RGA, RFLP, SCAR and morphological markers. Theor Appl Genet 110:766–777 Yokoya K, Roberts AV, Mottley J, Lewis R, Brandham PE (2000) Nuclear DNA amount in roses. Ann Bot 85:557–561

III.2 Carnation M. Moyal-Ben Zvi and A. Vainstein1

1 Introduction Carnation, native to the Mediterranean coastal region, is a member of the family Caryophyllaceae and belongs to the genus Dianthus, which contains more than 300 species. The perpetual flowering carnation first appeared in France in the early nineteenth century (Holly and Baker 1991) as a result of intense breeding, which involved at least two species of Dianthus, namely D. caryophyllus and D. sinensis. Further crosses within D. caryophyllus resulted in today’s numerous leading cut-flower varieties, which can be grouped into the phenotypical categories “standard” (flowers with up to 120 petals) and “spray” (smaller flowers). In addition to the cut flowers, crosses between D. chinensis and D. barbatus produced the varieties that comprise the more recent commercial niche of pot carnation. Carnation, together with rose and chrysanthemum, make up more than 50% of the world cut-flower market. Within the European market, carnation accounts for 20% of extra-EU fresh cut-flower imports, and is second only to rose. The main distribution destinies are the United Kingdom, the Netherlands and Germany, and the demand is mainly supplied by Colombia, Kenya, Turkey and Morocco, although new suppliers regularly appear (www.coleacp.org). High commercial value, together with continual consumer demand for new varieties, act as the driving force for carnation breeding. Altered plant and flower morphology, varieties and color combinations, enhanced fragrance and long vase-life are some of the most appealing traits from the consumer’s point of view and, as such, the focus of many ornamental breeding programs. Growers’ preferences also dictate breeding goals, leading to the creation of new varieties with improved agronomic traits, such as high production and propagation yield, and resistance to biotic and abiotic stresses. For many years, carnation breeders relied solely on classic breeding for the introduction of new traits, i.e., crossing and selecting for progeny with desirable traits. Broad genetic variation, vegetative propagation by cuttings, short flowering cycle and perpetual flowering are advantageous in classic breeding and are all characteristics of carnation. These virtues make carnation breeding a relatively dynamic process in comparison with that of other ornamentals, and 1 The

Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel, e-mail: [email protected]

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allow carnation breeders to respond rapidly to new trends. However, rapidly accumulating advances in the field of molecular breeding offer new tools to supplement classic approaches. Ornamentals, carnation in particular, lack saturated genetic maps, and molecular genetic markers are used mainly for the identification of varieties and the analysis of inter- and intraspecific genetic relatedness (Debener 2002; Smulders et al. 2003). It is clear that DNA markers can also be harnessed for tagging traits of interest, thereby paving the way to the establishment of a marker-assisted selection system and the isolation of the gene(s) of interest. There is a range of DNA markers, among which RAPD/RFLP markers are the most simple to produce and account for much of the knowledge accumulated to date regarding ornamentals (Debener 2002). Advances in genetic engineering approaches have opened up new possibilities in breeding programs, enabling the circumvention of some of the hurdles of classic breeding. For example, high heterozygosity amongst parents makes it almost impossible to specifically interfere with or modify a single trait, as numerous genes segregate in the progeny of such crosses. Genetic engineering enables specific modifications of an otherwise successful cultivar, irrespective of its heterozygosity, by manipulating a single gene. Furthermore, genetic engineering enables almost unlimited extension of the plant’s genetic pool, as it allows the transfer of genes across species, genera and even different kingdoms, thereby overriding sterility and inbreeding limitations. Considerable progress has been made in recent years in the improvement of carnation transformation and regeneration procedures (Zuker et al. 1998, 2001a). Moreover, new genes of horticultural importance to ornamentals, including carnation, are constantly being isolated, and the underlying molecular processes regulating traits of interest are under intensive study. This accumulating knowledge, coupled with technological advances, is required to fulfill the aforementioned potential of genetic engineering in upgrading breeding. Here, we describe some of the work conducted in our laboratory on carnation molecular breeding and highlight the latest achievements/developments from other groups in the field.

2 Recent Developments in Carnation Biotechnology 2.1 Flower Initiation Carnation mutates spontaneously at high frequency (Holly and Baker 1991), and this characteristic has been exploited extensively in classic breeding for the generation of new carnation variants. In an ongoing breeding program, we isolated mutants with an increased number of bracteoles formed prior to flower formation. This repetitive bracteole formation resulted in a spike-like structure subtending each flower. Based on genetic analysis, the mutation was ascribed to a single locus which was termed evergreen (e; Scovel et al. 2000).

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The semi-dominant nature of this mutation was displayed by the segregation of the selfed mutant’s offspring, which consisted of three phenotypical classes, namely wild type (ee), flowering with repetitive bracteoles (Ee), and non-flowering with repetitive bracteoles (EE). Arrest of flower initiation in EE segregants was complete, regardless of bud position along the stem. Selfing of flowering Ee mutants with dichotomous or alternate branching yielded non-flowering EE mutant segregants with dichotomous and alternate branching, respectively, which suggests that inflorescence formation is independent of allele E. In accordance with these findings, we postulated that evergreen is involved in regulating the initiation/execution of flower organogenesis. The semi-dominant nature of mutation E is unique in comparison with the recessive leafy (Arabidopsis), floricaula (Antirrhinum), aberrant leaf and flower (Petunia) and unifoliata (pea) mutants (Franks and Liu 2001), and permits an analysis that can discriminate between processes specifically involved in either inflorescence development or flower initiation. 2.2 Flower Architecture/Structure The differentiation, development and spatial arrangement of sepals, petals, stamens and carpels determine flower architecture (Zik and Irish 2003). Whereas the regulation of floral organ identity and development by homeotic genes has been studied extensively in Arabidopsis and Antirrhinum (Coen and Meyerowitz 1991; Zik and Irish 2003), the isolation of genes involved in these processes in carnation has only just commenced, with the isolation of the first MADS-box gene CMB2 (Baudinette et al. 2000), putatively encoding a class “B” floral homeotic gene. The basic flower structure, common to all carnation varieties, is composed, as in many other flowering plants, of the four floral organ types arranged in a series of concentric whorls. However, carnation varieties can be categorized into three phenotypical groups, distinguished by their petal number as “single”, “semi-double” and “double” flowers (Scovel et al. 1998). Single flowers are composed of five petals, semi-double flowers have between 20 and 80 petals per flower, and double flowers have up to 120 petals. The five-petal singleflower carnation varieties are commercially negligible, while semi-double and double flower carnations dominate most of the market and usually belong to the spray and standard varieties, respectively. Using segregating populations, it was shown that flower phenotype is controlled by a single locus (termed D) and that single-flower phenotypes are the result of the homozygous recessive genotype dd (Scovel et al. 1998). As such, flower doubleness is manifested by a dominant mutant allele (D), and DD gives a fully double flower. Interestingly, there seems to be a connection/linkage between the evergreen gene and the double-flower phenotype, as revealed by the ca. 30% reduction in petal number in semi-double (dD) and double (DD) Ee mutant flowers versus their ee flower counterparts. Since petal number was unaffected in single-flowered Ee

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mutants, E was suggested to exert its effect on flower doubleness via negative regulation of the functionality of D. Alternatively, E and D may regulate a common target gene for petal formation. Direct transcriptional activation of floral organ identity genes AGAMOUS and APETALA1 has been demonstrated (Franks and Liu 2001). The importance of flower doubleness as a breeding characteristic led to the generation of an RAPD DNA marker linked to the wild-type “d” allele capable of discriminating between double and semi-double phenotype (Scovel et al. 1998). Based on this RAPD fragment, an RFLP marker was generated and, like the former, demonstrated 100% reliability in discriminating the double flower lines from the single and semi-double flower lines. To date, more than 100 genotypes, including those not genetically related to the original-line-derived segregating progeny, have been analyzed, and in all cases, 100% co-segregation has been observed. Discrimination of flower phenotype was successful with carnations of both Mediterranean and American origin. Hence, these markers are applicable to spray (dD) and standard (DD) carnation variety breeding by screening, respectively, for or against semi-double flower genotype. Furthermore, these markers have opened the way to the isolation of the gene(s) involved in the determination of flower phenotype in carnation. 2.3 Flower Yield In ornamentals in general, carnation in particular, increased flower yield is a highly desirable trait from the grower’s point of view. Reduced apical dominance and increased branching may contribute to achieving this trait. Among the different genes applied to genetically engineer these characteristics, the Agrobacterium rhizogenes rol genes have been the most widely and successfully employed (reviewed by Casanova et al. 2005). Four genes in the T-DNA segment (rolA, rolB, rolC, rolD) are responsible for the morphological effects of the bacterium on plants. Although largely unknown, the mechanism by which these genes exert their morphological effects on plants presumably acts either through manipulating the plant’s endogenous hormone concentrations or hormone metabolism, or by influencing the plant cell’s sensitivity to these hormones. Reduced apical dominance and increased branching, dwarfed plants and male sterility are all phenotypic alterations ascribed to various 35S-rolCtransgenic plants (Casanova et al. 2005). To assess rol’s potentially beneficial effect on carnation, D. caryophyllus cv. White Sim was transformed with rolC under the control of the CaMV35S promoter and the transgenic carnation lines were analyzed under commercial glasshouse conditions (Zuker et al. 2001b). The reduction in plant apical dominance that occurred in these transgenic carnation lines did not result in an undesirable compact form or low growth rate, as reported for other 35SrolC-transgenic crops, but rather manifested itself in enhanced lateral shoot development, i.e., an up to 50% higher yield of stem cuttings per mother

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plant and a ca. 3-fold higher yield of flowering stems per mother plant relative to control plants. Moreover, the transgenes exhibited improved rooting ability, with up to five times the root dry weight of control plants. Thus, rolCtransgenic carnation plants demonstrated improved agronomic performance in terms of increases in both flower and propagation yield. This phenotype was retained during a 2-year glasshouse test, confirming the stability of rolC and the beneficial phenotype it exerts in transgenic plants. To study the mechanism by which rolC exerts its morphological effect, in vitro organogenic and rhizogenic capacity of transgenic carnation explants were assessed along with their endogenous concentrations of the auxin indole3-acetic acid (IAA), isoprenoid cytokinins (iP), zeatin (Z), dihydrozeatin (DHZ) and their 9-ribosides (iPR, ZR and DHZR, respectively). Explants of 35S-rolCtransgenic carnation exhibited enhanced shoot regeneration and rooting capability in vitro. Shoot regeneration from petals correlated with iP levels, but did not correlate with either the total cytokinins value or the cytokinins to auxin ratio. IAA concentrations remained unaffected in the transgenes. Based on these studies, it was suggested that the in vivo and in vitro cytokinin and auxin dual effect of rolC on transgenic carnation could be attributed to greater iP concentrations and an increase in auxin sensitivity in these plants, respectively, relative to control plants (Casanova et al. 2004). 2.4 Vase Life For many years, carnation flowers have been used as a model system in studies of relationships between ethylene and flower senescence and it is well established that endogenous ethylene production is one of the factors triggering senescence of carnation flowers. As a result, in commercial practice, carnations are routinely subjected to a post-harvest chemical treatment which interferes with ethylene perception, leading to increased flower longevity. The toxicity of the chemicals, coupled with advances in understanding the molecular processes leading to floral senescence, catalyzed the development of carnation varieties with improved vase life via genetic engineering. In fact, the first trait introduced into carnation via genetic engineering approaches was improved vase life. Down-regulation of endogenous ethylene production is a proven approach for achieving postponed flower senescence. This method was applied through antisense and co-suppression of ACC oxidase, and through ACC synthase cosuppression, both coding for enzymes of the ethylene biosynthetic pathway (Savin et al. 1995). An alternative approach was used by Bovy et al. (1999) to obtain carnation which was insensitive to both endogenous and exogenous ethylene. A mutated Arabidopsis ethylene receptor gene (Etr1-1) under its own promoter, a CaMV35S constitutive promoter, or the flower-specific floral binding protein promoter (FBP1), was introduced into carnation. As compared with control, non-transgenic flowers, vase life was increased up to 3-fold in the

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transgenic flowers, which also exceeded that of flowers chemically pretreated with silver thiosulfate (Bovy et al. 1999). It should be noted that it is advantageous to confine ethylene insensitivity to the flower organs as opposed to the whole plant, due to potential impairment of the plant defense response as a result of interfering with its ethylene perception throughout (Lorenzo et al. 2003). It should also be noted, however, that the commercialization of genetically engineered cultivars with long vase life is hindered by the consumers’ fear of the unknown and limited environmental awareness, rendering investment in the development of such cultivars economically questionable. 2.5 Flower Color In a range of plants, flower color is determined by flavonoids, of which anthocyanins are the most conspicuous group. The anthocyanin biosynthesis pathway is detailed very well genetically and biochemically (Holton and Cornish 1995; Springob et al. 2003), with both structural and regulatory genes of the pathway having been isolated from various plant species, including carnation (Ben-Meir et al. 2002; Forkmann et al. 1995). Two types of anthocyanin pigments are produced in carnation flowers, namely, pelargonidin derivatives responsible for orange, pink or red colors, and cyanidin derivatives for red or magenta colors. The absence of native blue and purple flowers in carnation is attributed to a lack of the flavonoid 3 ,5 -hydroxylase (F3 5 H) gene, which is responsible for the production of delphinidin derivatives (Mol et al. 1999). In view of the influence of flower color on consumer preference and the high commercial value of carnations, it is hardly surprising that the world’s first commercial transgenic flower was a novel-colored carnation. Designed by Suntory, the “Moon” series of transgenic violet-flowered carnations are marketed across North America, Australia and Japan and are currently the only transgenic flower crop released for commercial distribution. The violet color in “Moon” transgenic carnations was achieved through the introduction of a heterologous F3 5 H gene together with a petunia dihydroflavonol reductase (DFR) gene into a DFR-deficient white carnation (Mol et al. 1999). Similarly, introduction of petunia F3 5 H along with the cytochrome b5 gene into a cyanidin-accumulating carnation variety resulted in transgenic flowers showing a color shift from variegated pink and red to variegated mauve and purple (Tanaka et al. 2005). Chalcones, representing another flavonoid subgroup, impart the yellow and orange flower hues found in such plant species as cosmos, dahlia, peony, periwinkle and carnation. Chalcones possess free 2 - and 6 -hydroxyl groups via which they are converted to a colorless flavanone, through either enzymatic or spontaneous isomeration. Hence, in all of the aforementioned species, yellow flowers are generated due to the existence of a metabolic route to chalcone stabilization and subsequent accumulation. One such route that has already been harnessed for the generation of yellow petunia flowers by means of genetic

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engineering (Tanaka et al. 2005) is the 6 -deoxylation of tetrahydroxychalcone (THC), catalyzed by polyketide reductase (PKR). The molecular basis of an alternative route represented in yellow carnation flowers was recently revealed with the isolation of the genes encoding 2 -glucosyltransferase (C2 GT) responsible for the generation of THC 2 -glucoside (isosalipurposide, ISP); (Ishida et al. 2003; Okuhara et al. 2004). Interestingly, a deficiency in chalcone isomerase (CHI) activity was positively correlated with the levels of ISP accumulating in carnation flowers (Forkmann and Dangelmayr 1980; Itoh et al. 2002). However, as the conversion of THC to colorless flavanone occurs spontaneously as well as enzymatically, it is apparent that merely abolishing CHI activity is not sufficient for the accumulation of stable yellow chalcone and that the stabilization of THC is a prerequisite. Knowledge regarding factors leading to the generation of yellow carnation flowers was further elaborated by Zuker et al. (2002), who utilized f3h for the successful generation of a transgenic yellow-flowered carnation. Transformation of carnation cv. Eilat with an antisense f3h construct resulted in a strong color modification from orange/reddish to yellow/cream flowers in the transgenic plants, concomitant with almost completely abolished f3h transcript accumulation as well as enzyme activity. The observed phenotype of the yellow/cream flowers in the anti-f3h-transgenic plants was correlated with a dramatic decrease in pelargonidin levels relative to wild-type plants. ISP levels, in contrast, remained unaffected. Thus, the yellow/cream flower color in these transgenic plants may be attributed to unmasking of the yellow ISP as a result of the decrease in pelargonidin levels following anthocyanin biosynthesis blockage. These results suggest a role for both ISP accumulation and its unmasking in the process of generating carnation yellow flowers. It should be feasible to generate true yellow flowers in species lacking stable chalcone derivatives via the introduction of C2 GT and down-regulation of the anthocyanin pathway’s genes, allowing both diversion of the metabolic flow towards ISP accumulation and its unmasking. 2.6 Floral Scent Despite its enormous importance as one of the key traits influencing consumer preference, today’s cut-flower varieties typically possess only faint, if any, fragrance. The fact that the molecular and biochemical infrastructure leading to floral-scent biosynthesis and emission is still largely regarded as “terra incognita” calls for the application of available tools for large-scale transcriptome and metabolome profiling and screening in search of the genes involved in these processes. Nevertheless, such reports are scarce (Aharoni et al. 2000; Guterman et al. 2002). However, encouraged by consumer demand for the restoration of fragrance to existing cut-flower varieties, research into this subject is gradually gaining momentum, and several structural genes involved in flower-fragrance

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production have been cloned and characterized (Dudareva and Pichersky 2000; Vainstein et al. 2001; Dudareva et al. 2004), the first being the Clarkia breweri linalool synthase (lis) gene, the product of which catalyzes the conversion of geranyl diphosphate to the monoterpene, S-linalool, in a single step (Dudareva et al. 1996). In carnation cv. Eilat, as in Dianthus in general, there are no detectable concentrations of monoterpenes, one of the major groups of scent compounds produced and emitted by many fruits and flowers. Therefore, with the aim of broadening the fragrance spectrum of carnation flowers, cv. Eilat was transformed with lis (Lavi et al. 2002). Transgenic carnation expressing lis emitted linalool, which was detected in the headspace of leaves, intact flowers and detached petals at various developmental stages. In addition to linalool, its derivatives, cis- and trans-linalool oxide, were detected in the headspace of transgenic carnation. In accordance with the production of linalool in flowers of some representatives of the genus Dianthus, this linalool oxidation might be due to a residual endogenous monoterpene biosynthetic pathway. However, non-specific linalool oxidation is also plausible. In any case, emission of linalool from transgenic carnation cv. Eilat did not result in detectable olfactory changes in flower scent. Insufficient concentrations of linalool and/or its masking by other volatiles are among the possible reasons for the current lack of success in modifying carnation flower scent via the introduction of lis. Another scenario which could account for these results is the glycosylation of linalool, converting it to a non-volatile form, as has been shown to occur in lis-transgenic petunia (Lucker et al. 2001). An alternative approach to enhancing flower fragrance was revealed in our laboratory, when an olfactory change was detected in anti-f 3h-transgenic carnation plants (Zuker et al. 2002). An analysis of fragrance compounds in these transgenic plants revealed 5- to 7-fold higher concentrations of the benzenoids methyl benzoate and 2-hydroxy methyl benzoate, as compared with control, non-transgenic flowers. The concentrations of analyzed fragrance compounds representing other metabolic pathways were not affected in the transgenes, and so it is reasonable to conclude that the elevated concentrations of the benzenoid compounds were responsible for the enhanced fragrance in the transgenic plants. As both benzoic acid derivatives and anthocyanins originate from the phenylpropanoid pathway, the elevated concentrations of benzenoid compounds detected in the transgenic plants are most likely to result from a diversion of the metabolic flow from anthocyanin to benzoic acid production. This remarkable link between the three secondary metabolic pathways leading to color and scent production seems to have an evolutionary rationale, as both pathways are used by plants to lure pollinators and seed dispersers, thereby ensuring species survival. Moreover, these findings are in agreement with recent studies emphasizing the importance of substrate (benzoic acid) availability in methyl benzoate production/emission (Dudareva et al. 2000; Kolosova et al. 2001), further proving that the diver-

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sion of metabolic flow via modulation of related biosynthetic pathways is an innovative strategy that can be applied to the modification of flower fragrance. The isolation of enzymes catalyzing the biosynthesis of volatile compounds in plants can be harnessed for their manufacture, based on either cultured whole plant cells or simply enzyme templates (Oksman-Caldentey and Inze 2004). The latter approach was demonstrated by Schade et al. (2003), who immobilized an enzyme template of carnation petals to a stationary matrix. Upon addition of linoleic acid, the green-note volatile hexanal, a highly important aroma compound, was produced in this packed-bed bioreactor. 2.7 Disease Resistance Fusarium wilt is a major disease causing yearly losses in carnation crops. This disease is soil-borne and caused by the fungus F. oxysporum. Methods currently used to control this fungus are hazardous, ineffective and costly, and phenotypic selection for resistance in carnation varieties is proving difficult and inefficient. With the aim of generating Fusarium resistance, a combination of tobacco antifungal genes encoding osmotin and chitinase (pXBOC) were introduced into the highly Fusarium-susceptible carnation cv. White Sim (Zuker et al. 2001c). Carnations transgenic for osmotin and chitinase were propagated in the glasshouse via cuttings and evaluated for resistance to Fusarium. Selected transgenic lines of cv. White Sim showed a high level of resistance to Fusarium oxysporum f. sp. dianthi, race 2, following greenhouse testing. As early as 4 weeks after inoculation, more than 90% of the control plants showed strong symptoms, whereas disease symptoms in the transgenic lines were apparent in only 16% of the plants. Furthermore, relative to control plants, disease symptoms in the transgenic lines were markedly less severe. Similar

Fig. 1. Resistance of transgenic carnation to Rhizoctonia solani. Disease severity in two independent lines of carnation cv. White Sim transgenic for tobacco-derived osmotin and chitinase genes (left) and in control, non-transgenic carnation plants (right). Bar 1 cm

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results were recorded in these lines with respect to resistance to Rhizoctonia solani (Fig. 1). To date, however, this study has not been applied to commercial carnation production.

3 Conclusions The advances in molecular breeding of carnation described herein reflect the continuous effort being made by ornamental breeders to meet the demands of both producers and customers for high-quality varieties. Indeed, the first transgenic ornamentals to be commercialized were violet carnation varieties. It is apparent today that molecular approaches other than transgenesis can also strongly advance the progress of breeding programs, e.g., the application of DNA markers. Again, carnation breeders were among the first to apply markers for cultivar identification, as well as for the efficient and early selection of a desirable trait. However, despite the impressive advances made by molecular breeders, there are still many bottlenecks to fully realizing the approach’s inherent potential. For example, interactions between genes and pathways (flower initiation and architecture, color and scent), differences in the functionality of genes across genera, and the complexity of traits that are governed by several genes, impede the generation of a desirable final product. Integrative breeding approaches which combine traditional and molecular tools, based on advances in research aimed not only at isolating single genes but also at delineating whole networks leading to a desirable phenotype, should lead, in the foreseeable future, to a new generation of top-notch carnation flowers. Acknowledgements. Work in the authors’ laboratory is supported by Research Grant Number US-3437-03 from BARD, the Israeli Ministry of Agriculture, the Israeli Ministry of Science, the Israel Science Foundation and the Hebrew University Intramural Research Fund Basic Project Awards.

References Aharoni A, Keizer LCP, Bouwmeester HJ, Sun ZK, Alvarez-Huerta M, Verhoeven HA, Blaas J, Houwelingen AMML van, De Vos RCH, Voet H van der, Jansen RC, Guis M, Mol J, Davis RW, Schena M, Tunen AJ van, O’Connell AP (2000) Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays. Plant Cell 12:647–661 Baudinette SC, Stevenson TW, Savin KW (2000) Isolation and characterisation of the carnation floral-specific MADS box gene, CMB2. Plant Sci 155:123–131 Ben-Meir H, Zuker A, Weiss D, Vainstein A (2002) Molecular control of floral pigmentation: anthocyanins. In: Vainstein A (ed) Breeding for ornamentals: classical and molecular approaches. Kluwer, Dordrecht, pp 155–196 Bovy AG, Angenet GC, Dons HJM, Altvorst AC van (1999) Heterologous expression of the Arabidopsis etr1-1 allele inhibits the senescence of carnation flowers. Mol Breed 5:301–308

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Casanova E, Valdes AE, Zuker A, Fernandez B, Vainstein A, Trillas MI, Moysset L (2004) rolCtransgenic carnation plants: adventitious organogenesis and levels of endogenous auxin and cytokinins. Plant Sci 167:551–560 Casanova E, Trillas MI, Moysset L, Vainstein A (2005) Influence of rol genes in floriculture. Biotechnol Adv 23:3–39 Coen ES, Meyerowitz EM (1991) The war of the whorls – genetic interactions controlling flower development. Nature 353:31–37 Debener T (2002) Molecular markers as a tool for analyses of genetic relatedness and selection in ornamentals. In: Vainstein A (ed) Breeding for ornamentals: classical and molecular approaches. Kluwer, Dordrecht, pp 329–345 Dudareva N, Pichersky E (2000) Biochemical and molecular genetic aspects of floral scents. Plant Physiol 122:627–633 Dudareva N, Cseke L, Blanc VM, Pichersky E (1996) Evolution of floral scent in Clarkia: novel patterns of S-linalool synthase gene expression in the C. breweri flower. Plant Cell 8:1137–1148 Dudareva N, Murfitt LM, Mann CJ, Gorenstein N, Kolosova N, Kish CM, Bonham C, Wood K (2000) Developmental regulation of methyl benzoate biosynthesis and emission in snapdragon flowers. Plant Cell 12:949–961 Dudareva N, Pichersky E, Gershenzon J (2004) Biochemistry of plant volatiles. Plant Physiol 135:1893–1902 Forkmann G, Dangelmayr B (1980) Genetic control of chalcone isomerase activity in flowers of Dianthus caryophyllus. Biochem Genet 18:519–527 Forkmann G, Dedio J, Henkel J, Min BW, Wassenegger M (1995) Genetics, biosynthesis and molecular biology of flower color of Dianthus caryophyllus (carnation). Acta Hortic 420:29–31 Franks RG, Liu Z (2001) Floral homeotic gene regulation. Hortic Rev 27:41–77 Guterman I, Shalit M, Menda N, Piestun D, Dafny-Yelin M, Shalev G, Bar E, Davydov O, Ovadis M, Emanuel M, Wang J, Adam Z, Pichersky E, Lewinsohn E, Zamir D, Vainstein A, Weiss D (2002) Rose scent: genomics approach to discovering novel floral fragrance-related genes. Plant Cell 14:2325–2338 Holly WD, Baker R (1991) Carnation production. Kendall/Hunt, Dubuque Holton TA, Cornish EC (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7:1071–1083 Ishida M, Ogata J, Yoshida H, Itoh Y, Ozeki Y (2003) Isolation of a cDNA for the chalcone 2 glucosyltransferase gene and its expression profile in carnation flowers. Plant Cell Physiol 44:S158 Itoh Y, Higeta D, Suzuki A, Yoshida H, Ozeki Y (2002) Excision of transposable elements from the chalcone isomerase and dihydroflavonol 4-reductase genes may contribute to the variegation of the yellow-flowered carnation (Dianthus caryophyllus). Plant Cell Physiol 43:578–585 Kolosova N, Gorenstein N, Kish CM, Dudareva N (2001) Regulation of circadian methyl benzoate emission in diurnally and nocturnally emitting plants. Plant Cell 13:2333–2347 Lavi M, Zuker A, Lewinsohn E, Larkov O, Ravid U, Vainstein A, Weiss D (2002) Linalool and linalool oxide production in transgenic carnation flowers expressing the Clarkia breweri linalool synthase gene. Mol Breed 9:103–111 Lorenzo O, Piqueras R, Sanchez-Serrano JJ, Solano R (2003) Ethylene response factor 1, integrates signals from ethylene and jasmonate pathways in plant defense. Plant Cell 15:165–178 Lucker J, Bouwmeester HJ, Schwab W, Blass J, van der plas LHW, Verhoeven HA (2001) Expression of Clarkia S-linalool synthase in transgenic petunia plants resulted in the accumulation of S-linalyl-beta-D-glucopyranoside. Plant J 27:315–324 Mol J, Cornish E, Mason J, Koes R (1999) Novel colored flowers. Curr Opin Biotechnol 10:198–201 Oksman-Caldentey KM, Inze D (2004) Plant cell factories in the post-genomic era: new ways to produce designer secondary metabolites. Trends Plant Sci 9:433–440 Okuhara H, Ishiguro K, Hirose C, Gao M, Togami J, Nakamura N, Ono E, Ochiai M, Fukui Y, Yanaguchi M, Tanaka Y (2004) Molecular cloning and functional expression of tetrahydroxychalcone 2 -glucosyltransferase genes. Plant Cell Physiol 45:S133

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Savin KW, Baudinette SC, Graham MW, Michael MZ, Nugent GD, Lu C-L, Chandler SF, Cornish EC (1995) Antisense ACC oxidase RNA delays carnation petal senescence. HortScience 30:970– 972 Schade F, Thompson JE, Legge RL (2003) Use of a plant-derived enzyme template for the production of the green-note volatile hexanal. Biotechnol Bioeng 84:265–273 Scovel G, Ben-Meir H, Ovadis M, Vainstein A (1998) RAPD and RFLP markers tightly linked to the locus controlling carnation flower type. Theor Appl Genet 96:117–122 Scovel G, Altshuler T, Liu Z, Vainstein A (2000) The evergreen gene is essential for flower initiation in carnation. J Hered 91:487–491 Smulders MJM, Noordijk Y, Rus-Kortekaas W, Bredemeijer GMM, Vosman B (2003) Microsatellite genotyping of carnation varieties. Theor Appl Genet 106:1191–1195 Springob K, Nakajima J, Yamazaki M, Saito K (2003) Recent advances in the biosynthesis and accumulation of anthocyanins. Nat Prod Rep 20:288–303 Tanaka Y, Katsumoto Y, Brugliera F, Mason J (2005) Genetic engineering in floriculture. Plant Cell Tissue Organ Cult 80:1–24 Vainstein A, Lewinsohn E, Pichersky E, Weiss D (2001) Floral fragrance. New inroads into an old commodity. Plant Physiol 127:1383–1389 Zik M, Irish VF (2003) Flower development: initiation, differentiation, and diversification. Annu Rev Cell Dev Biol 19:119–140 Zuker A, Aharoni A, Tzfira T, Ovadis M, Itzhaki H, Shklarman E, Ben-Meir H, Vainstein A (1998) Application of an integrative system based on microprojectile bombardment and Agrobacterium tumefaciens to generate transgenic carnation plants with novel characteristics. Int Congr Plant Tissue Cell Cult 9:35 Zuker A, Tzfira T, Aharoni A, Shklarman E, Ovadis M, Itzhaki H, Ben-Meir H, Vainstein A (2001a) Transgenic Dianthus spp (carnation). In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 48. Springer, Berlin Heidelberg New York, pp 123–136 Zuker A, Tzfira T, Scovel G, Ovadis M, Shklarman E, Itzhaki H, Vainstein A (2001b) RolCtransgenic carnation with improved agronomic traits: quantitative and qualitative analyses of greenhouse-grown plants. J Am Soc Hortic Sci 126:13–18 Zuker A, Shklarman E, Scovel G, Ben-Meir H, Ovadis M, Neta-Sharir I, Ben-Yefet Y, Weiss D, Watad A, Vainstein A (2001c) Genetic engineering of agronomic and ornamental traits in carnations. Acta Hortic 560:91–94 Zuker A, Tzfira T, Ben-Meir H, Ovadis M, Shklarman E, Itzhaki H, Forkmann G, Martens S, Neta-Sharir I, Weiss D, Vainstein A (2002) Modification of flower color and fragrance by antisense suppression of the flavanone 3-hydroxylase gene. Mol Breed 9:33–41

III.3 Chrysanthemum P.B. Visser, R.A. de Maagd, and M.A. Jongsma1

1 Introduction Chrysanthemum, Chrysanthemum x morifolium Ramat., is one of the leading cut flowers and potted plants in the international market and is grown worldwide, both in glasshouses and in the field. The Netherlands is the largest producer after Japan and Italy (Table 1) and the world’s main exporter of chrysanthemum. In 2003, turnover of chrysanthemums on the Dutch flower auctions was 336 × 106 Euro for cut- and 29 × 106 Euro for pot-flower varieties (source: Flower Council of Holland). Fast altering trends drive chrysanthemum breeders to create a large number of new and attractive varieties each year. The growers demand that these varieties are uniform and reliable in production, easy to handle in a partly mechanized production system, and resistant to pests and diseases. In turn, wholesalers and retailers expect a good shelf-life, resulting in a satisfactory vase life of both leaves and flowers for the consumer (de Jong 2001). It is of increasing importance that these varieties are compatible with environmentally sound and sustainable production systems, including the use of less energy and growth regulators in glasshouses, and the implementation of integrated pest management. By introducing such novel production systems, growers might certify their produce according to international standards like Eurepgap or MPS (www.eurep.org; www.st-mps.nl) which give them access to new markets dominated by powerful retailers. Cross-breeding and mutation-breeding are the basic tools for the generation of new cultivars. Genetic variation in aesthetic traits, like flower color and shape and plant architecture, as well as production traits, such as time-toflowering and yield, is widely available in Chrysanthemum x morifolium Ramat. germplasm, or otherwise easy to obtain via physical or chemical mutagenesis (Rout and Das 1997). Bringing in new genetic variation through crosses with related species is also relatively easy, provided that the genotypes are chosen carefully based on the compatibility of the species’ genomes (Fukai et al. 2000; Spaargaren 2002). Progress in breeding is slow, however, since back1 Business

Unit Bioscience, Plant Research International, Wageningen University and Research Centre, P.O. Box 16, NL-6700 AA Wageningen, The Netherlands, e-mail: [email protected]

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Table 1. Worldwide production area of cut-flower chrysanthemums (Spaargaren 2002)

Country

Production area (ha; glasshouse or tunnels)

Japan The Netherlands Italy Colombia Germany France United States Great Britain Israel

2,950 753 655 500 219 187 92 32 21

Production area (ha; field)

524 124

crossing to an ideotype chrysanthemum is virtually impossible due to its out-crossing nature, high heterozygosity, varying ploidy, and the polygenic control of many important traits. Thus, controlled combination of various desired traits into a single cultivar can only be achieved by tedious selection from siblings originating from large numbers of cross combinations between elite lines. Despite the large advances made in recent years for various biotechnological approaches in research laboratories (Rout and Das 1997; Zuker et al. 1998; Jong 2001; Deroles et al. 2002; Teixeira da Silva 2004; Rout et al. 2006), these have hardly been rewarding to the chrysanthemum industry to date. Contrary to other ornamental crops, in vitro micropropagation via somatic embryos or regenerated shoots for large-scale production is not used, no varieties on the market are known to be generated from somatic hybrids engineered in the laboratory, cryopreservation is not commonly used for the conservation of varieties, and no transgenic varieties are yet on the market. Regarding the slow progress in the classic breeding of chrysanthemums, the advantages of genetic engineering to introduce novel traits are obvious; a single gene can be inserted in the background of an established cultivar, and it is possible to stack multiple genes for various traits into a single cultivar. In addition, genes for the introduction of a new variation can be obtained from the entire plant and animal kingdom, surpassing any natural crossing barrier. Due to the high costs of bringing genetically engineered plants to the market, mostly generic traits, i.e., unique traits that can be applied to improve the performance of a wide range of varieties, rather than non-generic traits like flower color or changes in plant habit, may turn out to be the most commercially interesting. Examples of generic traits are product yield, pest and disease resistance, or drought and cold resistance, specifically for garden “mums”. In this review we discuss recent advances in the introduction of novel traits by genetic transformation and pay special attention to recent work on conferring insect and disease resistance in chrysanthemum.

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2 Chrysanthemum Transformation Agrobacterium-mediated transformation is the most commonly used method of introducing target genes into chrysanthemum. The success of transformation is cultivar-dependent, both in susceptibility to Agrobacterium infection and in vitro regeneration methods (Deroles et al. 2002; Teixeira da Silva 2004). The choice of Agrobacterium strain, binary vector, and type of explant are important factors determining the efficiency of transformation. Several groups have carried out extensive experiments to identify the best combination suitable for a range of different cultivars (Robinson and Firoozabady 1993; Urban et al. 1994; Seiichi et al. 1995; Dolgov et al. 1997; Sherman et al. 1998a; Yepes et al. 1999; Kudo et al. 2001). The relationship of the in vitro regeneration capacity of different cultivars relative to different explant types and hormone combinations has been reported, amongst others, by Kaul et al. (1990), Takatsu et al. (1998) and Annadana et al. (2000). The latter group was able to efficiently regenerate 23 of the 37 cultivars tested, as a result of a systematic assessment of the response of either stem or leaf explants to different media. In our work to assay the effect of transgenes in chrysanthemum, we are using a transformation protocol that has proved very efficient in the cultivar “1581”, as described in detail by de Jong (2001). We use pedicels from flowering plants as explants and transform with bacterium strain Agl0 harboring the binary vector pBINPLUS (Engelen et al. 1995) that carries the nptII gene under the control of the nos promoter for selection of transformed shoots on kanamycin. The transformation efficiency, defined as the number of transgenic shoots compared with the number of explants infected, is typically 25%, which allows us to generate large numbers of independent lines in a single experiment. 2.1 Promoter Studies During the mid-1990s it became increasingly clear that the CaMV35S promoter was almost 100-fold less active in chrysanthemum than in, for example, tobacco (Outchkourov 2003). Apparently, chrysanthemum and other Compositae do not possess the correct transcription factors or not in sufficient quantities for this commonly used promoter. This frustrated many attempts to over-express a broad variety of different genes and led Dutch and Japanese laboratories towards the isolation and testing of a series of alternative promoters for use in chrysanthemum leaves and flowers (Annadana 2002a, b; Outchkourov 2003; Aida et al. 2004, 2005). Table 2 gives an overview of the differences in activity of the different promoters fused to the β-glucoronidase (GUS) gene and expressed in chrysanthemum. Promoters tested were CaMV35S, the Cab promoters from potato (Lhca3.St1) and chrysanthemum (Cab), the rubisco small subunit (RbcS1) and ubiquitin extension protein (UEP1) promoters from chrysanthemum, the chalcone synthase (ChsA) and zinc finger transcription factor (EPF2) promoter

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Table 2. Activity of different promoters in chrysanthemum expressing the GUS enzyme. n.d. Not determined

Promoter

CaMV35S CaMV35S CaMV35S with MARs CaMV35S Lhca3.St1 Lhca3.St1 with MARs RbcS1 UEP1 ChsA EPF2 CER6 PMC Cab

GUS activity (nmol min−1 mg 1−1 ) Leaves Ray florets Maximum Average Maximum 0 1.7 2.0

0 0.1 0.2

3.0 142.0 228.0 82.0 1.4 0.3 1.0 1.6 1.5 31.0

Reference

n.d. n.d. n.d.

Outchkourov et al. (2003) Annadana et al. (2002a) Annadana et al. (2002a)

n.d. 25.0 26.0

21.0 n.d. 7.8 (mean)

Aida et al. (2004) Annadana et al. (2001) Annadana et al. (2001)

17.0 0.9 0.2 0.6 0.7 0.9 n.d.

n.d. 16.5 8.5 4.0 14.5 8.3 0.1

Outchkourov et al. (2003) Annadana et al. (2002a) Annadana et al. (2002a) Annadana et al. (2002a) Annadana et al. (2002a) Annadana et al. (2002a) Aida et al. (2004)

from petunia, the eceriferum promoter from Arabidopsis (CER6), and the multicystatin promoter from potato (PMC). Only the Cab and RbcS1 promoters were able to express proteins in the range of activity known for CaMV35S in other plants. Interestingly, the CaMV35S promoter expressed reasonably well in ray florets (Aida et al. 2004). Ray floret specificity was higher with the UEP1 promoter, however. The rubisco promoter from chrysanthemum is available commercially at www.impactvector.com as a range of ten different expression vectors. 2.2 Target Genes for Chrysanthemum Transformation The development of efficient chrysanthemum transformation systems has often been combined with the transfer and assessment of target genes for traits such as flower color, plant architecture, and resistance. The use of suboptimal transformation protocols and expression vectors resulted in a number of cases in the assessment of only a handful of independent lines or low expression of the transgene, rendering unclear or doubtful reports on the true effects of the inserted genes. A summary of all reported relevant traits is given in Table 3. Those papers are discussed in greater detail in the following sections. 2.2.1 Flower Color Manipulation of flower color is an obvious target as it dictates for a large part the aesthetic value in ornamentals. Blue tones, which are absent in the wide natural

Trait

Gene used

Phenotypic effects

Reference(s)

Color

Chrysanthemum CmCCD4a RNAi

Suppression of the carotenoid oxygenase gene results in white to yellow conversion Suppression of flower color, paler flowers

Ohmiya et al. (2006)

Antirrhinum antisense chalcone synthase (CHS) Maize flavonoid regulatory cDNA Lc Chimeric flavonoid 3 ,5 -hydroxylase Chrysanthemum chalcone synthase (CHS) Plant architecture

Ipomoea batatas ibMADS4 Arabidopsis gai Tobacco phytochrome B1 Rice phyA and Arabidopsis phyB Agrobacterium rhizogenes rolC

Flowering time Abiotic stress Viroid and virus resistance

No effect, presumably due to low transcript levels No effect reported Pink to white conversion of flowers and delayed flowering time Abundant branching and reduction in plant height; no flowering Reduction in plant height Reduction in plant height No effect, presumably due to low expression Dwarf bush habit with smaller flower in more compact flower stems Both early flowering and delayed lines obtained Freezing tolerance CSVd resistance correlated to expression levels

TSWV viral nucleocapsid gene

Complete TSWV resistance in three out of 20 lines No phenotype reported No effect, presumably due to low expression No effect, presumably due to low expression

Viral nucleocapsid genes TSWV viral nucleocapsid gene TSWV viral nucleocapsid gene

Mitiouchkina et al. (2000) Boase et al. (1998) Kim and Kim (1998) Courtney-Gutterson et al. (1994) Aswath et al. (2004) Petty et al. (2003) Zheng et al. (2001) Petty et al. (2000) Mitiouchkina and Dolgov (2000); Kubo et al. (2006) Shao et al. (1999) Hong et al. (2006) Ishida et al. (2002); Toguri et al. (2003) Sherman et al. (1998b) Yepes et al. (1999) Yepes et al. (1995) Urban et al. (1994)

257

Arabidopsis Leafy (LFY) Arabidopsis DREB1A dsRNA-specific ribonuclease (pacI)

Chrysanthemum

Table 3. Chrysanthemum transformation studies using genes to alter various traits

258

Table 3. continued

Trait

Gene used

Phenotypic effects

Reference(s)

Fungus resistance Insect resistance

Rice chitinase gene RCC2 Modified Bt gene (CryIC)

Moderate resistance to Botrytis Several lines with complete resistance to Spodoptera exigua Growth retardation of S. exigua larvae Complete resistance to Helicoverpa armigera Complete resistance to H. armigera No clear effects on Frankliniella occidentalis larvae count, possibly due to relatively low transgene expression 80% reduction of F. occidentalis larvae compared with wild type; deterrence of adults 75% deterrence Myzus persicae and 90% deterrence of F. occidentalis adults

Takatsu et al. (1999) de Jong 2001; this paper

Modified Bt gene (CryIAb) Modified Bt gene (CryIAb) Modified Bt gene (CryIAb) Potato multicystatin

Engineered seven-domain protein inhibitor gene Strawberry linalool synthase gene

Dolgov et al. (1995) Shinoyama et al. (2002) Shinoyama et al. (2003) Annadana et al. (2002c)

P.B. Visser, R.A. de Maagd, and M.A. Jongsma

Jongsma (2004); this paper Jongsma (2004); this paper

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palette of chrysanthemum colors, might be introduced into chrysanthemum via biotechnology, in a way similar to that achieved in carnation and rose by Suntory–Florigene (Mol et al. 1999; Tanaka and Mason 2003). To achieve blue colors, carotenoids responsible for the yellow-bronze colors need to be absent in the host cultivar and the pathway of a pink flower normally producing cyanidins needs to be modified to produce delphinidins. This could be achieved by suppressing the expression of flavonoid-3 -hydroxylase and replacing it with flavonoid-5 ,3 -hydroxylase, but this has not been reported to date. In fact, the first application of genetic transformation of chrysanthemum was the development of a white flowering variant from the pink flowering cv. Moneymaker by blocking the formation of 2 ,4 ,6 ,4-tetrahydroxychalcone, 6-steps upstream of the final product cyanidin (Courtney-Gutterson et al. 1994). Both sense and antisense constructs containing the homologous CHS gene were effective. However, up to 12% of the cuttings prepared from the altered plants also produced pink flowers, indicating that suppression of the CHS gene was not complete or was unstable. A similar effect was found when the Antirrhinum CHS gene was inserted into chrysanthemums; pink to white conversion was not complete and only a paler flower color was obtained. Attempts with other genes implicated in the flavonoid biosynthesis pathway have so far failed. The reverse project turning white flowers into yellow (not purple) ones was recently found to be possible by blocking (using RNAi) the carotenoid cleavage dioxygenase CmCCD4a gene which is active only in white flowers. This gene actively degrades the carotenoids which are responsible for the yellow petal color (Ohmiya et al. 2006; see Table 3). 2.2.2 Plant Architecture The modification of plant architecture in chrysanthemum is a relevant target investigated by a number of groups. First, pot or garden varieties could be developed that do not require chemical growth retardants to achieve the desired plant stature. Application of growth retardants is undesirable because of their costs and potential threat to the environment. Second, new, interesting cutflower varieties may be created with altered flowering habits or branching patterns, or spray chrysanthemums that do not need manual pruning of the axillary bud. Several different genes have been tested in chrysanthemum to change plant architecture (see Table 3). An example that is closest to commercial application might be chrysanthemums transformed with the tobacco phytochrome B1 gene (Zheng et al. 2001). Transgenic lines exhibited growth reductions very similar to those caused by commercial growth retardants, while production characteristics such as leaf area, canopy diameter or night-break sensitivity were hardly altered. Introduction of the A. rhizogenes rolC gene (Mitiouchkina and Dolgov 2000) and the ibMADS4 gene from Ipomoea batatas (Aswath et al. 2004) also rendered smaller plants, but these were accompanied by strong

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undesirable pleiotropic effects, such as lack of flowering, poor growth, and abundant branching. Chrysanthemums transformed with the gai gene also showed a dwarfed phenotype, but here flower size and number were also significantly reduced as compared with the wild type (Petty et al. 2003). 2.2.3 Resistance to Abiotic Stress The Arabidopsis DREB1A gene, encoding a stress-inducible transcription factor, driven by the cauliflower mosaic virus 35S (CaMV) promoter or by the stress-inducible rd29A promoter, was transferred into chrysanthemum plants (Hong et al. 2006). When exposed to 2 ◦ C, expression of DREB1A was enhanced in the roots of young transgenic 35S:DREB1A plant lines, and induced in transgenic rd29A:DREB1A lines. Electrolyte leakage in leaves of rd29A:DREB1A plant lines was significantly lower than in 35S:DREB1A lines. Compared with control plants, superoxide dismutase activities and proline contents increased slowly in transgenic plants at the start of the cold stress treatment and remained at high levels during later periods, especially in rd29A:DREB1A transgenic lines. Young plants of the rd29A:DREB1A line could tolerate –8 ◦ C for 12 h, with a survival rate of 37.5%. No survival was observed in 35S:DREB1A lines or in wild-type plants. These results indicate that a combination of the stress-inducible rd29A promoter and the DREB1A gene enhanced the tolerance of ground-cover chrysanthemum plants to cold stress through a transgenic approach. 2.2.4 Resistance to Viruses and Viroids Chrysanthemums are subject to a large number of virus and viroid diseases, of which chrysanthemum virus B (CVB), tomato aspermy virus (TAV), and chrysanthemum stunt viroid (CSVd) are considered to be the most important (Bouwen and van Zaayen 1995). However, the incidence of several tospoviruses is increasing worldwide, both in mother stocks and flower production and coincides with the spread of various thrips species, which are their natural vectors (Matteoni and Allen 1989; Matsuura et al. 2004). As no cure for virus-diseased plants exists, infection should be avoided by strict phytosanitary measures in the production chain, including the eradication of infected plants and their vectors, or the creation of resistant varieties. For the latter, the transgenic approach has proven to be efficient. Several groups employed the “nucleocapsid (N) gene approach” to confer resistance to TSWV (see Table 3), an approach that was shown to be effective for many different viruses and many different crops, but with the disadvantages of being isolate-specific in most cases, not conferring absolute immunity, and being prone to resistance breaking (Lomonossof 1995). Sherman et al. (1998b) introduced three TSWV N genes corresponding to three different virus isolates into chrysanthemum, encoding either truncated or antisense versions of

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these genes. Both sense and antisense constructs were found to be effective in yielding resistance, exhibiting a lack of symptoms and no virus accumulation in a small percentage of the transformed plants. A new concept of finding broad N gene-mediated resistance to tospoviruses was demonstrated recently by Rudolph et al. (2003). Instead of using complete or randomly truncated N genes, they transformed Nicotiana benthamiana with the GUS gene fused to a sequence encoding a 29-amino-acid peptide that was selected with the yeast two-hybrid system based on its strong interaction with the full-length TSWV N gene. The transgenic plants showed strong resistance to TSWV, tomato chlorotic spot virus (TCSV), groundnut ring spot virus (GRSV), and chrysanthemum stem necrosis virus (CSNV). As an alternative approach, transgenic chrysanthemums with enhanced resistance to CSVd were produced by expressing the dsRNA specific ribonuclease (dsRNase) Pac I protein from the yeast Schizosaccharomyces pombe (Ishida et al. 2002; Toguri et al. 2003). Pac I is an enzyme that specifically recognizes and degrades double-stranded RNA molecules, intermediates of virus and viroid replication. Resistance was found to be correlated with the expression level of Pac I. The highest expressors were free of symptoms and CSVd could be detected only in 20% of the tested plants, compared with 100% in low expressors. 2.2.5 Resistance to Fungi In chrysanthemum, the main fungal problems are white rust (Puccinia horiana) and botrytis (Botryotinia fuckeliana, Botrytis cinerea). Other common problems include Verticillium, Fusarium, Pythium, and Phoma spp. Against white rust, resistant germplasm exists and is used in new cultivars (de Jong and Rademaker 1986; Rademaker and de Jong 1987). The trait is monogenic but difficult to maintain in the hexaploid background of chrysanthemum. Currently, there are no efforts known to the authors to clone the gene. The Rpg1 gene providing resistance against Puccinia graminis in barley was cloned and characterized as a receptor kinase (Brueggeman et al. 2002). Expressed sequence tags with good homology to the Rpg1 coding sequence were found in wheat and with fair homology in sorghum, maize, barrel medic, and tomato. These data suggest that Rpg1 homologues may have a function in other species and possibly chrysanthemum against white rust. In that case, cloning by homology could be a feasible approach. No resistant germplasm has been found against Botrytis. This constraint can be controlled by cultivation conditions and fungicides, but is often still a serious post-harvest problem. In Japan, transgenic chrysanthemum plants were generated which over-expressed the RCC2 rice chitinase gene under the control of the CaMV35S promoter (Takatsu et al. 1999). This gene resulted in resistance to Botrytis in strawberry, cucumber and tobacco. Also in chrysanthemum a clear correlation could be established between the expression of the RCC2 gene and the degree of resistance. The plants with greatest expression

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showed only very slight symptoms compared with the control. The correlation with expression suggests that, if the RCC2 gene is expressed using a stronger promoter, symptom-free plants may be generated. A different approach to contemplate for fungal control is the over-expression of terpene compounds which are known to possess antifungal properties. One of the genes used for this is the resveratrol synthase gene from grape. This gene has shown resistance to Botrytis and Phoma in grape and alfalfa (Hipskind and Paiva 2000). Over-expression of terpene synthases has been successful in chrysanthemum (Jongsma 2004), so such approaches may well lead to resistance. 2.2.6 Resistance to Insects Chrysanthemum is subject to a broad array of common insect pests of glasshouses. The major ones which are most difficult to control are the western flower thrips (Frankliniella occidentalis) and leafminers (Liriomyza trifolii, L. huidobriensis). Other insect pests include aphids, the moth Spodoptera exigua, whitefly, and spider mite. In The Netherlands, their chemical control comprises anywhere up to 5% of the auction value obtained by the grower. The western flower thrips is one of the most important scourges of horticultural crops worldwide and is hosted by over 200 crops, including chrysanthemum. The insect causes growth defects and silver damage on the leaves. Growers of chrysanthemum consider the growth defects the most serious as they can lose an entire stem. Furthermore, for chrysanthemum this thrips is an important vector of tospoviruses (van de Wetering et al. 1999; Matsuura et al. 2002). The insect is difficult to control and rapidly develops resistance against many different insecticides. The difficulty in controlling leaf miners is related to the fact that the pupae and older larvae are insensitive to chemical treatments and reinfect treated plots (Spaargaren 2002). Resistance against growth and silver damage of thrips (de Jager et al. 1997) and damage by leafminers (de Jong and van de Vrie 1987) is found within chrysanthemum species and even among cultivars, but currently few of the available cultivars are resistant like cv. Penny Lane. One reason for the lack of breeding effort is the high cost of testing for thrips resistance amongst the breeding material at an early stage. Another reason is the complex inheritance of this quantitative and multigenic trait in the background of a hexaploid genome. Thus, there is a clear need for resistance genes which could be introduced into transgenic cultivars. So far, three categories of genes Bacillus thuringiensis (Bt) toxins, protease inhibitors, monoterpene synthases have been tested with success against moths and thrips in chrysanthemum. Bt Toxin Approaches Against Lepidopteran Pests Transgenic tobacco plants with resistance to lepidopteran pests were first produced in 1987 (Barton et al. 1987; Fischoff et al. 1987; Vaeck et al. 1987) and, since that time, many different plant species have followed, including chrysanthemum. So far, all com-

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mercialized transgenic insect-resistant crop varieties contain genes encoding insecticidal crystal proteins or delta-endotoxins from the bacterium Bacillus thuringiensis (Bt; de Maagd et al.1999). B. thuringiensis is a common bacterium found in soil and on plant surfaces and Bt has a long history of safe use as a biopesticide in agriculture, mainly for lepidopteran (and coleopteran in one case) larvae attacking crop plants. Different strains of this bacterial species produce different crystal proteins specifically active against one or a few insect species. The Bt toxins are produced in a crystal form and need to be ingested by the insect to be active. In the gut of the insect, the crystals dissolve and are proteolytically activated by gut enzymes to form a smaller toxic core protein. This binds to the gut epithelial cell membranes, in which it forms pores, killing the cells and, eventually, the insect (de Maagd et al. 2001). Early transgenic plants expressed a 3 -truncated wild-type toxin gene comprising the part encoding the toxic core. Likewise, the first example of chrysanthemum transformation with a Bt gene used a 3 -truncated version of the cry1Ab gene. Chrysanthemum calli transformed with this gene under transcriptional control of the Agrobacterium tumefaciens TR-2 promoter and the CaMV 35S terminator were toxic to larvae of the tobacco budworm (Heliothis virescens) and, in some cases, completely arrested larval development although no direct mortality was observed (van Wordragen et al. 1993). Cry1Ab transcript levels in these calli were low and only detectable by RT-PCR. Low transgene expression and incomplete protection (growth retardation instead of mortality), or protection against only very Bt toxin-sensitive insect species, is typical for this first generation Bt crops where an unmodified bacterial gene was used. A high A/T content and the presence of plant-specific mRNAinstability motifs in the bacterial gene were probably responsible for this. It was subsequently shown for various crops that removal of such instability or premature polyadenylation sequences significantly enhanced expression of various Bt toxin genes. Even further expression optimization was achieved by production of synthetic genes with plant species-specific codon usage (de Maagd et al. 1999). In the first published report on regenerated Bt-chrysanthemum, Dolgov et al. (1995) described two chrysanthemum plants expressing the truncated, unmodified cry1Ab gene under the control of the CaMV35S promoter. They gave growth retardation when fed to beet armyworm (Spodoptera exigua) larvae, but no further information on protein expression levels was reported. Surprisingly, Shinoyama et al. (2003), using a similarly truncated, unmodified Cry1Ab gene under the control of the double CaMV 35S-promoter, found Cry1Ab expression levels up to 0.08% of total soluble leaf protein. This is unusually high for any unmodified Bt gene in any plant. The expression was sufficient to inhibit the feeding of Helicoverpa armigera larvae, which were relatively insensitive to Cry1Ab. A version of the Cry1Ab gene that was optimized for codon usage of the family Compositae gave only slightly higher expression levels, up to 0.16% of soluble leaf protein. Levels of approximately 0.1% and higher were sufficient to achieve 100% mortality in feeding H. armigera larvae.

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At CPRO-DLO, later Plant Research International, we have been working towards the production of Spodoptera exigua-resistant chrysanthemum. S. exigua is a cosmopolitan, polyphagous pest on many crops in subtropical and tropical areas. In the Netherlands, it occurs as sporadic infestations in glasshouses with chrysanthemum. The larvae feed preferentially on flower buds and young leaves of chrysanthemum plants. Initially, we transformed a Cry1Ca gene with limited modifications under control of the 35S promoter, which previously had given some S. exigua-resistance in tobacco and tomato (van der Salm et al. 1994). In transgenic chrysanthemum, this yielded only two plants (out of 38 tested) with significant growth reduction (50%) of feeding larvae. We then switched to the use of a synthetic, plant codon-optimized Cry1Ca gene provided by Dr. Nikolay Strizhov, which had been shown to confer complete S. exigua resistance in various other plants (Strizhov et al. 1996; Cao et al. 1999). Under transcriptional control of the 35S promoter, this gene gave readily detectable protein expression and insect resistance in transgenic tobacco, but in transgenic chrysanthemum the expression of Cry1Ca did not reach more than 0.01% of total soluble protein and, in most cases, protein expression was below the detection limit. Thus, only three out of 38 plants yielded 100% mortality among second instar larvae, while the other plants gave varying degrees of growth retardation and/or partial mortality (Jong 2001; Fig. 1). As described in Section 2.1 and Table 22, an endogenous chrysanthemum rubisco small subunit promoter (rbcS1) performed considerably better (Outchkourov et al. 2003) and was, therefore, selected for driving expression of the Cry1Ca gene in a new series of transgenic plants. As can be seen in Fig. 1, all transgenic plants containing the new construct performed better than their counterparts with the 35S promoter, and more than half (27 out of a total of 50 tested plants) gave 100% mortality of S. exigua larvae in a detached-leaf assay (Fig. 2A). Overall protein expression levels as detected by Western blotting were still relatively low (up to 0.016%), indicating that there were still limiting factors for Bt protein expression in chrysanthemum to be discovered, but apparently expression levels over 0.01% of total soluble protein were sufficient to confer complete protection. High-expressing plants were grown in the glasshouse till flowering and infested with several egg packets of S. exigua to mimic a natural infestation. Whereas untransformed control plants were devastated, the tested transgenic plants were protected completely (Fig. 2B). Protease Inhibitors Against Thrips Protease inhibitors are known to interfere with protein digestion, causing stunted growth, increased mortality, and reduced fecundity. They were applied successfully for the first time in transgenic tobacco in 1987 against the moth Heliothis zea. This initiated a rush of research to employ these commonly found genes in plants as insect-resistance traits. However, it became quite evident that over-expression of most plant protease inhibitors was rather ineffective and resulted, at the most, in a minor slowdown of growth rate. In 1995 it was demonstrated that, in response to dietary inhibitors, insects were able to induce protease genes which were insensitive to them (Jongsma et al. 1995). Recently, we published a detailed

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Fig. 1. Results of detached leaf feeding assays with L2 larvae of Spodoptera exigua for two sets of transgenic chrysanthemum cv. 1581. Gray bars indicate larval weights for plants containing the CaMV35S-synthetic cry1Ca construct, solid black bars for plants containing the RbcS1 promotercry1Ca constructs. Weights of the sum of four feeding larvae per leaf, averaged for four repeats, are shown. Zero weights indicate 100% mortality. Hatched bars indicate the weights for leaves of two different non-transformed controls. Note that the total number of tested plants as indicated below the X-axis is different for the two sets

Fig. 2. Detached leaf (A) and glasshouse whole-plant assays (B) comparing non-transformed and rbcS1-cry1Ca expressing plants. A Right: detached leaves placed on water-agar infested with four L2 larvae of S. exigua and photographed after 5 days. Left: untransformed control. B Plants grown in the glasshouse till flowering, infested with S. exigua eggs by attaching several egg packages to the lower side of the top leaves. Right: non-transformed control. Left: rbcS1-cry1Ca expressing plant

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analysis of how these resistant enzymes evolved from their sensitive ancestral genes (Volpicella et al. 2003). It was clear that there was a need to find inhibitors still effective against such “resistant” enzymes. As a source of such inhibitors, it was proposed to use protease inhibitors from the animal kingdom in tests against insects. A large range of known serine, cysteine, and aspartic protease inhibitors were tested in vitro against the enzymes and in vivo against several different types of insects, including western flower thrips (Annadana et al. 2002a). Cysteine protease inhibitors reduced the fecundity of thrips, but did not affect the mortality of adults, at least not over the duration of the assay (6 days). Transgenic plants were made initially with available single- and multidomain inhibitors. Transgenic chrysanthemum over-expressing potato multicystatin did not have a clear effect on the fecundity of the insects (Annadana et al. 2002c). However, potato plants which expressed a different set of inhibitors of animal origin were much more effective (Outchkourov et al. 2004a). Combining equistatin with a number of different cystatins in the form of fusion proteins of four to seven independent domains resulted in improved activity against thrips when compared with any of the single domains. Glasshouse trials, which monitored the survival of adult insects and the number of offspring produced during the first 14 days, demonstrated that the multidomain transgenic potato plants had fewer adults and 80% less offspring (Outchkourov et al. 2004b). In this review, we also present very similar results for chrysanthemum over-expressing the seven-domain inhibitor (Fig. 3). From the data, it can be predicted that the population may eventually die out; and Fig. 4 shows that the damage level was reduced from intermediate to low. In addition, the effect of the inhibitors is not only to reduce fecundity. Choice assays indicated that protease inhibitors are also strongly deterrent to adult insects in a dose-dependent fashion (Outchkourov et al. 2004a). Deterrence or repellence may prove an effective, additional way of protecting plants against herbivores such as thrips. Monoterpenes Against Sucking Insect Pests Volatile organic compounds emitted by plants are known to provide strong cues to predators and parasites of herbivores to locate their prey. Aharoni et al. (2003) demonstrated in choice assays that aphids are deterred from Arabidopsis plants that constitutively produce high concentrations of linalool. Further corroboration of these results was obtained for other plant species such as potato and chrysanthemum. Figure 5 shows that over-expression of the same linalool synthase gene from strawberry in chrysanthemum under the control of the rubisco promoter resulted in high emission of linalool and dimethylnonatriene. Given a choice, 90% of adult thrips preferred the control over the transgenic plants. Applications of these genes may, therefore, be an effective way of keeping chrysanthemum free from these insects. Moreover, some elements of this line of defense first need to be more fully understood. At low concentrations, linalool is not repellent, but attractive to these insects. Apparently, only the high emission levels achieved in these plants create the repellent effect. This may be due to the fact the insects can be killed by high vapor concentrations. Possibly, these classes

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Fig. 3. Effect of over-expression of an engineered seven-domain protease inhibitor in chrysanthemum on the average number of larvae found on the plants 2 weeks after inoculation with females. Tests were performed in the glasshouse with individual caged plants. Data are based on a replicate of six plants. P values represent significance by Student’s t-test

Fig. 4. Diagrams demonstrating the effectiveness of the multidomain inhibitor EIMKACP in reducing reproduction and silver damage on plants in the glasshouse in a non-choice experimental set-up. Significant differences were calculated by Student’s t-test and are indicated with their P value. The error bars indicate the standard error. Experiments were performed in 6-fold replication with individual plants in plastic tubular cages

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Fig. 5. Effect of over-expression of the monoterpene linalool in chrysanthemum based on the linalool synthase gene from strawberry. Right: the transgenic line T58-39 emits a large peak of linalool, which is not detectable in the control. Left: as a result, thrips given a choice between the transgenic and the control line prefer (in a large majority; 84%) the control line without linalool. Effects are significant after the first 15 min of orientation by the insects

of compounds are recognized as toxins at certain threshold concentrations, which would make them more interesting for applications in plant protection.

3 Future Outlook In chrysanthemum, many novel traits have been introduced by genetic modification over the past twelve years. Traits have ranged from flower color, plant architecture, and flowering time to virus, fungal, and insect resistance. The potential of insect, virus, and disease resistance appears to be most ready for application in the case of chrysanthemum. Half of the publications deal with those aspects and they seem to be successful in achieving their goals. From contacts with breeders, it also seems that those traits are of greatest interest, because they complement the current breeding aims, which are mainly for different floral shapes and colors. The direct economic benefit of resistance traits for the grower will be a reduced production cost of around 5% of the auction value. Since the margins in flower production are narrow, an attractive resistant variety may win a grower’s favor against an equally attractive non-resistant variety. Similarly, an ecological image will be important in a market which is more and more dominated by large retailers. For them, a variety which can be grown without pesticides may be favored over a variety with high residues. Finally, in intensive growing areas like The Netherlands, the government is restricting the use of pesticides, forcing the sector to respond accordingly.

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Thus, the need to find durable solutions has a multipronged socioeconomic motivation which may finally win breeders to adopt these new biotechnological methods. The pioneering work of companies like Suntory–Florigene will also help to demonstrate the success of engineered traits on the market in Europe. Blue carnations recently went on offer in Europe, apart from the United States, Japan, and Australia. It seems only a matter of time until the first transgenic chrysanthemum varieties appear on the market.

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Sherman JM, Moyer JW, Daub ME (1998b) Tomato spotted wilt virus resistance in chrysanthemum expressing the viral nucleocapsid gene. Plant Dis 82:407–414 Shinoyama H, Komano M, Nomura Y, Nagai T (2002) Introduction of delta-endotoxin gene of Bacillus thuringiensis to chrysanthemum [Dendranthema x grandiflorum (Ramat.) Kitamura] for insect resistance. Breed Sci 52:43–50 Shinoyama H, Mochizuki A, Komano M, Nomura Y, Nagai T (2003) Insect resistance in transgenic chrysanthemum (Dendranthema x grandiflorum (Ramat.) Kitamura) by the introduction of a modified delta-endotoxin gene of Bacillus thuringiensis. Breed Sci 53:359–367 Spaargaren JJ (2002) Jaarrondchrysanten: Teelt en achtergronden. Spaargaren, Aalsmeer Strizhov N, Keller M, Mathur J, Konczkalman Z, Bosch D, Prudovsky E, Schell J, Sneh B, Koncz C, Zilberstein A (1996) A synthetic cryIC gene, encoding a Bacillus thuringiensis delta-endotoxin, confers Spodoptera resistance in alfalfa and tobacco. Proc Natl Acad Sci USA 93:15012–15017 Takatsu Y, Tomotsune H, Katsumi M, Sakuma F (1998) Differences in adventitious shoot regeneration capacity among Japanese chrysanthemum [Dendranthema grandiflorum (Ramat.) Kitamura] cultivars and the improved protocol for Agrobacterium-mediated genetic transformation. J Jpn Soc Hortic Sci 67:958–964 Takatsu Y, Nishizawa Y, Hibi T, Akutsu K (1999) Transgenic chrysanthemum Dendranthema grandiflorum (Ramat.) Kitamura expressing a rice chitinase gene shows enhanced resistance to gray mold (Botrytis cinerea). Sci Hortic 82:113–123 Tanaka Y, Mason J (2003) Manipulation of flower colour by genetic engineering. In: Singh RP, Jaiwal PK (eds) Plant genetic engineering. SCI Tech, Houston, pp 361–385 Teixeira da Silva JA (2004) Ornamental chrysanthemums: improvement by biotechnology. Plant Cell Tissue Organ Cult 79:1–18 Toguri T, Ogawa T, Katitani M, Tukahara M, Yoshioka M (2003) Agrobacterium-mediated transformation of chrysanthemum (Dendranthema grandiflora) plants with a disease resistance gene (pac1). Plant Biotechnol 20:121–127 Urban LA, Sherman JM, Moyer JW, Daub ME (1994) High frequency shoot regeneration and Agrobacterium-mediated transformation of chrysanthemum (Dendranthema grandiflora). Plant Sci 98:69–79 Vaeck M, Reynaerts A, Höfte H, Jansens S, Beuckeleen MD, Dean C, Zabeau M, Montagu MV, Leemans J (1987) Transgenic plants protected from insect attack. Nature 328:33–37 Volpicella M, Ceci LR, Cordewener J, America T, Gallerani R, Bode W, Jongsma MA, Beekwilder MJ (2003) Properties of purified gut trypsins from Helicoverpa zea, adapted to proteinase inhibitors. Eur J Biochem 270:10–19 Wetering F van de, Posthuma K, Goldbach R, Peters D (1999) Assessing the susceptibility of chrysanthemum cultivars to tomato spotted wilt virus. Plant Pathol 48:693–699 Wordragen MF van, Honee G, Dons HJ (1993) Insect-resistant chrysanthemum calluses by introduction of a Bacillus thuringiensis crystal protein gene. Transgenic Res 2:170–180 Yepes LM, Mittak V, Pang SZ, Gonsalves C, Slightom JL, Gonsalves D (1995) Biolistic transformation of chrysanthemum with the nucleocapsid gene of tomato spotted wilt virus. Plant Cell Rep 14:694–698 Yepes LM, Mittak V, Pang SZ, Gonsalves D, Slightom JL (1999) Agrobacterium tumefaciens versus biolistic-mediated transformation of the Chrysanthemum cvs. polaris and golden polaris with nucleocapsid protein genes from three tospovirus species. Acta Hortic 482:209–218 Zheng ZL, Yang ZB, Jang JC, Metzger JD (2001) Modification of plant architecture in chrysanthemum by ectopic expression of the tobacco phytochrome B1 gene. J Am Soc Hortic Sci 126:19–26 Zuker A, Tzfira T, Vainstein A (1998) Genetic engineering for cut-flower improvement. Biotechnol Adv 16:33–79

III.4 Orchids H. Yu1 and Y. Xu2

1 Introduction Orchids are members of the Orchidiaceae, one of the largest families of flowering plants. With more than 20,000 species, its members occupy a wide range of ecological habitats in all parts of the world and exhibit highly diverse morphological and physiological characteristics (Dressler 1990). A large diversity of orchids is found in tropical regions, with almost all epiphytic species being limited to tropical or subtropical areas. With the exception of using some orchids as herbs, most orchids are grown mainly for floricultural purposes because of their attractive flowers. Orchids have the typical flowers of monocotyledonous plants, which consist of four whorls of floral organs, namely petaloid sepals, petals, anthers, and pistils (Goh and Arditti 1985). Most orchid species have several unique floral characteristics that distinguish them from other flowering plants (Fig. 1). First, there is only one functional stamen, fused with styles and stigmas into a structure called the column or gynostemium in most orchid species. Second, pollen grains are bound together by viscin threads in masses and packaged as a pollinium for effective pollination. Third, the median petal located opposite to a column is often modified to form a gaudy and variable labellum or lip, which can serve as a landing platform for insect pollinators. These special features facilitate pollinators to move pollen grains from one flower to another, which might be one of the main reasons contributing to the success of the Orchidiaceae during the evolutionary development of flowering plants. It is of importance and practical value for the orchid industry to continuously generate novel varieties with improved floral characters to satisfy human fancy and desire for something new. Although classic breeding of orchids by sexual hybridization and selection of variants and polyploids is still the mainstay of developing new varieties, the inability to obtain specific traits and the time-consuming hybridization process are major hurdles to the production of orchids with commercial value. There was some major progress on 1 Plant Functional Genomics Group, Temasek Life Sciences Laboratory, 1 Research Link, National

University of Singapore, Singapore 117604, and Plant Functional Genomics Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Science Drive 4, Singapore 117543, e-mail: [email protected] 2 Plant Functional Genomics Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Science Drive 4, Singapore 117543 Biotechnology in Agriculture and Forestry, Vol. 61 Transgenic Crops VI (ed. by E.C. Pua and M.R. Davey) © Springer-Verlag Berlin Heidelberg 2007

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Fig. 1. Dendrobium “Chao Praya” Smile flower showing unique floral characteristics, with the fused structure of stigmas, styles and stamens (column), pollinium packaged by pollen grains, and the variable median petal (labellum or lip)

orchid biotechnology to manipulate important floral traits and other relevant desirable characteristics, such as flowering time, flower morphology, color, fragrance, vase-life and resistance to diseases and pests. During the past decade, intensive and rapid development in molecular biology opened up new avenues for the molecular breeding of orchids, which overcome the limitations of conventional breeding methods. Indeed, the application of molecular techniques to orchid improvement represents a typical trend of current orchid biotechnology. The significant advantage of this new technology is its ability to mark and achieve desirable traits by genetic engineering of specific genes into orchids, thus being a major driving force in the development of orchid research and industry. The core component of molecular breeding of orchids is to create efficient and reproducible gene transformation systems for different species. These systems are more or less established for the available tissue culture methods in the corresponding species. Coupled with advances in generating gene transfer systems, the considerably expanded number of genes identified either from orchids or from other plant species provide ample resources for molecular breeding of orchids for desired traits. The purpose of this chapter is to summarize the methodology of genetic transformation of orchids, to discuss the various factors affecting the transformation process and to briefly introduce the application of some interesting genes in genetic engineering of orchids.

2 Genetic Transformation of Orchids Genetic transformation of plants aims to incorporate exogenous genes into plant chromosomes and to change their traits either by the regulated expression

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of exogenous genes or by the interruption of endogenous gene expression. During orchid transformation, various gene transfer methods are utilized to deliver nucleic acids into appropriate starting materials, which are followed by a series of processes to isolate and regenerate transgenic plants under specific selection conditions. A number of factors such as transformation methods, various starting materials and selection markers have been identified for developing simple, efficient and reproducible transformation systems. 2.1 Transformation Methods Transformation methods used for orchid species include particle bombardment, Agrobacterium-mediated transformation, pollen tube pathway and electrophoresis (Kuehnle 1997; Mudalige and Kuehnle 2004). The former two methods are more frequently employed. 2.1.1 Particle Bombardment Particle bombardment, also known as biolistic transformation or microprojectile bombardment, is the most effective way to directly introduce nucleic acids into plant cells via high-velocity microprojectiles (Klein et al. 1987). Its basic procedures involve coating microcarriers with DNA and propelling the coated microcarriers into target plant cells, usually by gas acceleration. Two groups of parameters exert major effects on the efficiency of particle bombardment. One is the physical parameters associated with non-biological materials; the other is the biological parameters associated with the targeted tissues and foreign DNAs. Physical parameters mainly include the microcarrier variables (e.g. type, amount, size) and the acceleration parameters (e.g. gas pressure, distances between the rupture disk, macrocarrier, stopping screen and target tissue). The two most important parameters are the size of microcarriers and the target distance between the stopping screen and the target tissue. Other physical parameters have relatively low impact on transformation efficiency (Sanford et al. 1991, 1993; Christou 1994). The biological parameters include the nature of the target tissues and the expression vectors used for transformation. The physiological state of the target tissues greatly influences their competence for stable DNA uptake and shoot regeneration. This state can be regulated by the selection of appropriate explants for transformation and the optimization of pre- and post-bombardment culture conditions. The nature and concentration of the expression vectors for bombardment may also be important for transformation efficiency, although it has not been widely tested in particle bombardment (Sanford et al. 1991, 1993; Christou 1994). The introduction of foreign genes into orchids by particle bombardment was first reported in Vanda and Dendrobium, two distinct genera in terms of

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their taxonomic group and physiological and genetic characters (Chia et al. 1990; Kuehnle and Sugii 1992). As particle bombardment bypasses several key barriers, for example, poor plant regeneration from protoplasts or less susceptibility to Agrobacterium infection (Kuehnle and Sugii 1992) as encountered in other orchid transformation systems, it was the most widely used method of gene transfer in orchids during the past decade (Table 1). Two main challenges encountered in the process of particle bombardment of orchids are to reduce the frequency of chimerism in transformants and to increase the selection efficiency. In order to obtain stable and uniform transformants, more attention needs to be paid to the investigation of explant characteristics before and after bombardment. Although information on the fate of plant cells after being transformed with accelerated particles is rare, meristematic tissues of orchids have been used. The starting materials used in the first two reports on particle bombardment of orchids were Vanda embryos and Dendrobium protocorms (Chia et al. 1990; Kuehnle and Sugii 1992). Chimerism was found to be a major problem in both studies, because non-transformed Table 1. Examples of genetic transformation of orchids by particle bombardment. Bar Bialaphos resistance gene, CymMV-CS-CP Cymbidium mosaic virus Taiwan isolate capsid protein, DOH1 Dendrobium orchid homeobox 1, GFP green fluorescent protein, GUS β-glucuronidase, HPT hygromycin phosphotransferase, LUC luciferase, NPT II neomycin phosphotransferase, PFLP sweet pepper ferredoxin-like protein, PLB protocorm-like body, PRV-CP papaya ringspot virus coat protein

Orchid genus

Explant

Vanda Dendrobium Dendrobium Dendrobium Phalaenopsis Cymbidium Dendrobium Brassia Cattleya Doritaenopsis Dendrobium Phalaenopsis

Embryos Protocorms PLBs Protocorms Calli PLBs Protocorms PLBs Protocorms PLBs PLBs PLBs

Cymbidium Cymbidium Dendrobium Dendrobium Oncidium Dendrobium

Genes introduced

LUC PRV-CP, NPT II LUC GUS, NPT II GUS, NPT II GUS, NPT II GUS, HPT Bar Bar Bar DOH 1, NPT II Bar, NPT II, GUS, soybean β-1,3-endogluconase PLBs GUS Petals GUS PLBs GUS, NPT II Calli and PLBs GUS, HPT PLBs PFLP, GFP-GUS, HPT PLBs CymMV-CS-CP, HPT

Reference Chia et al. (1990) Kuehnle and Sugii (1992) Chia et al. (1994, 2001) Nan and Kuehnle (1995) Anzai et al. (1996) Yang et al. (1999) Yu et al. (1999) Knapp et al. (2000) Knapp et al. (2000) Knapp et al. (2000) Yu et al. (2000) Anzai and Tanaka (2001)

Boase et al. (2001) Peters et al. (2001) Yu et al. (2002) Men et al. (2003a) You et al. (2003) Chang et al. (2005)

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cells might not be effectively eliminated during direct plant regeneration from such embryos and protocorms. To recover non-chimeric transgenic plants, a series of further optimizations of orchid transformation were designed to regenerate plantlets with an intervening callus or organogenic phase, thus eliminating non-transformed cells in the presence of selection markers. In most of these studies, calli, protocorms, or protocorm-like bodies (PLBs) were sliced into smaller pieces and served as the target tissues for bombardment (Chia et al. 1994; Knapp et al. 2000; Yu et al. 2000, 2002; Men et al. 2003a; Yang et al. 2003). The regeneration of PLBs and shoots from these sliced tissues proceeds with an organogenic phase, which effectively discriminates between putatively transformed and non-transformed tissues. The selection efficiency of particle bombardment is determined by multiple factors that need to be coordinated in a comprehensive selection system. The timing of selection and the choice of selection markers are two critical factors. It was shown that the timing of selection had a significant impact on transformation efficiency in different orchid systems (Yang et al. 1999; Men et al. 2003a). Plant tissues used as target materials are susceptible to damage by particle bombardment, and thus may require a healing period after bombardment under special culture conditions without selective pressure (Christou 1994). However, delayed selection may cause the production of chimeric plants, as bombarded cells are usually injured and eventually fail to regenerate shoots in competition with unbombarded cells on culture medium free of selection agents (Men et al. 2003a). Therefore, the timing of selection, which varies amongst different orchid species, is always optimized by a compromise between gaining time for the recovery of the wounded, bombarded cells and limiting the propagation of unbombarded cells. For example, most of the bombarded protocorms of Dendrobium hybrid “Mihua” gradually died when selection was initiated 2 weeks after bombardment; the eventual transformation efficiency, however, was rather high when selection was delayed for up to 3 months (Yu et al. 1999). In contrast, in both D. phalaenopsis Banyan Pink and D. nobile, early selection commencing 2 days after bombardment achieved a much better transformation efficiency than selection delayed for up to 30 days (Men et al. 2003a). The selection marker is another important factor that greatly influences the selection efficiency in all stable gene transfer systems, including particle bombardment and Agrobacterium-mediated transformation. It is discussed later as a separate topic in this chapter. 2.1.2 Agrobacterium-Mediated Transformation Agrobacterium tumefaciens, a soil bacterium pathogenic to a range of plant species, is able to transfer a discrete portion of its T-DNA into the nuclear genomes of host plant cells by the combined action of virulence (vir) genes in the same strain (Hellens et al. 2000). Agrobacterium-mediated transformation takes advantage of this natural mechanism by incorporation of the T-DNA

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region carrying a gene-of-interest into the genome of host plant cells. This method has some significant advantages over other transformation methods, such as a high transformation efficiency, a potentially low copy number of transgenes inserted into target plant cells, the capacity to transfer large DNA fragments and relatively low cost (Hiei et al. 1994). Moreover, after about 30 years of intensive research on the molecular genetic mechanisms underlying T-DNA transfer in tumorigenesis, the components of Agrobacterium-mediated transformation can now be flexibly manipulated and developed according to specific demands and expectations, thus making this method the most userfriendly and common choice for plant transformation. The procedures of Agrobacterium-mediated transformation include three basic steps, namely, cloning of genes-of-interest into the T-DNA, introduction of T-DNA into Agrobacterium strains and integration of T-DNA into plant genomes via Agrobacterium-mediated infection of host plants (Hooykaas and Schilperoort 1992). These processes are affected by two groups of parameters related to host plants and non-host characteristics, respectively. Despite the broad host range of A. tumefaciens encompassing a large number of dicotyledons, some monocotyledons and gymnosperms, many agronomically important plant species are recalcitrant to this transformation method (Gelvin and Liu 1994). This is partially due to the different amounts of plant phenolic compounds, such as acetosyringone, coumaryl alcohol and sinapyl alcohol, in the host plants, which can induce vir gene expression during Agrobacteriummediated transformation. Non-host parameters are mainly determined by the selection of Agrobacterium strains and tumor-inducing (Ti) plasmids that contain T-DNA and/or the vir region. Orchids, like many other monocotyledons, are usually considered as plants recalcitrant to Agrobacterium infection. However, the presence of coniferyl alcohol, the vir gene inducer in Dendrobium orchids, provides a fundamental element required for successful Agrobacterium-mediated transformation (Nan et al. 1997). Nan et al. (1998) reported the first example of Agrobacterium-mediated transformation of orchids, in which they documented the incorporation of partial T-DNA into the Dendrobium genome. Since then, numerous efforts have been made to optimize Agrobacterium-mediated transformation systems in orchids (Table 2). There are several factors that contribute significantly to the success of Agrobacterium-mediated transformation of orchids. Amongst them, the concentration of phenolic compounds in the transformation system and the period of co-cultivation of Agrobacterium with orchid explants are two of the most important factors. Despite the endogenous production of a certain concentration of phenolic signal compounds in orchids (Nan et al. 1997; Belarmino and Mii 2000), considerable evidence shows that exogenous application of acetosyringone (AS), an essential phenolic compound for transformation of some monocotyledons (Hiei et al. 1994; Ishida et al. 1996; Suzuki et al. 2001), dramatically increases the transformation efficiency in orchids (Belarmino and Mii 2000; Liau et al. 2003; Men et al. 2003b). Agrobacterium-

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Table 2. Examples of Agrobacterium-mediated transformation of orchids. DOH1 Dendrobium orchid homeobox 1, DSCKX1 Dendrobium Sonia cytokinin oxidase gene, GUS β-glucuronidase, HPT hygromycin phosphotransferase, NPT II neomycin phosphotransferase, PFLP sweet pepper ferredoxin-like protein, PLB protocorm-like body

Orchid genus

Explant

Genes transformed

Reference

Dendrobium Phalaenopsis Dendrobium Oncidium Dendrobium Oncidium Dendrobium

PLBs Calli PLBs PLBs PLBs PLBs PLBs

GUS, HPT GUS, NPT II, HPT DOH1 GUS, HPT GUS, HPT PFLP, GFP-GUS, HPT DSCKX1 (sense and antisense)

Nan et al. (1998) Belarmino and Mii (2000) Yu et al. (2001) Liau et al. (2003) Men et al. (2003b) You et al. (2003) Yang et al. (2003)

mediated transformation of Dendrobium, Phalaenopsis and Oncidium orchids was enhanced by the addition of an appropriate concentration of AS in the co-cultivation medium at pH 5.0–5.5, the optimum condition for AS to activate the vir gene of the Ti plasmid and to initiate T-DNA transfer (Stachel et al. 1986). The co-cultivation period should be balanced between maximal gene transfer from Agrobacterium to explant cells and a minimal explant necrosis by manipulating the growth of Agrobacterium in the co-cultivation medium (Yu et al. 2001). In both Dendrobium “Madame Thong-In” and Oncidium “Sherry Baby cultivar OM8” orchids, division of the co-cultivation period into two consecutive stages contributed greatly to the success of transformation (Yu et al. 2001; Liau et al. 2003). The first stage was the 3-day co-cultivation of sliced PLBs with Agrobacterium on antibiotic-free medium. This was followed by the second stage of co-cultivation of explants with slow-growing Agrobacterium for a period of 1 month on medium containing low concentrations of antibiotics for the suppression of Agrobacterium growth. Since the foreign DNA from Agrobacterium may keep being transferred into actively growing explants during co-cultivation, an extension of this process can effectively improve transformation efficiency. However, optimization of a definite co-cultivation period is subject to the species and types of explants used for transformation. For example, when suspension cells derived from friable calli of a Phalaenopsis orchid were used for transformation, extension of the co-cultivation period from 3 days to 7 days did not increase transient transformation efficiency, but instead resulted in necrosis and cell death (Belarmino and Mii 2000). 2.2 Explants Used for Orchid Transformation During orchid transformation and regeneration, the response of explants is influenced by factors such as defense mechanisms that cause phenolic production, cell division frequency and shoot regeneration through competence of the

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cells at the wound sites. An explant that is highly responsive to transformation may not efficiently regenerate shoots. This could be one of the major reasons why the frequency of transient expression of transgenes in orchid explants after transformation is usually much higher than that of the production of stable transformants derived from these explants. Therefore, it is important to select the appropriate explants that are competent for both transformation and regeneration. The intensive research on tissue culture of orchids for mass propagation provides useful information on the suitability of the starting materials for gene transformation. Orchid was one of the first horticultural crops successfully mass-propagated via tissue culture. The first attempt was initiated with the intention to produce virus-free Cymbidium Plant (Morel 1960). Since then, various explants have been used for orchid propagation through tissue culture, with most of them being meristematic tissues with the capacity of regenerating into plants. Thin sections or chopped tissues of PLBs are the most widely used explants reported for successful gene transfer in orchids by particle bombardment and Agrobacterium-mediated transformation (Chia et al. 1994; Knapp et al. 2000; Yu et al. 2000, 2001; Liau et al. 2003; Men et al. 2003a; Yang et al. 2003; You et al. 2003). There are several obvious advantages for using PLBs as starting materials for transformation. First, generation of uniform PLBs directly from explants at a high frequency could be achieved (e.g. Park et al. 1996, 2000, 2002), thus serving as a good basis for large-scale gene transformation experiments. Second, PLBs in early stages of development consist of meristematic cells in an active stage of division, which is important for both transformation and regeneration of shoots (Kuehnle and Sugii 1992; Park et al. 2002; Liau et al. 2003). Third, it was reported that, at least in Dendrobium orchids, the amount of coniferyl alcohol, a vir gene inducer, is higher in PLBs than in other tissues. The production or stability of coniferyl alcohol in PLBs was found to be enhanced by light (Nan et al. 1997). These advantages suggest that PLBs are ideal starting materials for both particle bombardment and Agrobacterium-mediated transformation. It is noteworthy that direct application of PLBs in transformation is prone to generate chimeric plants because PLBs themselves are able to form shoots directly without an intervening organogenic phase (Kuehnle and Sugii 1992). This problem is overcome practically by the use of thin sections or chopped portions of PLBs, because these treated tissues regenerate young shoots with an organogenic phase, during which non-transformed cells are effectively eliminated under selection pressure. Orchid calli are another type of starting material suitable for transformation (Anzai et al. 1996; Belarmino and Mii 2000; Men et al. 2003a). Calli contain actively dividing cell clumps that are usually more competent for transformation because the active state of cell division and DNA synthesis in host plant cells might be important for the integration of exogenous DNA into the host genome (Binns and Thomashow 1988). Furthermore, infected callus cells are exposed

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directly and immediately to the selection agents, which can effectively prevent the regeneration of chimeric plants. However, transformation of orchid callus is not widely used at present because orchid callus culture has proven to be very difficult and successful only in exceptional cases (Chang and Chang 1998; Men et al. 2003a). 2.3 Selectable Markers The selectable marker is an integral part of a plant transformation system. With the introduction of a particular marker gene into plant materials, putative transformants can be identified by a specific selection agent that corresponds to the selection marker. The sensitivity of plant cells to a selection agent is affected by the genotype and developmental stage of plant materials, the transformation and regeneration processes and the activity of selection marker genes. The currently available selection markers for screening transgenic orchids are antibiotic-, herbicide-, or pathogen-resistant genes and visual reporter genes. 2.3.1 Antibiotic Selection Neomycin phosphotransferase, or NPTII, encoded by the nptII gene, is the most widely used selectable marker for plant transformation. Through phosphorylation, NPTII can inactivate a number of aminoglycoside antibiotics, such as kanamycin, neomycin, geneticin and paromomycin, amongst which kanamycin is the most common selective agent, and is normally used in concentrations ranging over 50−700 mg l−l (Angenon et al. 1994; Chia et al. 1994). Although some orchids are known to be resistant to kanamycin selection, this antibiotic has been used for selecting transformants in different orchid genera (Kuehnle and Sugii 1992; Nan and Kuehnle 1995; Anzai et al. 1996; Yang et al. 1999; Yu et al. 2000, 2001). However, in most of these studies, high concentrations of kanamycin had to be applied for stringent selection, which not only inhibited plant regeneration, but also increased the cost of the process. Moreover, the transformation strategy by kanamycin selection in orchids is labor-intensive and time-consuming, because it usually takes a relatively long time to perform several rounds of screening on kanamycin selective media. For example, when transforming Dendrobium “Madame Thong-In”, putative transformants could only be identified after 6–8 months of selection with monthly subculture on the medium containing 200 mg l−l kanamycin (Yu et al. 2000). Excessive tissue culture with such a selection process is always associated with somaclonal variation (Chen et al. 1998) that could result in the development of undesirable characteristics in the culture. The hpt gene encoding hygromycin phosphotransferarse (HPT) has been used as the hygromycin B resistance marker for plant transformation. Hygromycin B is an aminocyclitol antibiotic interfering with protein synthesis, this antibiotic usually being more toxic than kanamycin and eliminating sen-

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sitive cells more quickly (Angenon et al. 1994). In orchid transformation, hygromycin was more effective than kanamycin in discriminating between transformed and non-transformed cells (Nan et al. 1998; Yu et al. 1999; Belarmino and Mii 2000; Liau et al. 2003; Men et al. 2003a, b; You et al. 2003). In the reported protocols, hygromycin concentrations of 5−50 mg l−l were used for selection of transformants from Dendrobium, Phalaenopsis and Oncidium orchids. Compared with kanamycin selection, application of hygromycin in transformation and regeneration significantly reduces the selection time and the frequency of generating chimeric plants, and is thus considered as a more stringent antibiotic for orchid transformation. 2.3.2 Herbicide Selection Phosphinothricin (PPT) is a glutamate analog and is able to inhibit glutamine synthetase in a wide range of organisms, resulting in the accumulation of inhibitory levels of ammonium. PPT, together with two other l-alanine residues, make up the tripeptide antibiotic bialaphos. The bialaphos resistance gene (bar) from Streptomyces hygroscopicus encodes a phosphinothricin acetyl transferase that can detoxify PPT and its alanine derivative, bialaphos, by acetylating their free amino residues (De Block et al. 1987; D’Halluin et al. 1992). Thus, plants transformed with the bar gene can survive on selection medium containing PPT or bialaphos. Herbicides such as BASTA or Finale (Bayer CropScience Pty, Australia) that contain PPT can be used to select transgenic plants with the bar gene. However, some non-specific growth inhibitors in these commercial herbicides prevent them from being used in agar medium. For selection in agar-solidified media, the purified form of PPT or bialaphos may have to be used, which results in extra cost for the selection process. Several widely varied and unrelated orchid genera, such as Doritaenopsis, Brassia, Cattleya (Knapp et al. 2000) and Phalaenopsis (Anzai and Tanaka 2001), have been transformed by particle bombardment using pure bialaphos as a selective agent for the bar gene in transformants. Compared with antibiotic selection, herbicide selection requires a lower concentration of bialaphos (e.g. 1−3 mg l−l ) and less selection time (e.g. 1 month). As a result, the inhibitory effects of exogenous chemicals on plant growth and somaclonal variation due to excessive tissue culture can be reduced. However, the necessity to use pure bialaphos for selection of transformants in agar-solidified medium makes these orchid transformation systems comparatively expensive. It was reported recently that l-methionine sulfoximine (MSO) has a similar structure to PPT and acts as a highly effective alternative selective agent for bar gene expression in plants (Maughan and Cobbett 2003). In the selection of transgenic Arabidopsis plants, MSO is at least 40 times more effective and 10 times less costly than PPT. Moreover, MSO is readily available from major laboratory chemical companies. Thus, MSO seems to be a more potent

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and economical agent for selection of transformants in agar-solidified medium (Maughan and Cobbett 2003). The application of MSO in orchid transformation is being investigated in our laboratory (Chia et al., unpublished data). Preliminary experiments show that 10 μM MSO is sufficient to kill non-transformed shoots of Dendrobium “Sonia” within 1 month (Fig. 2), which is in contrast to selection using a high concentration of kanamycin at 200 mg l−l for several months (Yang et al. 2003). Further studies of the effects of MSO on the growth of orchid transformants and the introduction of MSO into a variety of transformation systems are required to assess its potential as an efficient and economical selective agent in the genetic engineering of orchids. 2.3.3 Visual Selection The firefly luciferase gene (LUC) has been successfully used as a non-invasive reporter gene for Dendrobium orchid transformation (Chia et al. 1994). With the application of a photon-counting video camera–photomultiplier system, transgenic tissues can be readily identified by monitoring bioluminescence emitted upon the addition of a minute amount of a non-toxic substrate (luciferin). In this system, non-transformed tissues can be selectively removed and the transformed ones can be maintained during each round of screening, thus increasing the efficiency and reproducibility of the transformation process. However, this method is costly because of the necessity to utilize

Fig. 2. Sensitivity of Dendrobium “Sonia” wild-type calli to different concentrations of lmethionine sulfoximine (MSO) in half MS medium with 2% sucrose and 5 mg l−l benzyladenine 1 month after incubation. A 0 μM MSO, B 1 μM MSO, C 5 μM MSO, D 10 μM MSO

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photon-counting imaging technology and its related facilities. In addition, the need for manpower to manually monitor and isolate transformed tissues in rounds of selection makes this method labor-intensive. Therefore, such a visual selection method is, so far, not widely applied to orchid transformation. 2.3.4 Pathogen Resistance Selection Pathogen resistance selection is a novel method for the selection of transgenic orchids utilizing the sweet pepper (Capsicum annuum) ferredoxin-like protein (pflp) gene as a selectable marker and the pathogen Erwinia carotovora as a selection agent (You et al. 2003). The pflp gene encodes a peptide with antimicrobial activity that shows delayed hypersensitive response in non-host plants via the release of a harpin proteinaceous elicitor. Oncidium orchids transformed with the pflp gene showed enhanced resistance to E. carotovora, even when the whole plant was challenged with this pathogen, while nontransformed plants could not survive under similar conditions. E. carotovora causes soft-rot disease, which is one of the most severe diseases of orchids. The application of the pflp gene in the transformation of Oncidium orchids not only protects the plants from pathogen infection, but also provides a useful selection agent for genetic engineering strategies. There are several advantages for adopting this new system over other available systems (You et al. 2003). First, putative transformants can be identified after 2 weeks, which is significantly faster than other methods. Second, in contrast to the inhibitory effect of antibiotics on orchid growth, this method seems to cause fewer side-effects on plant development. In addition, the presence of antibiotic- and herbicide-resistant marker genes in transformants is always a matter of concern regarding the toxicity or allergenicity of their gene products to the environment and humans. The pflp gene originates from plants and its application in plant transformation raises less concern in this respect. Lastly, because of the removal of the large cassette for antibiotic gene expression, it is more feasible to include economically important genes combined with the pflp gene into vectors for orchid transformation. Although this system is not widely tested in different orchid genera thus far, the above-mentioned advantages make it an excellent choice to perform gene transformation for orchids that are not natural hosts of E. carotovora.

3 Potential Genes for Genetic Engineering of Orchids Potential genes that are useful for genetic engineering of orchids can be selected either from orchids or from other organisms. So far, there are more than 70 genes cloned from seven orchid genera, namely Dendrobium, Phalaenopsis, Doritaenopsis, Aranda, Bromheadia, Vanilla and Cymbidium (Mudalige and Kuehnle 2004; Xu et al. 2006). Based primarily on sequence similarity with other known genes and the expression patterns, these orchid

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genes are divided into different groups with possible functions in cell division and structure, primary metabolism, flower induction and development, flower color, flower senescence and disease or stress response, respectively. Although time-consuming and inefficient orchid transformation systems have hampered the investigation of orchid gene function and regulation in vivo, a few studies have been carried out with improved transformation systems. Dendrobium orchid homeobox 1 (DOH1), a class 1 knox gene, is one of the orchid genes successfully studied and applied to the genetic engineering of orchids (Yu et al. 2000, 2001). Northern analysis and in situ hybridization showed that DOH1 mRNA accumulates in meristem-rich tissues, and its expression is greatly down-regulated during floral transition. In Dendrobium orchids transformed with the sense construct of the DOH1 gene driven by the cauliflower mosaic virus 35S promoter, over-expression of DOH1 completely suppressed shoot organization and development. Transgenic orchid plants expressing antisense mRNA for DOH1 showed multiple shoot apical meristem (SAM) formation and early flowering. These findings suggest that DOH1 plays a key role in maintaining the basic plant architecture of orchids through control of the development of SAM. Furthermore, the precocious flowering phenotype exhibited by DOH1 antisense transformants is coupled with the early expression of DOMADS1, a MADS-box gene involved in orchid floral transition (Yu and Goh 2000). This indicates that down-regulation of DOH1 in the SAM is required for floral transition in orchids. Many orchid species need several years to develop from juvenile to the reproductive phase, and this greatly limits orchid production and research. Despite numerous studies involving physiological aspects of flowering time control in orchids, the molecular understanding of orchid flowering remains poor. Since the molecular genetic pathways involved in the control of flowering time in the model plant Arabidopsis are being elucidated, it is plausible to use characterized flowering time genes in Arabidopsis or their orchid orthologs for genetic engineering of orchids. The successful regulation of DOH1 expression in Dendrobium provided the first example of genetic engineering of orchids for early flowering. Dendrobium Sonia cytokinin oxidase (DSCKX1) is another endogenous gene that has been used for orchid transformation (Yang et al. 2003). The DSCKX1 gene appears to have three alternative splicing forms and its expression was induced in a tissue-specific manner by cytokinins. In orchid transformants over-expressing DSCKX1, the up-regulation of cytokinin oxidase activity was accompanied by a reduction of cytokinin content. These plants exhibited slow shoot growth with numerous long roots in vitro. Their calli exhibited decreased capability for shoot formation. In contrast, antisense orchid transformants coupled with a higher endogenous cytokinin content than wild-type plants showed rapid proliferation of shoots and inhibition of root growth. Thus, DSCKX1 activity plays an important role in the regulation of cytokinin metabolism and its related developmental programs in orchids,

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demonstrating a novel way to control orchid development by affecting the hormone signaling pathway through transgene expression. There are several studies using exogenous genes to produce transgenic orchids with new characters, such as improved disease or pathogen resistance, novel flower color and increased vase life. Transgenic orchids with overexpression of the pflp gene exhibited significantly enhanced resistance against soft-rot disease caused by the bacterium Erwinia carotovora (You et al. 2003). However, the antimicrobial properties of the pflp gene remain unknown. Transformation of Dendrobium orchids with the viral coat protein of Cymbidium mosaic virus (CymMV; Chia 1999) generated transgenic orchids that exhibited only partial resistance to viral infection. Following successful production of Dendrobium plants carrying the firefly luciferase gene, Chia et al. (2001) made the transgenic orchids glow in the dark after spraying the plants with a suitable luciferin. During the past decade, intensive molecular genetic studies in the model plants Arabidopsis and rice have elucidated many gene regulation processes during development. Knowledge in this field is being further enhanced with the complete sequencing of the rice genomes of these plants and some ongoing sequencing projects in other plant species. These studies provide orchid biologists with new information for investigation of the function of orchid orthologs, which will provide, in turn, more gene resources for the genetic engineering of orchids for new traits.

References Angenon G, Dillen W, Montagu MV (1994) Antibiotic resistance markers for plant transformation. In: Gelvin SB, Schilperoort RA (eds) Plant molecular biology manual II. Kluwer, Dordrecht, C1:1–13 Anzai H, Tanaka M (2001) Transgenic Phalaenopsis (a moth orchid). In: Bajaj YPS (ed) Transgenic crops III. (Biotechnology in agriculture and forestry, vol 48) Springer, Berlin Heidelberg New York, pp 249–264 Anzai H, Ishii Y, Schichinohe M, Katsumata K, Nojiri C, Morikawa H, Tanaka M (1996) Transformation of Phalaenopsis by particle bombardment. Plant Tissue Cult Lett 13:265–271 Belarmino MM, Mii M (2000) Agrobacterium-mediated genetic transformation of a Phalaenopsis orchid. Plant Cell Rep 19:435–442 Binns AN, Thomashow MF (1988) Cell biology of Agrobacterium infection and transformation of plants. Annu Rev Microbiol 42:575–606 Boase MR, Peters TA, Spencer MA, Bendall MJ (2001) Factors affecting transient expression of the GUS A reporter transgene in Cymbidium protocorm-like bodies transformed biolistically via a particle inflow gun. Tissue culture and biotechnology in New Zealand. Crop Food Res Rep 16:47 Chang C, Chang WC (1998) Plant regeneration from callus culture of Cymbidium ensifolium var. misericors. Plant Cell Rep 17:251–255 Chang C, Chen YC, Hsu YH, Wu JT, Hu CC, Chang WC, Lin NS (2005) Transgenic resistance to Cymbidium mosaic virus in Dendrobium expressing the viral capsid protein gene. Transgenic Res 14:41–46

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Chen WH, Chen TM, Fu YM, Hsieh RM, Chen WS (1998) Studies on somaclonal variation in Phalaenopsis. Plant Cell Rep 18:7–13 Chia TF (1999) DNA technology and genetic engineering of orchids. Proc Asia Pac Orchid Conf 6:1–4 Chia TF, Chan YS, Chua NH (1990) Large-scale screening of Cymbidium mosaic and Odontoglossum ringspot viruses in cultivated orchids by nucleic acid spot hybridization. In: Bonham OG, Kernohan J (eds) Proceedings of the 13th world orchid conference. WOC Proceedings Trust, Auckland, p. 284 Chia TF, Chan YS, Chua NH (1994) The firefly luciferase gene as a non-invasive reporter for Dendrobium transformation. Plant J 6:441–446 Chia TF, Lim AYH, Luan Y, Ng I (2001) Transgenic Dendrobium (orchid). In: Bajaj YPS (ed) Transgenic crops III. (Biotechnology in agriculture and forestry, vol 48) Springer, Berlin Heidelberg New York, pp 95–106 Christou P (1994) Gene transfer to plants via particle bombardment. In: Gelvin SB, Schilperoort RA (eds) Plant molecular biology manual II. Kluwer, Dordrecht, A2:1–15 De Block M, Botterman J, Vandewiele M, Dockx J, Thoen C, Gossele V, Movva NR, Thompson C, Van Montagu M, Leemans J (1987) Engineering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO J 6:2513–2518 D’Halluin K, De Block M, Denecke J, Janssens J, Leemans J, Reynaerts A, Botterman J (1992) The bar gene as selectable and screenable marker in plant engineering. Methods Enzymol 216:415–426 Dressler RL (1990) The orchids: natural history and classification. Harvard University Press, Cambridge, Mass. Gelvin SB, Liu CN (1994) Genetic manipulation of Agrobacterium tumefaciens strains to improve transformation of recalcitrant plants species. In: Gelvin SB, Schilperoort RA (eds) Plant molecular biology manual II. Kluwer, Dordrecht, B4:1–13 Goh CJ, Arditti J (1985) Orchidaceae. In: Halevy AH (ed) Handbook of flowering. CRC, Boca Raton, pp 309–336 Hellens R, Mullineaux P, Klee H (2000) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5:446–451 Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of T-DNA. Plant J 6:271–282 Hooykaas PJJ, Schilperoort RA (1992) Agrobacterium and plant genetic engineering. Plant Mol Biol 19:15–38 Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency of transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750 Klein T, Wolf E, Wu R, Sanford J (1987) High velocity microprojectiles for delivering nucleic acids into living cells. Nature 327:70–73 Knapp JE, Kausch AP, Chandlee JM (2000) Transformation of three genera of orchid using the bar gene as a selectable marker. Plant Cell Rep 19:893–898 Kuehnle AR (1997) Molecular biology of orchids. In: Arditti J, Pridgeon AM (eds) Orchid biology: reviews and perspectives VII. Kluwer, Dordrecht, pp 75–115 Kuehnle AR, Sugii N (1992) Transformation of Dendrobium orchid using particle bombardment of protocorms. Plant Cell Rep 11:484–488 Liau CH, You SJ, Prasad V, Hsiao HH, Lu JC, Yang NS, Chan MT (2003) Agrobacterium tumefaciensmediated transformation of an Oncidium orchid. Plant Cell Rep 21:993–998 Maughan SC, Cobbett CS (2003) Methionine sulfoximine, an alternative selection for the bar marker in plants. J Biotechnol 102:125–128 Men S, Ming X, Wang Y, Liu R, Wei C, Li Y (2003a) Genetic transformation of two species of orchid by biolistic bombardment. Plant Cell Rep 21:592–598 Men S, Ming X, Liu R, Wei C, Li Y (2003b) Agrobacterium-medium genetic transformation of a Dendrobium orchid. Plant Cell Tissue Organ Cult 75:63–71 Morel G (1960) Producing virus-free Cymbidiums. Am Orchid Soc Bull 29:495–497

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Mudalige RG, Kuehnle AR (2004) Orchid biotechnology in production and improvement. HortScience 39:11–17 Nan GL, Kuehnle AR (1995) Factors affecting gene delivery by particle bombardment of Dendrobium orchids. In Vitro Cell Dev Biol 31:131–136 Nan GL, Tang CS, Kuehnle AR, Kado CI (1997) Dendrobium orchids contain an inducer of Agrobacterium virulence genes. Physiol Mol Plant Pathol 51:391–399 Nan GL, Kuehnle AR, Kado CI (1998) Transgenic Dendrobium orchid through Agrobacteriummediated transformation. Malay Orchid Rev 32:93–96 Park SY, Kakuta S, Kano A, Okabe M (1996) Efficient propagation of protocorm-like bodies of Phalaenopsis in liquid medium. Plant Cell Tissue Organ Cult 45:79–85 Park SY, Murthy HN, Paek KY (2000) Mass multiplication of protocrom-like bodies (PLBs) using bioreactor system and subsequent plant regeneration in Phalaenopsis. Plant Cell Tissue Organ Cult 63:67–72 Park SY, Yeung EC, Chakrabarty D, Paek KY (2002) An efficient direct induction of protocorm-like bodies from leaf subepidermal cells of Doritaenopsis hybrid using thin-section culture. Plant Cell Rep 21:46–51 Peters TA, Boase MR, Nielsen KM, Spencer MA, Lewis DH (2001) Promoter expression studies in Cymbidium petal tissue using biolistic-mediated transformation. Tissue culture and biotechnology in New Zealand. Crop Food Res Rep 16:58 Sanford JC, Devit MJ, Russell JA, Smith FD, Harpending PR, Roy MK, Johnston SA (1991) An improved, helium driven biolistic device. Technique 3:3–16 Sanford JC, Smith FD, Russell JA (1993) Optimizing the biolistic process for different biological applications. Methods Enzymol 217:483–509 Stachel SE, Nester EW, Zambryski PC (1986) A plant cell factor induces Agrobacterium tumefaciens vir gene expression. Proc Natl Acad Sci USA 83:379–383 Suzuki S, Supaibulwatana K, Mii M, Nakano M (2001) Production of transgenic plants of liliaceous ornamental plant Agapanthus praecox ssp. orientalis (Leighton) Leighton via Agrobacteriummediated transformation of embryogenic calli. Plant Sci 161:89–97 Xu Y, Teo LL, Zhou J, Kumar PP, Yu H (2006) Floral organ identity genes in the orchid Dendrobium crumenatum. Plant J 46:54–68 Yang J, Lee H, Shin DH, Oh SK, Seon JH, Paek KY, Han K (1999) Genetic transformation of Cymbidium orchid by particle bombardment. Plant Cell Rep 18:978–984 Yang SH, Yu H, Goh CJ (2003) Functional characterisation of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid. Plant Mol Biol 51:237–248 You SJ, Liau CH, Huang HE, Feng TY, Prasad V, Hsiao HH, Lu JC, Chan MT (2003) Sweet pepper ferredoxin-like protein (pflp) gene as a novel selection marker for orchid transformation. Planta 217:60–65 Yu H, Goh CJ (2000) Identification and characterization of three orchid MADS-box genes of the AP1/AGL9 subfamily during floral transition. Plant Physiol 123:1325–1336 Yu H, Yang SH, Goh CJ (2000) DOH1, a class 1 knox gene, is required for maintenance of the basic plant architecture and floral transition in orchid. Plant Cell 12:2143–2160 Yu H, Yang SH, Goh CJ (2001) Agrobacterium-mediated transformation of a Dendrobium orchid with the class 1 knox gene DOH1. Plant Cell Rep 20:301–305 Yu H, Yang SH, Goh CJ (2002) Spatial and temporal expression of the orchid floral homeotic gene DOMADS1 is mediated by its upstream regulatory regions. Plant Mol Biol 49:225–237 Yu Z, Chen M, Nie L, Lu H, Ming X, Zheng H, Qu LJ, Chen Z (1999) Recovery of transgenic orchid plants with hygromycin selection by particle bombardment. Plant Cell Tissue Organ Cult 58:87–92

III.5 Gladiolus K. Kamo1 and Y.H. Joung2

1 Introduction Gladiolus is a floral bulb used as both a cutflower and as a garden plant. In 2002, the wholesale value of Gladiolus in the United States as cutflower was U.S.$ 26,708,000, and there were 126,285,000 flower spikes sold (USDA 2002). In the same year, the wholesale value of all floral crops was U.S.$ 4.9 × 109 , of which cutflowers were U.S.$ 4.1 × 108 . The main pathogens affecting Gladiolus are Fusarium oxysporum and viruses. Viruses are a major problem for crops such as Gladiolus because the plants are propagated each year by corms that harbor virus (Stein 1995). Gladiolus is susceptible to several viruses, among which bean yellow mosaic virus and cucumber mosaic virus are the most common ones. Infection by multiple viruses has more impact on the plant than that by a single virus. Viral symptoms include colorbreak of the flowers, mosaic of the leaves and deformed plants, making the flowers unattractive for sale (Stein 1995). The viruses can cause stunting of the plant, resulting in a decreased yield for the grower. Effective physical and chemical treatments are unavailable for virus elimination, and even if eliminated, the plants can become infected again with virus. Virus-resistant cultivars of Gladiolus are not available for breeding.

2 Tissue Culture Callus capable of regenerating plants has been induced from various explants, including inflorescence stalks, cormel slices, basal meristems and tips of shoots, ovaries and young leaf bases. A comparison using four cultivars of Gladiolus showed that the greatest induction of embryogenic callus was from corm slices (69–100%) as compared with whole plants (36–40%) or young leaf bases (22– 35%; Stefaniak 1994). The middle section of the cormel was preferred to the top or bottom sections for callus induction (Remotti and Loffler 1995). The addition of 5 mg l−1 adenine sulfate, 1 g l−1 casein hydrolysate, 1 mg l−1 thiamine and 80 mg l−1 NaH2 PO4 enhanced callus induction (Remotti and Loffler 1 Floral and Nursery Plants Research Unit, US National Arboretum, USDA, Beltsville, MD 20705,

USA, e-mail: [email protected] Science and Technology, Chonnam National University, Gwangju 500-757, Korea

2 School of Biological

Biotechnology in Agriculture and Forestry, Vol. 61 Transgenic Crops VI (ed. by E.C. Pua and M.R. Davey) © Springer-Verlag Berlin Heidelberg 2007

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1995). Most callus induction occurs using MS basal salts medium (Murashige and Skoog 1962) rather than other basal salts, as first demonstrated by Wilfret (1971). Although callus induction is genotype-dependent, induction has been reported for many cultivars including Aldebaran, Blue Isle, Firmament, Hector, Her Majesty, Hit Parade, Jenny Lee, Oscar, Peter Pears, Rosa Supreme, Spic and Span, Snow Princess, Topaz, Traveler and a South African species, G. garnierii (Simonsen and Hildebrandt 1971; Wilfret 1971; Bajaj et al. 1982; Stefaniak 1994; Kamo 1995; Remotti and Loffler 1995; Kasumi et al. 1998; Goo et al. 2003). Media supplemented with either 1−10 mg l−1 naphthaleneacetic acid (NAA), 0.5−2.0 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l−1 dicamba or 1 mg l−1 picloram have been used for callus induction and maintenance. Cell suspensions of Gladiolus were first reported using liquid MS medium supplemented with 0.1 mg l−1 NAA (Simonsen and Hildebrandt 1971). A well dispersed cell suspension is ideal for transformation using the gene gun (Kamo et al. 1995b). The callus induced in the presence of 2,4-D is typically friable and has been used to establish suspension cultures of the cultivars Jenny Lee and Peter Pears (Kamo et al. 1990; Kamo 1995; Remotti 1995). Suspension cells consisted of well dispersed clusters of 25–50 cells that doubled in packed cell volume in two weeks (Remotti 1995). Plants have been regenerated from gladiolus callus and suspension cells via primary and secondary somatic embryogenesis (Stefaniak 1994; Remotti 1995). Primary embryos developed from suspension cells cultured on MS medium supplemented with either zeatin or benzyladenine (BA; Remotti 1995). Regeneration from callus occurs on hormone-free MS medium and can be increased 2- to 3-fold for the cultivar Jenny Lee by the addition of 2 mg l−1 kinetin (Kamo 1994). Kumar et al. (1999) found that prolific shoot regeneration from callus occurred on MS medium following a heat shock at 50 ◦ C for 1 h. Optimal shoot regeneration could be induced from callus cultured in the presence of 1 μM BA and 10 μM NAA (Kumar et al. 1999). The antioxidant enzyme activities for catalase and peroxidase decreased during somatic embryogenesis, while superoxide dismutase increased (Gupta and Datta 2003). In comparison, the activity of superoxide dismutase decreased, while catalase and peroxidase increased during shoot organogenesis. The reports are varied of transplanting in vitro-grown plants either from micropropagation or regenerated from callus. Ziv (1979) had difficulty transplanting micropropagated plants of the cultivar Eurovision to soil until the plants were cultured on half-strength MS medium supplemented with a reduced sucrose concentration (1.5%), 0.4 mg l−1 thiamine, 0.5 mg l−1 NAA and 0.3% activated charcoal, and grown under a higher light intensity than used for maintaining the micropropagated plants. Lilien-Kipnis and Kochba (1987) found that the success of transplanting miniature hybrids of Gladiolus propagated in vitro depended on the extent of rooting, the latter being affected by the NAA concentration. Others have planted cormels formed in vitro to soil in the glasshouse rather than plants. The cormel size increased using an

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increased (6–10%) sucrose concentration (Dantu and Bhojwani 1987). Remotti (1995) reported that plants of the cultivar Peter Pears regenerated from suspension cells formed a well developed root system after being cultured on MS medium lacking hormones. Prior to transplanting in the glasshouse, the in vitro-grown plants with well developed roots were transferred to medium containing paclobutrazol to reduce stress after transplanting. All plants survived in the glasshouse, except for a few albino plants (Remotti 1995). A machine vision system was used to determine the possibility of sorting regenerated plants of Gladiolus based on their photometric behavior, using image analysis coupled with neural network algorithm (Mahendra and Gupta 2004). Two groups of plants were recognized, but the capacity of each group to survive acclimatization was not tested. Direct regeneration without a callus phase or a minimal callus phase occurs directly from cormel slices cultured on agar-solidified medium with 1 mg l−1 BA (Sutter 1986; Kamo 1995). Plants regenerate more vigorously and with fewer phenotypic abnormalities when they originate directly from the cormel slice as compared with regeneration from callus. Direct regeneration is not cultivar-dependent. Longitudinal corm sections resulted in more shoots (six per corm explant) than the shoot tips, basal plate or daughter corm explants (Nhut et al. 2004). These regenerated shoots were mass-propagated in liquid MS medium containing BA and sucrose. Gene gun bombardment of the cormel slices has been demonstrated using the uidA gene (Kamo et al. 1995a). A few of the transformed plants had completely blue leaves indicating that they were non-chimeric plants. However, most of the transformed plants showed leaves with blue horizontal stripes indicating their chimeric nature. The most devastating pathogen infecting Gladiolus is Fusarium oxysporum that causes corm rot, plants that are blind and deformed, and flowers that are disfigured (Wilfret 1992). There have been few successes where genetic engineering for antifungal resistance results in resistance in the field. An alternative approach is in the selection of cell lines of the Gladiolus cultivar Peter Pears that are resistant to fusaric acid (Remotti et al. 1997). Plants regenerated from resistant cells showed an increased tolerance to fusaric acid in vitro.

3 Genetic Transformation Genetic engineering for virus resistance could be important for bulb crops such as Gladiolus, as well as for other ornamental plants that are propagated vegetatively. Transformation of Gladiolus has been accomplished using either Agrobacterium (Graves and Goldman 1987; Babu and Chawla 2000) or biolistics (Kamo et al. 1995a). Graves and Goldman (1987) showed that Gladiolus corm slices infected with A. tumefaciens formed tumors that synthesized opines. Babu and Chawla (2000) confirmed A. tumefaciens infection of Gladiolus using shoot tips. Shoot tips that had been wounded by bombardment with gold

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particles using the gene gun rather than with a scalpel prior to incubation with A. tumefaciens showed more areas of GUS expression three weeks after infection. The availability of an efficient regeneration system from callus of several Gladiolus cultivars (Kamo et al. 1990; Remotti and Loffler 1995) has been utilized for transformation via biolistics. Regeneration has been verified to occur through both primary and secondary somatic embryogenesis (Stefaniak 1994; Remotti 1995). The efficiency of phosphinotricin and bialaphos on both selections of putatively transformed plants and for stimulating regeneration has contributed to the establishment of a transformation system for Gladiolus (Chauvin et al. 1997; Kamo and Van Eck 1997).

4 Promoters and Gene Expression Initial testing of several promoters using the gene gun for transient transformation revealed that most GUS expression was conferred by the dicotyledon promoters mas2 and rolD, rather than the maize Ubi1 and rice Act1 promoters derived from cereal monocotyledons (Kamo et al. 1995a). The activity of nine promoters was then characterized in Gladiolus plants using the uidA gene under control of either the cauliflower mosaic virus CaMV 35S, duplicated 35S, rice Act1, potato Ubi3, potato Ubi7, Arabidopsis UBQ3, rolD, mas2 or Arabidopsis translation elongation factor 1 subunit α promoters. GUS expression was least with the potato Ubi3 and Ubi7 promoters, and these promoters were not characterized further. Young leaves of plants with all promoters tested showed strong GUS expression, but only the rolD, UBQ3 and 35S plants showed strong GUS expression throughout the length of the older leaves (Kamo and Blowers 1999; Kamo et al. 2000). GUS expression was detected primarily in the vasculature of the leaves. Most GUS expression in leaves occurred with the 35S promoter, although this was contrary to the results with cereals where the 35S promoter expressed relatively poorly compared to promoters derived from cereal. The least levels of GUS expression in leaves were in Act1 plants, confirming the low GUS expression previously observed in transient transformation. Roots, especially at the tips, showed strong GUS expression in the rolD, EF-1α, 35S, duplicated 35S, UBQ3 and Act1 plants, whereas strong expression occurred throughout the length of the roots in mas2 plants. Successful genetic engineering of Gladiolus, as well as other floral bulb and corm crops, requires long-term expression because the crop is vegetatively propagated and requires two years of bulb growth for flowering. Transgenic Gladiolus plants with the bar-uidA gene under control of either the 35S, rolD, mas2 or UBQ3 promoters were selected for study of long-term gene expression following three seasons of dormancy (Kamo 2003). Transgene silencing has been reported in cereal for various genes, including uidA when under control of the 35S or other promoters (Iyer et al 2000). Transformation of cereals by biolistics sometimes results in a substantial portion of the population being

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silenced. Six of nine rice plants containing both the bar and uidA genes under control of the 35S promoter were silenced for either transgene (Oard et al. 1996). Kohli et al. (1999) reported that the trangenes of uidA, hpt and bar, each under control of the 35S promoter, were silenced in four of 12 transformed rice lines. In contrast, Gahakwa et al. (2000) reported that six transgenes, three marker genes and three insect resistance genes were expressed over four generations of rice plants, and gene silencing was rarely observed. In transgenic maize plants, more than 75% of the plants with both the bar and uidA genes, each under control of the 35S promoter, expressed GUS (Register et al. 1994). There is only one report of possible gene silencing in a floral monocotyledon, namely Easter lily (Watad et al. 1998). All five lily lines analyzed with uidA under control of the maize ubiquitin promoter failed to express GUS due to either gene silencing, or loss of the transgene following propagation of a chimeric plant. In our study of Gladiolus, all 23 plant lines expressed GUS following three seasons of dormancy. There was no difference in GUS expression for plants grown in vitro as compared with those grown in the glasshouse. GUS expression was greater in roots than shoots for all of the four promoters: 35S, rolD, mas2 and UBQ3. Transgene silencing often occurs with time in callus lines, which is not unexpected as callus growth induced in culture is an unnatural state of plant growth. The levels of silencing exceeded 50% in perennial ryegrass with uidA (van der Maas 1994), in wheat with nptII (Mueller et al. 1995) and in Pennisetum glaucum with both uidA and hph (Lambe et al. 1995). For all three plant species, the silencing was attributed to methylation. Silencing of a rice cell line growing in culture was shown to result from post-transcriptional gene silencing (Kanno et al. 2000). Seventeen callus lines of Gladiolus that initially showed high levels of GUS expression continued to express GUS after three years in culture, except for one line that had lost the uidA gene. All seven of the callus lines that contained the bar-uidA gene under control of the 35S promoter showed high GUS expression after three years in culture. The other seven callus lines were co-bombarded with bar and uidA each under the 35S promoter, and two of these co-bombarded lines showed only a few cells expressing GUS. GUS expression could not be reversed using 5-azacytidine with these two lines and Southern hybridization revealed that methylation of genomic DNA had not occurred. The results with the callus lines of Gladiolus were similar to that of the plants analyzed after three seasons of dormancy, in that transgene silencing is not a common occurrence in Gladiolus. Three ubiquitin promoters, namely GUBQ1, GUBQ2 and GUBQ4, were isolated from Gladiolus by screening a genomic DNA library with a rice RUBQ2 gene (Wang et al. 2000) and by PCR amplification of a genome walker library using a conserved region of the ubiquitin gene. Transient transformation following biolistics showed the greatest GUS expression with the GUBQ1 promoter as compared with the GUBQ2 and GUBQ4 promoters, and these levels were comparable with that of the 35S promoter in Gladiolus cells (Table 1). Unexpectedly, transient GUS expression was very low in other floral mono-

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Table 1. Comparison of gene promoter activities in suspension cells of Gladiolus ‘Jenny Lee’ 48 h after particle gun bombardment of the cells. Values represent nine plates of cells used for 2–3 bombardments. a, b Statistically significant similarity

Promoter

GUS/luciferase

None CaMV 35S GUBQ1 GUBQ2 GUBQ4

11.3 a 132.3 b 419.4 b 21.9 a 15.0 a

Relative GUS expression (35S-GUS set at 1.00) 0.08 1.00 3.17 0.17 0.11

cotyledons including calla lily, cannas, Easter lily and freesia, which is in the same family (Iridaceae) as Gladiolus. In plants other than Gladiolus, greater GUS expression occurred in tobacco and rose cells, followed by rice. The GUBQ1 promoter is 1.9 kb. The RNA transcript has highly conserved 5 and 3 splicing sites for the 1.2-kb intron. Deletion of the intron significantly decreases transient GUS expression in Gladiolus cells. The coding sequence of the first two ubiquitin genes of GUBQ1 shows the highest homology (88%) to the ubiquitin genes of Malus domestica, Oryza sativa RUBQ2 and Nicotiana tabacum (Joung and Kamo 2006).

5 Resistance to Bean Yellow Mosaic Virus Bean yellow mosaic virus (BYMV) infection occurs in almost all Gladiolus plants (Stein 1995). Gladiolus plants transformed with either the BYMV coat protein gene in sense or antisense orientations were developed using biolistics (Kamo et al. 2005). Transcription of the transgene occurred in three of the four coat protein lines and in five of the seven antisense lines, as determined by Northern hybridization. All 11 plant lines were challenged with BYMV using controlled aphid transmission. One month following aphid transmission, the transgenic plants were examined by immunoelectron microscopy for the presence of the virus. Several transgenic plant lines containing either of the antiviral transgenes showed a lower incidence of infection as determined by percent plants infected, as detected by immunoelectron microscopy one month after challenge. The next season, all transgenic plants that were previously challenged and found not to contain BYMV after one month were grown outdoors in clay pots from May through September and found to contain BYMV. Infection was delayed in the coat protein and antisense transgenic lines, but there was eventual infection with BYMV. The same BYMV coat protein gene in sense or antisense orientation was used to transform Nicotiana benthamiana (Hammond and Kamo 1995a). Trans-

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genic N. benthamiana plants expressing the BYMV coat protein gene showed less severe viral symptoms than control plants challenged with BYMV, and a few lines recovered from initial infection. One out of ten N. benthamiana plant lines transformed with the antisense orientation was highly resistant to the virus (Hammond and Kamo 1995b). There are two other reports of conferring resistance to BYMV by developing transgenic clover and N. benthamiana plants expressing the BYMV coat protein gene (Nakamura et al. 1994; Chu et al. 1999). It remains to be determined why this approach was successful in other crops, but not in Gladiolus.

6 Resistance to Cucumber Mosaic Virus Infection of Gladiolus with cucumber mosaic virus (CMV) may result in dramatic streaking throughout the flower petals and deformation of the flower. Transgenic Gladiolus plants were generated with either the CMV replicase gene of serotype 1 (Anderson et al. 1992), the CMV S strain which is a member of serogroup 2 coat protein (Dr. J. Kaper, USDA, personal communication), the CMV coat protein serotype 1 (Gielen et al. 1996), or combinations of the replicase gene with coat protein serotype 2, or coat protein serotype 1 with serotype 2. Transgenic and non-transgenic plants growing in vitro were challenged using biolistic inoculation. Biolistic inoculation of virus particles had been reported previously (Helleco-Kervarrec et al. 2002; Hoffmann et al. 2002; Valat et al. 2003) and this technique was adapted to Gladiolus (Aebig et al. 2005) using the hand-held Helios gene gun system (Bio-Rad, Hercules, Calif.). The gold particles coated with virus were shot into the basal meristem of the Gladiolus plant, the basal meristem being exposed by removing all leaves. The latter usually grow again after about one month and can be used for ELISA assays or immunostrips to determine whether virus infection has occurred. Only 2 μg of cucumber mosaic virus was used to prepare 35 cartridges for bombardment, resulting in 100% infection in non-transgenic Gladiolus cv. Peter Pears with 100% survival of the plants. In the cultivar Jenny Lee, 10 μg of the virus resulted in 100% infection of non-transgenic plants.

7 Future Studies Transgene silencing does not appear to be a common occurrence in Gladiolus. All 23 plant lines with the bar-uidA chimeric gene were not silenced after three seasons of dormancy. Fourteen callus lines with either of these genes, or with both the bar and uidA genes delivered on separate plasmids, were not silenced after three years. Silencing was not observed in the 11 transgenic lines expressing the bean yellow mosaic virus coat protein in either sense or

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antisense orientation. This lack of silencing may have been the reason why there was little virus resistance in plants transformed with the coat protein gene, as post-transcriptional gene silencing has been found to result in virus resistance (Mueller et al. 1995; Ingelbrecht et al. 1999). Further studies such as effect of gene dosage will be undertaken to examine transgene silencing in Gladiolus, and this information will be applied to develop virus-resistant plants.

References Aebig JA, Kamo K, Hsu H-T (2005) Biolistic inoculation of gladiolus with cucumber mosaic cucumovirus. J Virol Methods 123:89–94 Anderson JM, Palukaitis P, Zaitlin M (1992) A defective replicase gene induces resistance to cucumber mosaic virus in transgenic tobacco plants. Proc Natl Acad Sci USA 89:8759–8763 Babu P, Chawla HS (2000) In vitro regeneration and Agrobacterium mediated transformation in gladiolus. J Hortic Sci Biotechnol 75:400–404 Bajaj YPS, Sidhu MMS, Gill APS (1982) Some factors affecting the in vitro propagation of gladiolus. Sci Hortic 18:269–275 Chauvin JE, Hamann H, Cohat J, Le Nard M (1997) Selective agent and marker genes for use in genetic transformation of Gladiolus grandiflorus and Tulipa gesneriana. Acta Hortic 430:291– 297 Chu PWG, Anderson BJ, Khan MRI, Shukla D, Higgins TJV (1999) Production of bean yellow mosaiv virus resistant subterranean clover (Trifolium subterraneum) plants by transformation with the virus coat protein gene. Ann Appl Biol 135:489–490 Dantu PK, Bhojwani SS (1987) In vitro propagation and corm formation in Gladiolus. Gartenbauwissenschaft 52:90–93 Gahakwa D, Maqbool SB, FuX, Sudhakar D, Christou P, Kohli A (2000) Transgenic rice as a system to study the stability of transgene expression: multiple heterologous transgenes show similar behaviour in diverse genetic backgrounds. Theor Appl Genet 101:388–399 Gielen J, Ultzen T, Bontems S, Loots W, Schepen A van, Westerbroek A, Haan P de, Grinsven M van (1996) Coat protein-mediated protection to cucumber mosaic virus infections in cultivated tomato. Euphytica 88:139–149 Goo DH, Joung HY, Kim KW (2003) Differentiation of Gladiolus plantlets from callus and subsequent flowering. Acta Hortic 620:339–343 Graves ACR, Goldman SL (1987) Agrobacterium tumefaciens-mediated transformation of the monocot genus Gladiolus: detection of expression of T-DNA-encoded genes. J Bacteriol 169:1745–1746 Gupta SD, Datta S (2003) Antioxidant enzyme activities during in vitro morphogenesis of gladiolus and the effect of application of antioxidants on plant regeneration. Biol Plant 47:179–183 Hammond J, Kamo KK (1995a) Resistance to bean yellow mosaic virus (BYMV) and other potyviruses in transgenic plants expressing BYMV antisense RNA, coat protein, or chimeric coat proteins. In: Bills DD, Kung SD (eds) Biotechnology and plant protection: viral pathogenesis and disease resistance. World Scientific, Singapore, pp 369–389 Hammond J, Kamo KK (1995b) Effective resistance to potyvirus infection conferred by expression of antisense RNA in transgenic plants. Mol Plant Microbe Interact 8:674–682 Helleco-Kervarrec C, Riault G, Jacquot E (2002) Biolistic-mediated inoculation of immature wheat embryos with barley yellow dwarf virus-PAV. J Virol Methods 102:161–166 Hoffmann K, Verbeek M, Romano A, Dullemans AM, Heuvel JFJM van den, Wilk F van der (2002) Mechanical transmission of poleroviruses. J Virol Methods 91:197–201

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Inglebrecht IL, Irvine JE, Mirkov TE (1999) Posttranscriptional gene silencing in transgenic sugarcane. Dissection of homology-dependent virus resistance in a monocot that has a complex polyploid genome. Plant Physiol 119:1187–1197 Iyer LM, Kumpatla S, Chandrasekharan MB, Hall TC (2000) Transgene silencing in monocots. Plant Mol Biol 43:323–346 Joung YH, Kamo K (2006) Expression of a polyubiquitin promoter isolated from Gladiolus. Plant Cell Rep 25:1081–1088 Kamo K (1994) Effect of phytohormones on plant regeneration from callus of Gladiolus cultivar “Jenny Lee”. In Vitro Cell Dev Biol 30P:26–31 Kamo K (1995) A cultivar comparison of plant regeneration from suspension cells, callus, and cormel slices of Gladiolus. In Vitro Cell Dev Biol 31:113–115 Kamo KK (2003) Long-term expression of the uidA gene in Gladiolus plants under control of either the ubiquitin, rolD, mannopine synthase, or cauliflower mosaic virus promoters following three seasons of dormancy. Plant Cell Rep 21:797–803 Kamo K, Blowers A (1999) Tissue specificity and expression level of gusA under rolD, mannopine synthase and translation elongation factor 1 subunit α promoters in transgenic Gladiolus plants. Plant Cell Rep 18:809–815 Kamo K, Van Eck J (1997) Effect of bialaphos and phosphinothricin on plant regeneration from long- and short-term callus cultures of Gladiolus. In Vitro Cell Dev Biol Plant 33:180–183 Kamo K, Chen J, Lawson R (1990) The establishment of cell suspension cultures of Gladiolus that regenerate plants. In Vitro Cell Dev Biol Plant 26:425–430 Kamo K, Blowers A, Smith, Van Eck J (1995a) Stable transformation of Gladiolus by particle gun bombardment of cormels. Plant Sci 110:105–111 Kamo K, Blowers A, Smith F, Van Eck J, Lawson R (1995b) Stable transformation of Gladiolus using suspension cells and callus. J Am Soc Hortic Sci 120:437–352 Kamo K, Blowers A, McElroy D (2000) Effect of the cauliflower mosaic virus 35S, actin, and ubiquitin promoters on uidA expression from a bar-uidA fusion gene in transgenic Gladiolus plants. In Vitro Cell Dev Biol Plant 36:13–20 Kamo K, Gera A, Cohen J, Hammond J, Blowers A, Smith F, Van Eck J (2005) Transgenic Gladiolus plants transformed with either the Bean yellow mosaic virus coat protein gene in sense or antisense orientations. Plant Cell Rep 23:654–663 Kanno T, Naito S, Shimamoto K (2000) Post-transcriptional gene silencing in cultured rice cells. Plant Cell Physiol 41:321–326 Kasumi M, Takatsu Y, Tomotsune H, Sakuma F (1998) Callus formation and plant regeneration from developing ovaries in Gladiolus. J Jpn Soc Hortic Sci 67:951–957 Kohli A, Gahakwa D, Vain P, Laurie DA, Christou P (1999) Transgene expression in rice engineered through particle bombardment: molecular factors controlling stable expression and transgene silencing. Planta 208:88–97 Kumar A, Sood A, Palni LMS, Gupta AK (1999) In vitro propagation of Gladiolus hybridus Hort.: synergistic effect of heat shock and sucrose on morphogenesis. Plant Cell Tissue Organ Cult 57:105–112 Lambe P, Diana M, Matagne RF (1995) Differential long-term expression and methylation of the hygromycin phosphotransferase (hph) and β-glucuronidase (GUS) genes in transgenic pearl millet (Pennisetum glaucum) callus. Plant Sci 108:51–62 Lilien-Kipnis H, Kochba M (1987) Mass propagation of new gladiolus hybrids. Acta Hortic 212:631–638 Maas HM van der, Jong ER de, Rueb S, Hensgens LAM, Krens FA (1994) Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.). Plant Mol Biol 24:401–405 Mahendra VSSP, Gupta SD (2004) Trichromatic sorting of in vitro regenerated plants of gladiolus using adaptive resonance theory. Curr Sci 87:348–353 Mueller E, Gilbert J, Davenport G, Brigneti G, Baulcombe DC (1995) Homology-dependent resistance: transgenic virus resistance in plants related to homology-dependent gene silencing. Plant J 7:1001–1013

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Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–492 Nakamura S, Honkura R, Ugaki M, Ohshima M, Ohashi Y (1994) Nucleotide sequence of the 3 -terminal region of bean yellow mosaic virus RNA and resistance to viral infection in transgenic Nicotiana benthamiana expressing its coat protein gene. Ann Phytopathol Soc Jpn 60:295–304 Nhut DT, Teixeira da Silva JA, Huyen PX, Paek KY (2004) The importance of explant source on regeneration and micropropagation of Gladiolus by liquid shake culture. Sci Hortic 102:407– 414 Oard JH, Linscombe SD, Braverman MP, Jodari F, Blouin DC, Leech M, Kohli A, Vain P, Cooley JC, Christou P (1996) Development, field evaluation, and agronomic performance of transgenic herbicide resistant rice. Mol Breed 2:359–368 Register JC III, Peterson DJ, Bell PB, Bullock WP, Evans IJ, Frame B, Greenland AG, Higgs NS, Jepson I, Jiao S, Lewnau CJ, Stillick JM, Wilson HM (1994) Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment. Plant Mol Biol 25:951–961 Remotti PC (1995) Primary and secondary embryogenesis from cell suspension cultures of Gladiolus. Plant Sci 107:205–214 Remotti R, Loffler HJM (1995) Callus induction and plant regeneration from gladiolus. Plant Cell Tissue Organ Cult 42:171–178 Remotti PC, Loffler HJM, Van Vloten-Doting L (1997) Selection of cell lines and regeneration of plants resistant to fusaric acid from Gladiolus x grandiflorus cv. ‘Peter Pears’. Euphytica 96:237–245 Simonsen J, Hildebrandt AC (1971) In vitro growth and differentiation of Gladiolus plants from callus cultures. Can J Bot 49:1817–1819 Stefaniak B (1994) Somatic embryogenesis and plant regeneration of Gladiolus (Gladiolus hort.). Plant Cell Rep 13:386–389 Stein A (1995) Gladiolus. In: Loebenstein G, Lawson RH, Brunt AA (eds) Virus and virus-like diseases of bulb and flower crops. Wiley, New York, pp 281–292 Sutter EG (1986) Micropropagation of Ixia viridifolia and a Gladiolus x homoglossum hybrid. Sci Hortic 29:181–189 USDA (2002) Statistics for floriculture crops. Available at: usda.mannlib.cornell.edu/reports/nassr/other/zfc-bb/ Valat L, Mode F, Mauro MC, Burrus M (2003) Preliminary attempts to biolistic inoculation of grapevine fanleaf virus. J Virol Methods 108:29–40 Wang J, Jiang J, Oard JH (2000) Structure, expression and promoter activity of two polyubiquitin genes from rice (Oryza sativa L.). Plant Sci 156:201–211 Watad AA, Yun D-J, Matsumoto T, Niu X, Wu Y, Kononowicz AK, Bressan RA, Hasegawa PM (1998) Microprojectile bombardment-mediated transformation of Lilium longiflorum. Plant Cell Rep 17:262–267 Wilfret GJ (1971) Shoot-tip culture of gladiolus: an evaluation of nutrient media for callus tissue development. Proc Fla State Hortic Soc 84:389–393 Wilfret GJ (1992) Gladiolus. In: Larson RA (ed) Introduction to floriculture. Academic, San Diego, pp 144–154 Ziv M (1979) Transplanting Gladiolus plants propagated in vitro. Sci Hortic 11:257–260

III.6 Forsythia C. Rosati1 , A. Cadic2 , M. Duron2 , and P. Simoneau3

1 Botanical Origin and Genetic Information The genus Forsythia Vahl, belonging to the family Oleaceae, consists of 10–15 species. These include F. europaea Degen and Bald., F. giraldiana Lingelsh., F. japonica Makino, F. koreana Nakai, F. likiangensis Ching and Feng, F. mira Chang, F. mandshurica Uyeki, F. ovata Nakai, F. saxatilis, F. suspensa (Thunb.) Vahl, F. togashii Hara and F. viridissima Lindl. (Fig. 1). The number of species described in the literature varies with the authors. The plant was first classified as Syringa suspensa in 1784 by C.P. Thunberg, but was named Forsythia in 1804 by M. Vahl in memory of William Forsyth, Director of the Royal Gardens of Kensington (Cadic 1988). All species in this genus are Spring-flowering shrubs (2−5 m in height) with yellow flowers, originating from Northeast Asia, except for F. europaea, which is native to the Balkans. The two species F. likiangensis and F. mira were added recently to the Chinese flora, while F. togashii was separated from F. japonica (Chang et al. 1996). Following the introduction of oriental Forsythia species in western countries in the nineteenth century, selection and breeding efforts produced several varieties. The latter are grown widely in temperate climates because of their hardiness and abundant flowering in early Spring. The most popular varieties belong to F. x intermedia (putative interspecific hybrid between F. viridissima and F. suspensa, see below). F. x intermedia is grown most commonly; it is smaller with an upright habit and produces flowers with a bright color. F. suspensa var. sieboldii is a shrub, has a weeping growth habit and thus is suitable for banks, and has paler flowers. The INRA Station of Angers (France) hosts the largest existing repository of Forsythia accessions and maintains a Forsythia website (www.angers.inra.fr/forsy/indexeng.html). Forsythia is a vegetatively propagated shrub, highly heterozygous because of its heteromorphic autoincompatibility system (Rehder 1891). Mating experiments between F. x intermedia, F. suspensa, F. europaea and F. ovata genotypes showed that crosses are only possible between thrum and pin species or varieties, that is, genotypes carrying short and long styles (Sampson 1971). This 1 ENEA,

Trisaia Research Center, BIOTEC-GEN, S.S.106, km 419+500, 75026 Rotondella (MT), Italy, e-mail: [email protected] 2 INRA, C.R. Angers, UMR Génétique et Horticulture (GenHort) – INRA/INH/UA, BP 60057, 49071 Beaucouzé Cedex, France 3 UMR PaVé 77, Faculté des Sciences, Boulevard Lavoisier 2, 49045 Angers, France Biotechnology in Agriculture and Forestry, Vol. 61 Transgenic Crops VI (ed. by E.C. Pua and M.R. Davey) © Springer-Verlag Berlin Heidelberg 2007

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Fig. 1. Phylogenetic relationships of major Forsythia taxa reconstructed from the available literature. For each taxon, the country of origin and year of introduction/release are indicated

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characteristic had been described by Darwin (1877) and Hildebrand (1894). In horticultural practice, varieties are easily propagated vegetatively by softwood cuttings in Spring or hardwood cuttings in Fall, using commercial rooting hormones. 1.1 Phylogenetic Studies Classifications based on morphological descriptors were supported recently by molecular analyses. In the latest available report, Wallander and Albert (2000) proposed a revised classification of the family Oleaceae, including ca. 600 species in 25 genera. Forsythia is closest to the genus Abeliophyllum, based on cladistic analysis of DNA sequences from two non-coding chloroplast loci (rps16 intron, trnL-F region). Both genera are the only members of the newly proposed tribe Forsythieae, which replaces the previous subfamily Jasminoideae, including four tribes and now considered paraphyletic. The data of Wallander and Albert (2000) are in substantial agreement with previous non-molecular classification, e.g., relying on accumulation patterns of flavonoid glycosides (Harborne and Green 1980) and iridoid glucosides (Jensen et al. 1992). Further examples are given by Wallander and Albert (2000). Kim (1999) studied the phylogenetic relationships within the genus Forsythia, by analyzing chloroplast DNA polymorphisms. The author mainly addressed systematics between native species, defining four distinct groups, namely: (1) F. suspensa, (2) F. europaea – F. giraldiana, (3) F. ovata – F. japonica – F. viridissima and (4) F. koreana – F. mandshurica – F. saxatilis. Interestingly, F. x intermedia, a putative F. suspensa x F. viridissima hybrid, did not cluster with either supposed parent species, but rather with the fourth group. The presence of ribosomal gene and internal transcribed spacer sequences in the GenBank database (Table 1) and new molecular phylogenetic studies (Kim et al. 2004) should shed new light on the systematics of Forsythia species. 1.2 Cytometry Studies All accessions of Forsythia wild species are diploid (2n = 28; Taylor 1945), but polyploid varieties were obtained by breeders (see Section 2.1). The Forsythia site of INRA Angers (www.angers.inra.fr/forsy/bota/cytogen.html) reports the results of flow cytometry studies on a certain number of clones. According to further flow cytometry studies carried out at INRA Angers, the DNA amount (C-value) of selected Forsythia accessions was estimated to range between 1.69 pg and 1.87 pg (A. Cadic, unpublished data), a figure 10–12 times greater than that of Arabidopsis thaliana.

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Table 1. Forsythia gene sequences present in the GenBank database as of 27 May 2007 Annotation

Species

GenBank accession

Chloroplast gene

Ribosomal protein S16 (rps16), partial intron sequence

F. suspensa F. x intermedia F. x intermedia F. suspensa F. x intermedia F. europaea F. europaea F. x intermedia

AF225231 AF225232 AM398046 AF231823 AF231824 DQ673256 DQ673264 Y12489 Y07623 Y13435 X93225 Y09127 X93226 AF127218 AF078079 AY166180 AF514840

Ribosomal protein S4 (rps4 gene) tRNA-Leu (trnL) gene, partial intron and 3 exon, and trnL-trnF spacer

Flavonoid pathway

Homeotic gene Isoprenoid and carotenoid pathways

psaA-psbB fragment NADH dehydrogenase subunit F (ndhF) Anthocyanidin synthase (ANS) mRNA ANS mRNA, partial ANS gene promoter Chalcone synthase (CHS) DNA, partial Dihydroflavonol 4-reductase (DFR) mRNA Dihydroflavonol 4-reductase (DFR) DNA, partial Flavonoid 3-O-glucosyltransferase (FGT) mRNA Flavonoid 3-O-glucosyltransferase (FGT) mRNA, partial TCP gene 1-d-Deoxyxylulose 5-phosphate synthase (DXS) mRNA, partial 1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) mRNA, partial 4-Diphosphocytidyl-2-C-methyl-d-erythritol kinase (IspE) mRNA, partial Carotenoid isomerase (CRTISO) mRNA, partial LytB-like protein mRNA, partial

Forsythia spp F. x intermedia

AF514841 AF514842 AF514839 AF514843

C. Rosati, A. Cadic, M. Duron, and P. Simoneau

Classification

Classification

Annotation

Lignan biosynthesis

(+)-Pinoresinol/(+)-lariciresinol reductase F. x intermedia Dirigent protein (psd Fi1) Dirigent protein (psd Fi2) Secoisolariciresinol dehydrogenase Recombinant pinoresinol/lariciresinol reductases, recombinant dirigent proteins and methods of use (patent JP 2001507931) Recombinant secoisolariciresinol dehydrogenase and method of using the same (patent JP 2002512790)

Species

Recombinant dehydrodiconiferyl alcohol benzyl ether reductase and method of using the same (patent JP 2002521027) Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use (patent WO 0149833)

Recombinant pinoresinol/lariciresinol reductases, recombinant dirigent proteins and methods of use (patent JP 2003527842)

Phenylpropanoid Phenylcoumaran benzylic ether reductase homolog Fi1 mRNA pathway (plant defense) Phenylcoumaran benzylic ether reductase homolog Fi2 mRNA Protein synthesis Elongation factor 1-alpha gene DNA, partial Elongation factor 1-alpha gene mRNA, partial

F. x intermedia

F. x intermedia

F. x intermedia

GenBank accession

Forsythia

Table 1. continued

U81158 AF210061 AF210062 AF352735 BD064364 BD206058, BD206059, BD206060, BD206061, BD206062, BD206071 BD235281 AX191860, AX191862, AX191895, AX191897, AX191899, AX191901, AX191903, AX191905, AX191921 BD397443, BD397444, BD397458, BD397459, BD397460, BD397461, BD397462, BD397463, BD397472 AF242491 AF242492 X99979 X97131

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Table 1. continued Classification

Annotation

Species

GenBank accession

Ribosomal genes and ITS

18S ribosomal RNA gene, partial; ITS1, 5.8S ribosomal RNA gene and ITS2; 28S ribosomal RNA gene, partial

F. europaea

AF534814

F. giraldiana F. japonica F. japonica var. saxatilis F. nakaii F. ovata F. suspensa F. viridissima F. viridissima var. koreana F. x intermedia F. mandschurica

AF534809 AF534813 AF534807 AF534811 AF534812 AF534808 AF534810 AF534806 AF540073 DQ022428

C. Rosati, A. Cadic, M. Duron, and P. Simoneau

Internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence

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2 Genetic Resources and Breeding Programs 2.1 Early Breeding Programs The first Forsythia accessions were introduced from the Far East by prospectors and botanists in the nineteenth century. The first F. suspensa var. sieboldii was introduced to European botanical gardens as early as 1833, followed by F. viridissima in 1845 and other species. New varieties appeared after the discovery of a natural hybrid in the botanical garden of Göttingen in Germany. Forsythia x intermedia, first described in 1885 (Zabel 1885) as a spontaneous hybrid between F. suspensa and F. viridissima based on morphological characteristics, is the origin of many modern varieties (Wyman 1961). Forsythia x intermedia varieties ‘Vitellina’, ‘Spectabilis’ and ‘Primulina’ were obtained in the early 1900s. Early breeding attempts date to the early twentieth century, from crosses between genotypes. The main breeding programs were developed in the United States, Canada, Poland and Germany through conventional crosses and selection of spontaneous mutants. The main selection traits were winter hardiness (using F. ovata and F. mandshurica), growth habit and flower traits such as abundance, size and color. Interspecific hybrids were obtained in Poland from crosses F. ovata x F. suspensa (F. x variabilis) and F. europaea x F. suspensa (F. x kobendzae) crosses. Several varieties were bred such as ‘Maluch’ and ‘Helios’ from Poland, F. ovata ‘Ottawa’ from Canada and F. ovata ‘Sunrise’ from the United States. 2.2 Polyploidization K. Sax used colchicine to obtain the first polyploid Forsythia genotypes at the Arnold Arboretum of Harvard University (Jamaica Plain, Mass., USA; Taylor 1945). The first reported tetraploid variety was ‘Arnold Giant’. Such genetic material was also used subsequently to give rise to further tetraploid varieties, described by Hyde (1951). Among them, the Farrand hybrids, issued by backcrossing ‘Arnold Giant’ with F. x intermedia ‘Spectabilis’, were claimed to be triploid, but clones that were released later, namely ‘Beatrix Farrand’ and ‘Karl Sax’, were tetraploid (Marks and Beckett 1963). Other tetraploid varieties were then produced in several countries, namely, F. ovata ‘Tetragold’ in The Netherlands and ‘Tremonia’, ‘Parkdekor’ and ‘Korfor’ GOLDZAUBER in Germany. The last two varieties are remarkable for their improved large flower size and dark golden-yellow flower color. F. mandshurica and F. mandshurica ‘Vermont Sun’ are the only two triploid taxa known to date. F. mandshurica was introduced to Japan from Manchuria in 1929. In 1940, cuttings were sent from Japan to the Botanical Garden of Montreal, Canada. From there, cuttings were distributed to the University of Vermont in 1968, where a selection for frost resistance was made, giving rise to ‘Vermont Sun’ (Pellet and Brainerd 1984). The two clones introduced to France were shown to be triploid using flow cytometry.

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2.3 Spontaneous and Induced Mutants Natural Forsythia mutants have been selected to increase the range of varieties. For example, F. x intermedia ‘Lynwood Gold’ is a spontaneous mutant of ‘Spectabilis’ found in a private garden in Northern Ireland in 1935. The variety F. x intermedia ‘Spring Glory’ is a mutant of ‘Primulina’ which was first observed by M.H. Horvath in 1930 in Ohio and commercialized in the early 1940s. The first induced mutant released as a new variety was selected by Minier Nurseries in France. ‘Flojor’ MINIGOLD derives from F. x intermedia ‘Lynwood Gold’, whose buds had been irradiated with γ -rays by L. Decourtye at INRA Angers. This was the first achievement of a breeding program started in 1971, whose goals were the release of floriferous dwarf varieties and modification of flower color. Induced mutagenesis experiments using 60 Co γ-rays were carried out on winter cuttings of 12 Forsythia diploid and tetraploid species and varieties, with irradiation doses of 15–70 Grays. A protocol taking into account the phyllotaxy of Forsythia was developed to avoid the loss of mutants from sectorial chimeras (Cadic et al. 1980). The mutants obtained displayed variability for growth habit (reduced plant size, loss of apical dominance), leaf shape and color (striped leaves with partial loss of photosynthetic ability), flower shape and flowering time. Only a few mutants had a slightly paler yellow flower color but, in general, this program failed to produce new flower colors. From this experiment, a more compact and densely branched mutant of F. x intermedia ‘Lynwood Gold’ was released as ‘Courtalyn’ WEEK END. A very compact mutant of F. x intermedia ‘Vitellina’ was bred as ‘Courtadic’ MELISA, but quickly revealed its chimeric nature with frequent reversions to the very vigorous original variety. Amongst all mutants produced, one was observed bearing a large quantity of fruits, a rather infrequent characteristic of germplasm in the Angers collection. An open pollinated progeny from this fertile mutant was bred, showing a very broad phenotypic variability. Very dwarf and compact forms were then selected, giving rise to ‘Courtasol’ MAREE D’OR (= GOLD TIDE) and ‘Courtacour’ BOUCLE D’OR (= GOLDEN CURLS) in 1986, ‘Courtaneur’ MELEE D’OR (= GOLD CLUSTER) in 1987 and ‘Courdijau’ GOLDEN PEEP in 1998. Concomitantly, the University of Tennessee (USA) initiated an independent irradiation program on F. x intermedia ‘Lynwood’. A shoot with variegated foliage was found and propagated. Repeated pruning generated several mutants, all of them being chimeras. Finally, two mutants were registered as ‘Lemon-Screen’ and ‘Tinkle Bells’. A third mutant, with whitish blotches on leaves and a bright leaf color during Fall, completely degenerated while producing fruits. Seeds were sown and three new varieties with narrow leaves and a slow growth rate were registered as ‘Minikin’, ‘Fairy-Land’ and ‘Pigmy-Red’ (Van de Werken 1988).

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3 In Vitro Culture 3.1 Establishment of In Vitro Cultures First proposed as a quick and effective way for plant propagation and improvement of the sanitary status (Morel et al. 1968), in vitro methods evolved along with technology for large-scale production and automation (Honda et al. 2001) and are extensively used for plant genetic transformation. Conditions for the establishment of cultures from meristems of Forsythia genotypes were reported for F. x intermedia and F. suspensa (Duron 1977), F. koreana (Shim and Ha 1997) and F. ovata (Uosukainen 1987). In the initial study, in vitro culture was also successful for improving the sanitary status of plants. 3.2 Somaclonal Variation Mass propagation of F. koreana ‘Seoul Gold’, a clone with variegated leaves, led to the recovery of its somaclonal mutant ‘Suwon Gold’ (Shim and Ha 1997). This mutant has gold leaves and brighter leaf color than the original variety and displays increased shoot and root growth under shady conditions. No further genetic variation of this mutant was observed during micropropagation. 3.3 Regeneration and Early Genetic Transformation Studies Success in plant regeneration from in vitro cultures is a prerequisite for genetic transformation. The first example of plant regeneration from leaf explants of F. x intermedia ‘Spectabilis’ was reported by Roest and Bokelman (1980). At INRA Angers, experiments using leaf explants of different Forsythia genotypes did not give positive results (M. Duron, unpublished data) or lead to vitrification (Rosati et al. 1996). In contrast, stem internode explants of F. x intermedia ‘Spring Glory’ gave high regeneration percentages on basal regeneration medium, modified from MS medium (Murashige and Skoog 1962) and containing indole-3-acetic acid (IAA) and benzylaminopurine (BAP; Rosati et al. 1996). The presence of IAA was essential for plant regeneration, and 100% regeneration rate (i.e., at least one shoot per explant) was obtained with 1 mg l−1 BAP and 0.5 mg l−1 IAA. F. x intermedia ‘Spring Glory’ is the only genotype amenable to genetic transformation, using an Agrobacterium tumefaciens-based method (Rosati et al. 1996). Kanamycin-resistant transgenic plants containing a GUS-intron gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter were obtained with a 1% transformation rate (i.e., at one transgenic shoot every 100 co-cultured explants). The CaMV35S promoter was able to drive strong expression of β-glucuronidase (gus) in both vegetative and flower tissues.

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3.4 Oriental Pharmacopoeias and In Vitro Production Systems of Selected Polyphenols Forsythia extracts contain large amounts of compounds, many of which belong to polyphenol classes (e.g., Katayama et al. 1992), which empirically have long been used in traditional Chinese, Japanese and Korean pharmacopoeias. Polyphenols are a large class of plant natural compounds including lignans, tannins, anthocyanins and other flavonoids and phenylpropanoids, that are discussed in more detail in the following sections. Many reviews highlight the health benefits of polyphenols and other antioxidant compounds present in foods for the prevention of degenerative and cardiovascular diseases (KrisEtherton et al. 2002). The literature on the effects of Forsythia-based preparations is vast and often difficult to interpret, since most reports are in local journals, i.e., not available in English (or only the abstract in English), and with very limited information on preparation. Some selected examples report on the treatment of inflammatory diseases (Kim et al. 2003), antiviral action (Zhang et al. 2002) and antibacterial activity (Nishibe et al. 1982). Assays on selected phenolic compounds extracted from Forsythia species were reported by Nishibe (1994). Many of the above-mentioned preparations were shown to contain large proportions of lignans, a class of polyphenols shown to possess antioxidant, antiviral, bactericidal, antifungal and cytotoxic properties. Controlled synthesis of selected lignan compounds provides an alternative to environmentally and economically costly purification methods from lignified plant tissues. Forsythia species produce large quantities of lignans, and F. x intermedia ‘Spectabilis’ cell suspension cultures were used to obtain pinoresinol, the first product in the Forsythia lignan pathway (Fig. 2), and matairesinol (Schmitt and Petersen 2002a). (+)-Pinoresinol was dominant over (–)-pinoresinol, with up to a 3:1 ratio at the time-point of maximum accumulation. However, the enantiomeric composition of matairesinol could not be determined. Sucrose concentration in the culture medium influenced lignan production; 6% sucrose maximized lignan yield [0.8 mg l−1 dry weight (dw) pinoresinol, 2.7 mg l−1 dw matairesinol]. As plant defense compounds, lignans can be induced by environmental cues and elicitors. In a second investigation, Schmitt and Petersen (2002b) used methyl jasmonate to increase pinoresinol and matairesinol production 3- and


Fig. 2. Schematic representation of the phenylpropanoid, lignan and flavonoid pathways. Only

steps and compounds most significant to Forsythia are highlighted. For ease of reference, enzyme acronyms are listed in alphabetical order: 4CL 4-coumarate:CoA ligase, ANR anthocyanidin reductase, ANS anthocyanidin synthase, C3H p-coumarate 3-hydroxylase, C4H cinnamate-4hydroxylase, CAD cinnamyl alcohol dehydrogenase, CCOMT caffeoyl-CoA O-methyltransferase, CCR cinnamoyl-CoA reductase, CHI chalcone isomerase, CHS chalcone synthase, DFR dihydroflavonol synthase, DP dirigent protein, FGT flavonoid glycosyltransferase, FHT flavanone 3-hydroxylase, FLS flavonol synthase, LAR leucoanthocyanidin reductase, PAL phenylalanine ammonia-lyase

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7-fold respectively in cell suspension cultures. In addition, the addition of coniferyl alcohol, the precursor to lignan biosynthesis, enhanced the production of pinoresinol, but had no effect on matairesinol concentration (Schmitt

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and Petersen 2002b). These divergences suggest different synthesis and storage sites for pinoresinol and matairesinol in cell suspensions of Forsythia.

4 Forsythia Biotechnology Research As of 27 March 2007, 52 gene sequences from Forsythia spp were present in the GenBank database (Table 1). Many genes belong to the polyphenol–lignan– flavonoid and isoprenoid–carotenoid pathways. The most prominent results from ongoing research programs are discussed in the following sections. 4.1 Molecular Fingerprinting Molecular markers are used occasionally for ornamentals and other crops to resolve issues of violation of Plant Breeders’ Rights. Given the limited economic importance of Forsythia, such technology was not developed extensively for this genus. RAPD markers were used to discriminate a F. koreana somaclonal mutant from the original genotype (Shim and Ha 1997). Using primers that flanked the unique intron in the anthocyanidin synthase (ANS) gene, we were able to detect four intron size variants amongst 24 Forsythia genotypes (Duron, Rosati and Simoneau, unpublished data). Segregation analysis in progenies from experimental crosses confirmed that these different forms represent alleles of a single ans gene and not homologous sequences at different loci (Fig. 3A). Interestingly, the allele polymorphism patterns of some F. x intermedia genotypes and other Forsythia species (Fig. 3B) might agree with either the hypothesized hybrid origin (F. suspensa x F. viridissima) of this species, or the possibility that the maternal genome donor could rather belong to the F. koreana-manshurica-saxatilis clade as proposed by Kim (1999). Sequence comparisons of the four size variants showed that length polymorphism is due mainly to large insertion/deletion events at a conserved position (Fig. 3C). Besides intron length, single nucleotide polymorphisms were also found in alleles of similar size (Fig. 3C). Further screening of intron lengths in other flavonoid pathway structural genes revealed that, in Forsythia, this situation is not unique to the ans gene (data not shown). These molecular signatures may prove useful for cultivar fingerprinting or to validate the origin of putative hybrids.


Fig. 3. A Segregation patterns of ans alleles in two different crosses of Forsythia genotypes. Ians1, Ians2, Ians3 and Ians4 represent the four size variants of the ans gene intron. F. Maluch is an hypothetical hybrid of F. x intermedia ‘vitellina’ and F. ovata. B Polymorphism of ans alleles in different Forsythia genotypes. PCR products were amplified using the FOANS1 and FOANS2 primers (Rosati et al. 1999) from DNA of F. mandshurica (F.m), F. suspensa Sieboldii (F.ss), F. suspensa Nymans (F.sn), F. suspensa atrocaulis (F.sa), F. suspensa Fortunei (F.s), F. x intermedia (F.xi), F. viridissima (F.v.). The deduced genotypes for each plant at the ans locus are indicated above the gel. C Partial intron sequences of the four ans alleles of Forsythia. Homologous portions of the ans intron sequence were compared using the Clustal W alignment software. The lack of a corresponding nucleotide is indicated by a hyphen

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4.2 Flavonoids and Flower Color 4.2.1 Knowledge Context and Fundamental Studies in Forsythia Phenylpropanoids and flavonoids are a large class of plant polyphenols which lead to the synthesis of important molecules such as lignin, lignans and an-

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thocyanins. The biosynthetic pathways of such compounds involve the coordinated action of several structural genes (Fig. 2) regulated by transcription factors. The reader is referred to prominent articles for further reference (Davin and Lewis 2000; Forkmann and Martens 2001; Anterola and Lewis 2002). All Forsythia genotypes bear yellow flowers, due to the accumulation of carotenoids (xanthophylls) and the absence of anthocyanins in petals. The intensity of the yellow color is due to the concentration of carotenoids and, to a lesser extent, of yellowish flavonoids (flavonol glycosides). The lack of genetic variability for flower color, the increasing amount of sequence information of flavonoids genes and the development of genetic transformation procedures for F. x intermedia ‘Spring Glory’ gave momentum to biotechnology-based Forsythia breeding approaches at INRA Angers in the 1990s. Previous studies reported the presence of large amounts of specific flavonols, namely rutin (quercetin-3-rutinoside), in Forsythia leaves and floral organs. In contrast, the synthesis of anthocyanins, the major class of flavonoids pigments, was limited to sepals and vegetative tissues (stems, petioles, leaf veins, senescent leaves). Flavonoid metabolism in Forsythia flower organs was studied by combining biochemical and molecular investigations aimed at identifying the limiting steps for anthocyanin biosynthesis in petals (Rosati 1997). Flavonoid structural genes coding for chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and flavonoid 3-O-glucosyltransferase (FGT) were cloned by PCR-based approaches and characterized (Rosati et al. 1997, 1999, 2003). Analytical and biochemical studies were used to reconstruct the metabolism of flavonoids in Forsythia flowers (Rosati et al. 1998). The effect of a 35S::dfr transgene was studied in flowers of F. x intermedia ‘Spring Glory’. These studies highlighted the following points, critical for defining the most suitable strategies for genetic engineering of flower color (Rosati 1997; Rosati et al. 2003). 1. In petals, anthocyanin biosynthesis is prevented by a transcriptional block of ans expression and a low expression of dfr (detectable only by competitive RT-PCR), while in anthers, both dfr and ans genes are blocked at the transcriptional level. The ans promoter contains several putative binding sites for factors which might act as transcriptional activators/repressors of its expression (Rosati et al. 1999). Gel retardation analyses showed that factors related to SBF-1, a reported silencer/enhancer of a bean chs gene (Lawton et al. 1991), are likely to act as silencers/enhancers of anthocyaninless (petals, anthers)/anthocyanin-containing (sepals) ans transcription, respectively (Fig. 4). 2. As a consequence of the transcriptional block of ans in petals, the constitutive expression of a transgenic dfr gene did not lead to any change of flower color (Rosati et al. 1997). The ectopic expression of an ans transgene alone could also be unsuccessful, because of the low expression of the endogenous dfr in petals.

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Fig. 4. A Binding of nuclear factor(s) to a probe (core: TTA-GGT-GCG-GGTTAA-TAT-TGC-GTGAGA), containing the core-binding sequence (GGTTAA) for the SBF-1 factor found in the promoter of F. x intermedia ‘Spring Glory’ ans gene (Rosati et al. 1999). For gel retardation experiments, nuclear extracts of anthocyaninless (anthers, petals) and anthocyanin-containing (sepals) flower organs were incubated with end-labeled probe alone (lanes 2, 7), or in the presence of the indicated molar excess of homologous (CORE, lanes 3–6) or heterologous (HET, lanes 8–11) non-labeled competitors. The free and bound forms of the labeled probe are indicated by open and closed triangles, respectively. P Probe with no nuclear extract (lane 1). B Effect of dephosphorylation on binding activity. Calf intestinal alkalin phosphatase (CIAP) treatment either alone (lane 12) or in the presence of 10 mM EDTA (lane 13) was carried out on anthocyaninless organ samples prior to incubation with end-labeled probe

3. Forsythia genotypes accumulate large amounts of flavonols in petal tissues, e.g. up to 0.3% of petal fresh weight (Rosati et al. 1998). Rutin (quercetin-3rutinoside; Fig. 2) accounts for a large proportion (>80–90%) of the flavonol pool in both petals and sepals. Therefore, a strong competition of endogenous flavonol synthase against anthocyanin biosynthesis enzymes has to be taken into account in further metabolic engineering approaches. 4. Fgt is expressed in both petals and sepals and, consequently, it does not constitute a rate-limiting step for anthocyanin formation in petals. 5. Forsythia flowers accumulate mainly 3 ,4 -hydroxylated flavonoids. The most probable pigment(s) formed in petals upon transgene induction of anthocyanin biosynthesis are cyanidin derivatives (Fig. 2).

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Fig. 5. Close-up of flowers of double transformant dfr+ans (left) and wild-type (right) ‘Spring Glory’ at the onset of blooming. Orange flower phenotype and more intense stem pigmentation are due to the accumulation of cyanidin type anthocyanins. Bar 1 cm

4.2.2 Modification of Forsythia Flower Color Through Genetic Transformation Based on acquired biochemical and molecular information, further transformation experiments introduced a 35S::ans in both F. x intermedia ‘Spring Glory’ wild-type and 35S::dfr transformant backgrounds. The stacking of dfr and ans transgenes was achieved by changing the selection agent (hygromycin), which was less efficient (0.1% regeneration rate, i.e., 10-fold lower) than kanamycin, but allowed the recovery of a few ans and dfr plus ans transformants. Only double transformants displayed a modified floral phenotype, that is, a novel bronze-orange color given by the de novo synthesis of cyanidinderived anthocyanins in petals over the yellow carotenoid background (Rosati et al. 2003; Fig. 5). Ectopic expression of dfr and ans in simple and double transformants also affected the flavan-3-ol accumulation pattern in petals, in agreement with the latest findings on the late flavonoid pathway (Xie et al. 2003). 4.3 Further Developments: Engineering of Carotenoid Pathway for Flower Color The accumulation of xanthophylls lutein, antheraxanthin, violaxanthin and neoxanthin (Kuhn and Löw 1949; Rosati, unpublished data) make the carotenoid pathway an attractive target for engineering flower color. Genetic transformation strategies for the modification of the carotenoid content in

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Forsythia petals ideally require the use of flower- or organelle-specific promoters, since carotenoids play an essential role in photosynthesis. The tomato phytoene synthase (PDS) gene promoter drives chromoplast-specific gene expression (Corona et al. 1996). Thus, it was chosen to avoid transgene interference with leaf carotenogenesis. Transformed F. x intermedia ‘Spring Glory’ clones carrying a Pds promoter::ccs (capsanthin–capsorubin synthase; CCS) gene from pepper were obtained (Rosati et al. 2000). The CCS enzyme transforms the xanthophylls antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively, which are the two main pigments of red peppers (Bouvier et al. 1994). Flowers of ccs transformants showed an unchanged floral phenotype. A probable explanation of this failure is that chromoplasts of Forsythia petals lack lipoprotein fibrils (fibrillin) associated with carotenoid pigments in pepper chromoplasts (Deruere et al. 1994). It is likely that capsanthin and capsorubin, possibly formed in Forsythia petal chromoplasts, are not bound by fibrillin and eventually degrade. Further antisense gene constructs have been assembled to knock out the synthesis of pigmented carotenoid, to obtain a white flower phenotype. 4.4 Lignan Biosynthesis Forsythia is a very rich source of phenolic compounds. Among them, lignans, a large class of phenylpropanoid dimers, accumulate in stem tissues and prevent degenerative diseases (Davin and Lewis 2000, and references therein). The group of Norman Lewis at Washington State University elucidated some key biosynthesis steps of lignans. The enzyme involved in the first step of lignan biosynthesis was isolated from F. suspensa extracts, and was called dirigent protein (from dirigere, Latin, to drive) because it is able to catalyze the steroselective coupling of two molecules of coniferyl alcohol to form (+)-pinoresinol (Davin et al. 1997; Fig. 2). The (+)-pinoresinol/(+)-lariciresinol reductase involved in the reduction of (+)-pinoresinol to form other molecules in the pathway (Fig. 2) was characterized by biochemical and molecular studies (Chu et al. 1993; Dinkova-Kostova et al. 1996). The exploitation of such genes for the biotechnology-based production of lignans is highlighted by relevant patents (Table 1). 4.5 Modification of Auxin Metabolism Affects Growth Habit Preliminary transformation experiments were reported with a 35S::iaaL construct (Spena et al. 1991), coding for a bacterial IAA–lysine synthetase responsible for the conversion of IAA to an amino acid conjugate, 3-indole-acetyl-εl-lysine. Transgenic clones displayed reduced plant size, suppression of apical dominance, premature spring bud germination and inability to differentiate flower buds, consistent with altered auxin metabolism. Further analyses on these clones are required to study in more detail the effect of the transgene on plant physiology and auxin metabolism.

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5 Conclusions The present review highlights conventional breeding and biotechnological research of Forsythia. Despite its limited commercial importance in horticulture compared with top-selling flower plants such as Rosa, Chrysanthemum and Pelargonium, several Forsythia varieties have been created for urban landscaping and private gardens. Recent successful studies of in vitro culture and genetic engineering, for the production of lignans and modification of flower color, open the way to biotechnology-based applications for the pharmaceutical and ornamental industries. Acknowledgements. The authors thank Dr. Stefan Martens (Philipps-Universität Marburg, Germany) for critical reading of this review.

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Rosati C, Cadic A, Duron M, Ingouff M, Simoneau P (1999) Molecular characterization of the anthocyanidin synthase gene in Forsythia x intermedia reveals organ-specific expression during flower development. Plant Sci 149:73–79 Rosati C, Duron M, Simoneau P, Cadic A (2000) Towards novel flower colors in Forsythia by genetic engineering. Acta Hortic 508:45–48 Rosati C, Simoneau P, Treutter D, Poupard P, Cadot Y, Cadic A, Duron M (2003) Engineering of flower colour in forsythia by expression of two independently-transformed dihydroflavonol 4-reductase and anthocyanidin synthase genes of flavonoid pathway. Mol Breed 12:197–208 Sampson DR (1971) Mating group ratios in distylic Forsythia (Oleaceae). Can J Genet Cytol 13:368–371 Schmitt J, Petersen M (2002a) Pinoresinol and matairesinol accumulation in a Forsythia x intermedia cell suspension culture. Plant Cell Tissue Organ Cult 68:91–98 Schmitt J, Petersen M (2002b) Influence of methyl jasmonate and coniferyl alcohol on pinoresinol and matairesinol accumulation in a Forsythia x intermedia suspension culture. Plant Cell Rep 20:885–889 Spena A, Prinsen E, Fladung M, Schulze SC, Van Onckelen H (1991) The indoleacetic acidlysine synthetase gene of Pseudomonas syringae subsp. savastanoi induces developmental alterations in transgenic tobacco and potato plants. Mol Gen Genet 227:205–212 Uosukainen M (1987) Establishment of cultures without preconditioning. Acta Hortic 212:60 Shim KK, Ha YM (1997) New gold leaf cultivar of Forsythia koreana (‘Suwon Gold’) and its mass propagation in vitro. Acta Hortic 447:187–190 Taylor H (1945) Cyto-taxonomy and phylogeny of the Oleaceae. Brittonia 5:337–367 Van de Werken H (1988) Mutant offspring. Am Nurseryman 167:127–132 Wallander E, Albert VA (2000) Phylogeny and classification of Oleaceae based on rps16 and trnL-F sequence data. Am J Bot 87:1827–1841 Wyman D (1961) Forsythias. Am Hortic Mag 40:191–198 Xie DY, Sharma SB, Paiva NL, Ferreira D, Dixon RA (2003) Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science 299:396–399 Zabel H (1885) Forsythia intermedia (= F. suspensa x viridissima). Gartenflora 34:35–37 Zhang GG, Song SJ, Ren J, Xu SX (2002) A new compound from Forsythia suspensa (Thunb.) Vahl with antiviral effect on RSV. J Herb Pharmacother 2:35–40

Section IV Forages and Grains

IV.1 Alfalfa C. Sengupta-Gopalan, S. Bagga, C. Potenza, and J.L. Ortega1

1 Introduction and Economic Importance Alfalfa (Medicago sativa L.) is the most widely grown forage legume in the world and represents about 2.5% of the total agricultural hectarage. Besides being an important forage crop, alfalfa has the potential to be a dual-purpose plant, that is, it can be used both for fuel and for feed. Alfalfa can be grown as a renewable replacement resource for other petroleum-based products, such as plastics. It can be used to reduce the need for other non-renewable resources, such as phosphorus and nitrogen fertilizers, and also can be used to clean land and water of contaminants. Alfalfa is a challenging crop to breed because of its complicated genetics and self-breeding restrictions, and development of new cultivars is usually done as a population. Strategies include breeding for resistance to pests and diseases, resistance to abiotic stresses like drought, salinity and heat, and for increased biomass, protein content and forage quality. Alfalfa breeding programs now include the areas of genomics and transgenic technologies. Alfalfa is an autotetraploid species (2n = 4x = 32) and, due to its tetrasomic inheritance, it is difficult to perform molecular mapping. However, the use of Medicago truncatula, an annual relative of alfalfa, as a model legume for genomic research opens new research possibilities in alfalfa. The possibilities of plant genetic engineering did not end with those scientists who helped develop the technology of gene transfer over a generation ago. They clearly envisioned a powerful tool with an almost unlimited potential to improve all aspects of agriculture, leading to increased yields, balanced nutrition, decreased losses due to insect attack and disease, and the ability to grow crops in soils and environments that long ago were left barren by poor and desperate agricultural practices. Indeed, the promise of genetic engineering is being fulfilled today as the technology has begun to spread in a world where the human population is still growing at an alarming rate. Many developing nations realize that biotechnology can be used to help increase sustainable agriculture and decrease dependency on foreign food imports, which ultimately leads to increased economical development and stability. Being amenable to transformation, alfalfa has been subjected to extensive genetic engineering and there are many reports in the literature on transgenic alfalfa with different transgenes. 1 Department of Plant and Environmental Sciences, New Mexico State University, Las Cruces, NM

88003, USA, e-mail: [email protected] Biotechnology in Agriculture and Forestry, Vol. 61 Transgenic Crops VI (ed. by E.C. Pua and M.R. Davey) © Springer-Verlag Berlin Heidelberg 2007

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2 Breeding Alfalfa breeding is a relatively new area of plant improvement. Medicago is cross-pollinated, polyploid and perennial, which means that it gives a heterozygous, continually segregating population in which every plant is genetically different. Polyploidy further complicates the picture by providing more than two homologous chromosomes and subsequently more than two genes at a locus. Furthermore, each cultivar is a random, interbreeding population of plants with a characteristic frequency of traits. Alfalfa breeding is basically a cyclic system in which successively improved populations are developed by intermating selected plants until the desired levels of performance are attained. The mating system usually includes phenotypic recurrent selection within a population, or genotypic recurrent selection. For simple traits, phenotypic selection is used, while genotypic selection is used for more complex traits. Primary traits of interest in alfalfa are yield, nutritive value, disease resistance, persistence and winter hardiness. To be grown profitably, alfalfa needs to sustainhigh yields over several years. Alfalfa breeding programs in the United States have made significant progress in improving multiple pest resistance (Lamb et al. 2006). However, relatively little progress has been made in forage yield per se (Maureira et al. 2004). Genetic improvement of alfalfa forage yields prior to the 1950s appeared to result from the accumulation of favorable alleles, while improvement later resulted from the exploitation of non-additive types of gene action, including heterosis (Holland and Bingham 1994). Significant amounts of hybrid vigor in forage yield of alfalfa were documented (SegoviaLerma et al. 2004; Riday and Brummer 2006). Alfalfa improvement programs in North America made extensive utilization of nine original germplasm resources, namely African, Chilean, Flemish, Indian, Ladak, M. falcata, M. varia, Peruvian and Turkestan (Segovia-Lerma et al. 2004). These diverse germplasms possess a wide range of yield potentials and a varying degree of fall dormancy, a phenomenon where plants cease to grow in response to reduced photoperiod and low temperature as a winter adaptation mechanism (McKenzie et al. 1988). In alfalfa, non-fall-dormancy is a desired trait because it produces more herbage, and many breeding programs focus on this trait (Brummer et al. 2000). However, winter hardiness is important for survival and thus hybrids have been produced between parents possessing different fall dormancy responses, as well as winter hardiness responses, exhibiting greater forage yield advantage. Current alfalfa breeding methods are almost exclusively based on recurrent phenotypic selection, involving intercrossing selected parents to produce synthetic varieties (Hill et al. 1988). Implementing a hybrid breeding system requires the improvement of at least two independent and complementary populations which, in combination, produce heterosis. M. sativa subsp. falcata has been identified as a subspecies that shows heterosis in crosses with elite M. sativa subsp. sativa breeding material (Riday and Brummer 2002a, b; Riday et al. 2002, 2003).

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3 Genomics Because of the tetrasomic inheritance, most genetic maps of alfalfa were constructed in diploids (Brummer et al. 1993), while two genetic maps were constructed in tetraploid populations (Sledge et al. 2004). More recently, a molecular map of tetraploid alfalfa was constructed for the identification of drought tolerance quantitative trait loci (QTL). The map was constructed using simple sequence repeat (SSR) markers derived from expressed sequence tags (ESTs) and bacterial artificial chromosomes (BAC) inserts of M. truncatula on crosses made between a water-use-efficient M. sativa subsp. falcata genotype and a low water-use-efficient M. sativa subsp. sativa genotype (Sledge et al. 2004). The goal of this study was also to anchor this map to the diploid M. truncatula physical map that is currently under construction. The EST-SSR markers developed will provide an important resource for genetic mapping and marker-assisted selection in alfalfa, as well as for comparative genetic studies between M. sativa and other species like M. truncatula, or pasture legumes such as white clover. Efforts are now in place to use this map and the segregating progeny from which it was derived to detect QTL for drought tolerance in tetraploid alfalfa. Genes from M. truncatula share high sequence identity to their counterparts from alfalfa and so it serves as an excellent genetically tractable model for alfalfa. More recently, the availability of M. truncatula microarrays (Tesfaye et al. 2006) and proteome data (Lei et al. 2005) also contributed immensely towards identifying genes which have agronomic importance and can be used both as molecular markers and for introducing valuable traits by genetic engineering. However, some complex traits like biomass yield and winter hardiness cannot be thoroughly assessed in the autogamous, annual, diploid model species. These traits have to be studied in alfalfa.

4 Genetic Engineering The ability to transform alfalfa and to move desirable genes into elite lines by breeding with the transformed plants has opened new doors for alfalfa improvement. The genes used for transgenic research focused mainly on traits dealing with agronomic performance, forage quality and producing industrial/pharmaceutical proteins. Genetic engineering approaches require a good transformation system, desirable target genes based on basic understanding of the biochemical pathways leading to the desired traits, and appropriate promoters for fine-tuning the expression pattern of the transgenes. 4.1 Alfalfa Transformation An efficient alfalfa transformation system has been reported by inoculating leaf explants from a highly regenerable genotype Regen-SY with Agrobac-

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terium tumefaciens (Bingham 1991; Bagga et al. 1992; Austin et al. 1995). The transformation protocol takes about 3–4 months and plants can be moved out into the glasshouse, where they are clonally propagated (Bagga et al. 2004). The other alfalfa cultivar successfully transformed using A. tumefaciens is Rangelander. However, since it was not clonally propagated, and independent plants were used for transformation, variation was seen in the allelic distribution of some of the endogenous genes among the different plants, causing problems in the interpretation of results (Temple et al., unpublished data). Some effort was made to check other more elite alfalfa varieties for transformation, but with little success. However, once the transgene was introduced into the Regen-SY genotype, the gene could be moved easily into elite lines by breeding (Bagga et al. 2004). Selectable markers used for alfalfa transformation are the neomycin phosphotransferase II (nptII) gene for selection on kanamycin, the hygromycin phosphotransferase (hpt) gene for selection on hygromycin and the pat gene for selection on glufosinate (Wehrmann et al. 1996; Bagga et al. 2005). Two approaches are used for studies on co-expression of multiple genes or gene stacking in the same alfalfa plants. One is re-transformation employing A. tumefaciens strains with different selectable markers, while the other is sexual crosses between transformants with different transgenes (Bagga et al. 2004, 2005). Gene constructs containing dual promoters, the cauliflower mosaic virus (CaMV) 35S promoter or the cassava vein mosaic virus (CsVMV) promoter (Verdaguer et al. 1998), in the same cassette driving two different coding sequences, are also used for gene stacking in alfalfa (Klypina et al. 2005). 4.2 Promoters for Genetic Engineering of Alfalfa As the application of genetically engineered plants widened over recent decades, so did the need to develop methods to fine-tune the expression of integrated transgenes (Potenza et al. 2004). Specifically, the development of a broad spectrum of promoters increased the successful application of genetic engineering technologies in both research and industrial/agricultural settings. The CaMV 35S promoter has an almost universal capacity to express transgenes at high levels in most tested plants, using host nuclear RNA polymerase, without the necessity of any trans-acting viral gene products. However, this promoter was shown to drive expression at lower levels in alfalfa than when used in other, non-leguminous plants (Bagga et al. 2005). Another viral promoter developed for use in alfalfa is the CsVMV promoter, which showed up to 24-fold greater activity than the CaMV 35S promoter in the leaves of alfalfa (Samac et al. 2004). Both root-specific and nodule-specific promoters have been developed (Gregerson et al. 1994; Fang and Hirsch 1998; Winicov et al. 2004), but since much of the agricultural value of alfalfa lies in its green tissues (leaves and stems), there is more focus in developing light-inducible and green-tissue specific promoters. The alfalfa light-inducible promoters isolated include small

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subunit rbcS (Khoudi et al. 1997; Aleman 2001), chlorophyll a/b binding protein (Aleman 2001) and rubisco activase, the latter being the strongest of all (Aleman 2001). Thus far, those isolated lack the strength to drive transgenes to higher levels than that of viral constitutive promoters (Aleman 2001; Moore 2003; Samac et al. 2004). Nevertheless, if the aim is to obtain mesophyll-specific expression, the weaker light-inducible promoters are more desirable. The development of M. truncatula as a model legume and its genetic closeness to alfalfa can be exploited in the development of alfalfa promoters. This is especially true when studying gene expression patterns based on M. truncatula transcriptomics. By comparing M. truncatula microarray expression patterns under a number of conditions, there might be increased success for mining alfalfa promoters with similar spatial and temporal expression patterns. 4.3 Potential Target Traits for Alfalfa Improvement by Genetic Engineering Genetic engineering in alfalfa commenced in the same way as in other plants, with transgenes that encode for proteins that are directly responsible for a value-added trait. This was followed by more complex strategies to alter metabolism. The following section reviews most of the engineered traits. However, the major focus is in the area of improvement of forage quality. 4.3.1 Genes for Conferring Resistance to Pests and Herbicides Alfalfa transformants with the Bt gene were produced to resist alfalfa weevil and other coleopteran insects (Strizhov et al. 1996). Similarly, transgenic alfalfa plants with the fungal chitinase gene were developed for protection against fungal pathogens (Tesfaye et al. 2005). A synthetic gene with plant-preferred codons for the bacterial gene for atrazine chlorohydrolase was introduced into alfalfa, and these transformants were developed for remediation of atrazinecontaminated soil and water (Wang et al. 2005). In a joint venture between Monsanto and Forage Genetics, alfalfa plants carrying a bacterial gene that confers glyphosate tolerance, was developed recently for commercial use. Herbicide tolerant alfalfa is touted as a product that will help with the management of alfalfa stands, with the scheduled application of herbicides to specifically kill unwanted weeds and grasses. These weeds not only reduce yield, but more importantly can reduce the forage quality of alfalfa. The herbicide tolerant technology applied to alfalfa is similar to the technology available in many other crops, such that the herbicide can be applied over the top of a crop. Crops containing transgenes that confer herbicide tolerance have been grown commercially for more than a decade (Heck et al. 2005). The technology is based on genetically engineering alfalfa to carry a 5enol-pyruvylshikimate-3-phosphate synthase gene from Agrobacterium strain CP4 (CP4-EPSPS gene) that makes it tolerate the application of glyphosate. The CP4-EPSPS gene product is not inhibited by glyphosate, thus allowing

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continued formation of shikimate pathway reactants and subsequent important downstream products. The transgene used to create glyphosate tolerance in alfalfa is the same gene used in the development of commercially available herbicide-resistant soybean and cotton. One of the highest input costs in the production of alfalfa is in the establishment of an alfalfa stand and subsequent management. Stand life, that may normally run from three to seven years, can be substantially decreased with poor or even neglectful management. 4.3.2 Abiotic Stress Tolerance Under stress conditions, such as drought and high salinity, stomatal closure triggered by abscisic acid limits CO2 supply to the leaf, leading to overreduction of the photosynthetic electron transport chain. Therefore, enhancement of the enzyme activity involved in active oxygen scavenging systems may be a potent strategy to increase abiotic stress tolerance. A class of metalloproteins is the superoxide dismutases (SODs) that have the ability to detoxify oxygen-free radicals by converting them to hydrogen peroxide and molecular oxygen. Alfalfa transformed with genes for SOD showed increased tolerance to drought (Higbie 2002). The SOD transformants also exhibited increased vigor to freezing stress and increased winter survival under field conditions (McKersie et al. 1999, 2000). With respect to freeze tolerance, alfalfa was also engineered with a gene encoding for sucrose phosphate synthase (SPS; Shearer et al. 2002). SPS channels carbohydrate away from starch production and into sucrose accumulation. Sucrose is implicated to play a role as a cryoprotectant (Castonguay et al. 2006). An alternative strategy to scavenging active oxygen species under abiotic stress conditions may be the suppression of active oxygen production. Photorespiration may function as a possible route for the dissipation of excess light energy or reducing power. When the supply of CO2 to the leaves is cut off by closure of the stomata, as under abiotic stress, the photorespiratory carbon oxidation (C2) cycle does not work, the photosynthetic carbon fixation (C3) cycle cannot operate and light energy may be diverted to produce oxygen radicals. Increased photorespiratory capacity resulting from the efficient recycling of photorespiratory ammonia may drive the C2 cycle and protect plants from the damaging effects of active oxygen. Under photorespiratory conditions, ammonia accumulates and there is a gradual increase in the concentration of glutamine and serine, suggesting that the rate-limiting step in photorespiration is the reassimilation of ammonia catalyzed by chloroplastic glutamine synthetase (Kozaki and Takeba 1996). Alfalfa plants transformed with the GS2 gene driven by the CaMV 35S promoter have increased tolerance to salt stress and alfalfa transformants co-expressing both GS2 and an antioxidant gene are now being tested for abiotic stress tolerance (Sengupta-Gopalan, unpublished data). Two different root-specific genes were identified in salt-tolerant alfalfa lines that were selected in tissue culture, these being MsPRP2 encoding for a proline-

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rich cell wall protein and Alfin1 encoding for a zinc-finger transcription factor (Winicov and Bastola 1999). Over-expression of Alfin1 in alfalfa is accompanied by increased root growth and salinity tolerance (Winicov 2000). Alfalfa was also transformed with a gene for citric acid synthase (Rosellini et al. 2002) and with a gene for malate dehydrogenase (Tesfaye et al. 2001) to confer tolerance to aluminum toxicity, since alfalfa is sensitive to aluminum. 4.3.3 Nitrogen Use Efficiency An area of great interest with respect to any crop plant is to increase yield under low nitrogen conditions, and concerted efforts have been made toward this goal in alfalfa. Nitrogen is the inorganic nutrient that plants require in greatest quantity and that most frequently limits productivity in agricultural systems. Because of a heavy use of fertilizers, worldwide nitrogen pollution along with water shortage is becoming one of the major threats to human survival and the environment. Increasing the efficiency of nitrogen use by plants would thus have an impact both on the cost of producing fertilizers and in minimizing problems associated with pollution. It was suggested that plants are not limited in their uptake or reduction of nitrate. However, some plants appear to be limited in the incorporation of inorganic nitrogen into proteins. Thus, the development of crop plants with improved ability to assimilate nitrogen may be the solution. Coincidental locations were found for a QTL for yield trait and a structural gene for the cytosolic form of glutamine synthetase (GS1 ) in rice and in maize (Obara et al. 2004). These results suggest that GS1 could represent a key component of nitrogen use efficiency and yield. Efforts were made to understand the regulatory mechanism underlying the expression of GS1 and to modulate its expression both in a constitutive manner and in a green-tissue specific manner. Constitutive over-expression of GS1 in the model legume Lotus japonicus resulted in significant improvement in plant performance (Ortega et al. 2004). In alfalfa, a more significant improvement in plant performance was observed in plants over-expressing GS1 in a mesophyll-specific manner when compared with plants over-expressing GS1 in a constitutive manner (Seger 2005). The failure to increase GS1 activity in the nodules (Ortega et al. 2001, 2006), where GS1 plays a major role in the assimilation of fixed nitrogen, raised the question of the availability of enough carbon skeletons to assimilate the ammonia. Towards this goal, there is an effort to simultaneously manipulate GS1 and the key enzyme in sucrose synthesis, sucrose phosphate synthase (SPS; Aleman 2005). 4.3.4 Improvement of Forage Quality The major focus of improvement in alfalfa, being a forage crop, is in the area of protein quality and fiber digestibility.

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Increasing Methionine in the Protein Fraction Humans and monogastric animals cannot synthesize ten out of 20 essential amino acids and, therefore, must obtain them in their diet. Alfalfa provided as forage is an important source of protein for animal feed and could be considered the most important forage crop in the world (Hanson et al. 1988). However, it is deficient in sulfur (S)-amino acids (Kaldy et al. 1979). Efforts to use conventional breeding and cell selection techniques to improve the S-amino acid content of alfalfa met with little success (Reish et al. 1981) and as such efforts are now being made to use a genetic engineering approach. There are reports of the production of transgenic forage legumes expressing genes encoding different rumen by-pass proteins rich in S-amino acid, such as chicken ovalbumin, pea albumin, Brazil nut 2S albumin and sunflower seed albumin (Schroeder et al. 1991; Saalbach et al. 1994; Khan et al. 1996). In all cases, the accumulation of the protein was very low in the leaves, probably because the proteins were targeted to the protease rich vacuoles. To protect transgenic proteins from being degraded in the vacuoles, efforts were also made to retain the protein in the ER by engineering the ER retention signal (KDEL) in the carboxy terminal end of the protein. While the modified protein accumulated to reasonably high concentrations in subterranean clover, the accumulation was very low in alfalfa (Khan et al. 1996). More recently, efforts were made to engineer plants with the genes for the ERtargeted, methionine (Met)-rich, zein proteins from corn (Bagga et al. 1995, 2004). The genes encoding the β-zein (15 kDa) and the two δ-zeins (10 kDa, 18 kDa), with a Met content of 22% and 27%, respectively, were engineered behind the CaMV 35S promoter and introduced into alfalfa. The transformants showed high accumulation of the zein proteins, and the proteins were found to accumulate in unique ER-derived protein bodies (Bagga et al. 2004). Preliminary studies also indicated that the zein proteins are highly stable in the rumen of cows (Bagga et al. 2004). Thus, the β- and δ-zein proteins, rich in sulfur-containing amino acids, resistant to ruminant bacteria and stable in the vegetative tissues of transformed plants, appear to be ideal candidates for use in improving the Met content of alfalfa forage. To increase zein accumulation in vegetative tissues, multiple zein genes were introduced by re-transformation and by sexual crosses into the same plant; and analysis of these transformants showed a zein gene dose-dependent increase in the accumulation of bound Met (Bagga et al. 2004). This implies that the plant cells are able to handle accumulation of the zein protein in spite of the fact that the protein accumulates in the ER. Moreover, co-expression of the β-zein and the δ-zein genes yielded a five-fold increase in the accumulation of the δ-zein proteins; and the two zeins were co-localized in the same protein bodies. However, the accumulation was still not enough to make an impact on the amino acid balance in alfalfa. To check whether a limited supply of free Met is the basis for low accumulation of the Met-rich zein proteins, callus tissues from alfalfa transformants (β-zein) were incubated with Met and analyzed for zein accumulation. A three-fold increase in zein levels was seen following incubation with free Met (Bagga et al. 2005).

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The ultimate experimental approach undertaken to increase Met in alfalfa was to simultaneously increase both the free Met pools and a Met sink in the form of the zein proteins (Bagga et al. 2005). Several lines of evidence indicated that cystathionine gamma synthase (CgS) controls the rate of Met synthesis (Gakiere et al. 2000, 2002; Hacham et al. 2002; Kim et al. 2002; Di et al. 2003). Therefore, the Arabidopsis CgS (AtCgS) gene, driven by the CaMV 35S promoter, was introduced into alfalfa. The transformants showed not only an increased free Met pool, but also an even larger increase in S-methyl methionine (SMM), a transport form of Met, when compared with control plants. Moreover, alfalfa plants co-transformed with the AtCγ S and the β-zein genes driven by the CaMV 35S promoter showed an increase in zein concentration compared with that in the β-zein transformants. Another genetic engineering manipulation that is currently under progress to increase free Met pools in alfalfa is to down-regulate threonine synthase (TS) and S-adenosylmethionine synthase (SAMS; Barrow, unpublished data). Metabolic engineering is a difficult venture because of the high degree of networking among the different pathways. Improving Digestibility of Alfalfa Forage Forages are the primary source of fiber in ruminant diets. A minimum amount of dietary fiber is required for normal rumen function, animal health and milk fat content. However, fiber is the least digestible part of most dairy diets and has the lowest energy content. The component that contributes to non-digestibility of forage is lignin. It cross-links with cellulose to increase cell wall strength, but decreases the usefulness of cellulose as an extractable energy component of forage. Lignin is a phenolic compound found in most plant secondary cell walls and is the major structural component of secondarily thickened cell walls. It is a complex polymer of hydroxylated and methoxylated phenylpropane units. Dicotyledonous angiosperm lignins contain two major monomer species, namely guaiacyl (G) and syringyl (S) units. Lignin content increases as the plant matures and there is also change in the lignin composition with age toward a progressively higher S/G ratio (Buxton and Russell 1988). Both lignin content and the S/G ratio correlate negatively with forage digestibility. Lignin is derived from the phenyl propanoid pathway. The formation of the G and S units of lignin is catalyzed by O-methyl transferase enzymes. Caffeic acid 3-O-methyl transferase (COMT) converts caffeic acid to ferulic acid which then, via 5-hydroxy-ferulic acid, leads to the formation sinapic acid. Methylation of the caffeate moiety is also catalyzed by caffeoyl CoA 3-O-methyl transferase (CCOMT), which leads to the synthesis of coniferaldehyde via feruloyl CoA (Guo et al. 2001a). The enzyme cinnamyl alcohol dehydrogenase (CAD) catalyzes the formation of sinapyl alcohol from sinapaldehyde and coniferyl alcohol from coniferylaldehyde. Sinapyl alcohol is the precursor for the S unit and coniferyl alcohol is the precursor for the G unit. Alfalfa transformed with antisense gene constructs of CAD driven by the CaMV 35S promoter showed no change in lignin quantity, but the lignin com-

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position was altered with a lower S/G ratio and a lower S+G yield (Baucher et al. 1999). While these transformants were not tested for rumen digestibility, they showed increased digestibility over the control during in situ degradation tests (Baucher et al. 1999). Independent alfalfa transformants down-regulated in COMT and CCOMT were produced by gene-silencing and antisense approaches (Guo et al. 2001a). Strong down-regulation of COMT resulted in decreased lignin content, a reduction in total G lignin units and a near total loss of S units in monomeric and dimeric lignin. In contrast, strong downregulation of CCOMT led to reduced lignin content and a reduction in G units, without a reduction in S units (Guo et al. 2001a). Analysis of rumen digestibility of alfalfa forage in fistulated steers showed that both COMT and CCOMT down-regulated alfalfa had improved digestibility, the latter showing greater improvement (Guo et al. 2001b). Improving Efficiency of Protein Utilization Condensed tannins (CTs) or proanthocyanidins are implicated to have a role in the prevention of pasture bloat. CTs bind to protein and decrease the ruminal protein degradability of forage, but the bonds are reversible at the low pH found in the abomasums, where the protein is released. CTs are dimers or higher oligomers of flavan-3ol units and are powerful antioxidants (Hagerman et al. 1998). While alfalfa foliar tissues accumulate anthocyanins at senescence or locally under certain stress conditions, no known conditions induce proanthocyanidins in alfalfa leaves. However, proanthocynadins do accumulate in seed coats, suggesting that alfalfa does have the genes for the pathway. Since several of these genes have been isolated, it is feasible to express the key members in the CT synthesis pathway in the leaves and stem of alfalfa by engineering the genes behind the appropriate leaf/stem promoters. A second approach taken to target CTs is at the level of the regulatory genes in the flavanoid pathway. Several of the maize myc (R, B-Peru, Sn, Lc) and myb (Cl, P) flavonoid regulatory genes were tested for their ability to influence the accumulation of flavonoids or proanthocyanidins (PA) when expressed in heterologous plants (Bradley et al. 1998; deMajnik et al. 2000; Bovy et al. 2002; Ray et al. 2003; Robbins et al. 2003). The expression of the myc protein Lc in alfalfa was accompanied by changes in PA accumulation under high light intensity or cold (Ray et al. 2003). During the ensiling process, there is a heavy loss of proteins due to proteolytic degradation. For some ensiled forages, such as alfalfa, proteolytic losses are especially high, with degradation of up to ∼80% of the forage protein (Papadopoulos and McKersie 2005). In contrast, red clover has up to 90% less proteolysis than alfalfa during ensiling (Papadopoulos and McKersie 2005). The lower extent of post-harvest proteolysis in red clover is related to the presence of soluble polyphenol oxidase (PPO) and o-diphenol PPO substrates in the leaves. Both PPO and o-diphenol are absent or present in very low amounts in alfalfa (Jones et al. 1995). PPO catalyzes the oxidation of o-diphenols to o-quinones, which can bind and inactivate endogenous proteases. A cDNA

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clone for PPO, isolated from red clover and engineered behind the CaMV-35S promoter was introduced into alfalfa. These transformants showed increased PPO activity and the proteolysis was dramatically reduced in the presence of an o-diphenol compared to controls (Sullivan et al. 2004). 4.3.5 Production of Novel Compounds Since alfalfa is perennial and can be easily transformed, it is ideal for molecular pharming. In fact, a company named Medicago focuses on producing two major families of biopharmaceuticals: monoclonal antibodies and plasmatic proteins. Alfalfa can also be engineered to produce an edible vaccine (Tuboly et al. 2000) and to produce phytase, which can replace inorganic phosphorus in poultry and swine feed and thus reduce phosphate in the manure (Koegel et al. 1999), and can also produce cellulase and manganese peroxidase (Rishi et al. 2001).

5 Conclusions The different desirable traits to improve forage quality in alfalfa are being investigated independently. However, to produce the ideal alfalfa plant with all the desirable traits discussed earlier, the engineering manipulations have to be performed on the same plant. This entails stacking the different gene constructs and ensuring that they are driven by different promoters to avoid homology-driven gene silencing (Halpin 2005).

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Halpin C (2005) Gene stacking in transgenic plants – the challenge for 21st century plant biotechnology. Plant Biotechnol J 2:141–155 Hanson AA, Barnes DK, Hill RR (1988) Alfalfa and alfalfa improvements. (Agron Monogr 29) American Society of Agronomy/Crop Science Society of America/Soil Science Society of America, Madison, pp 1–1084 Heck GR, Armstrong CL, Astwood JD, Behr CF, Bookout JT, Brown SM, Cavato TA, DeBoer DL, Deng MY, George C, Hillyard JR, Hironaka CM, Howe AR, Jakse EH, Ledesma BE, Lee TC, Lirette RP, Mangano ML, Mutz JN, Qi Y, Rodriguez RE, Sidhu SR, Silvanovich A, Stoecker MA, Yingling RA, You J (2005) Development and characterization of a CP4 EPSPS-based, glyphosate-tolerant corn event. Crop Sci 44:329–339 Higbie SM (2002) Analysis of transformed alfalfa overexpressing the genes for manganese superoxide dismutase or ascorbate peroxidase. PhD thesis, New Mexico State University, Las Cruces Hill RR, Shenk JS, Barnes RF (1988) Breeding for yield and quality. In: Hanson AA, Barnes DK, Hill RR (eds) Alfalfa and alfalfa improvement (Agron Monogr 29) American Society of Agronomy/Crop Science Society of America/Soil Science Society of America, Madison, pp 809–825 Holland JB, Bingman ET (1994) Genetic improvement for yield and fertility of alfalfa cultivars representing different eras of breeding. Crop Sci 34:953–957 Jones BA, Hatfield RD, Muck RE (1995) Screening legume forages for soluble phenols, polyphenols oxidase and extract browning. J Sci Food Agric 67:109–112 Kaldy MS, Hanna MR, Smoliak S (1979) Amino acid composition of sanfoin forage. Grass Forage Sci 34:145–148 Khan MRI, Ceriotti A, Tabe I, Aryan A, McNabb W, Moore A, Craign S, Spencer D, Higgins TJV (1996) Accumulation of a sulfur-rich seed albumin from sunflower in the leaves of transgenic subterranean clover (Trifolium subterraneum L.). Transgenic Res 5:81–88 Khoudi H, Vezina LP, Mercier J, Castonguay Y, Allard G, Laberge S (1997) An alfalfa rubisco small subunit homologue shares cis-acting elements with the regulatory sequences of the RbcS-3A gene from pea. Gene 197:1–2 Kim J, Lee M, Chalam R, Martin MN, Leustek T, Boerjan W (2002) Constitutive overexpression of cystathionine γ -synthase in Arabidopsis leads to accumulation of soluble methionine and S-methylmethionine. Plant Physiol 128:95–107 Klypina N, Bagga S, Potenza C, Sutton D, Ghoshroy S, Hanson SF, Sengupta-Gopalan C (2005) The β-zein is not enough to stabilize δ-zein in transgenic tissues to high enough levels. In Vitro Cell Dev Biol-Plant 41:46A Koegel RG, Straub RJ, Austin-Phillips S, Cook ME, Crenshaw TD (1999) Alfalfa produced phytase for supplementation of poultry and swine rations. In: ASAE (ed) ASAE-CSAE-SCGR annual international meeting, Toronto, Ontario. ASAE, Toronto, paper 996127 Kozaki A, Takeba G (1996) Photorespiration protects C3 plants from photooxidation. Nature 384:557–560 Lamb JFS, Sheaffer CC, Rhodes LH, Sulc RM, Undersander DJ Brummer EC (2006) Five decades of alfalfa cultivar improvement: impact on forage yield, persistence and nutritive value. Crop Sci 46:902–909 Lei Z, Elmer AM, Watson BS, Dixon RA, Mendes PJ, Sumner LW (2005) A two dimensional electrophoresis proteomic reference map and systematic identification of 1367 proteins from a cell suspension culture of the model legume Medicago truncatula. Mol Cell Proteomics 4:1812–1825 Maureira IJ, Ortega F, Campos H, Osborn TC (2004) Population structure and combining ability of diverse Medicago sativa germplasms. Theor Appl Genet 109:775–782 McKenzie JS, Paquin R, Duke SH (1988) Cold and heat tolerance. In: Hanson AA, Barnes DK, Hill RR (eds) Alfalfa and alfalfa improvement. (Agron Monogr 29) American Society of Agronomy/Crop Science Society of America/Soil Science Society of America, Madison, pp 259–302 McKersie BD, Bowley SR, Jones KS (1999) Winter survival of transgenic alfalfa overexpressing superoxide dismutase. Plant Physiol 119:839–847

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McKersie BD Murnaghan J, Jones KS, Bowley SR (2000) Iron-superoxide dismutase expression in transgenic alfalfa increases winter survival without a detectable increase in photosynthetic oxidative stress tolerance. Plant Physiol 122:1427–1437 Moore RL (2003) Functional analysis of alfalfa (Medicago sativa) green-tissue specific promoters. MSc thesis, New Mexico State University, Las Cruces Obara M, Sato T, Sasaki S, Kashiba K, Nagano A, Nakamura I, Ebitani T, Yano M, Yamaya T (2004) Identification and characterization of a QTL on chromosome 2 for cytosolic glutamine synthetase content and panicle number in rice. Theor Appl Genet 110:1–11 Ortega JL, Temple SJ, Sengupta-Gopalan C (2001) Constitutive overexpression of cytosolic glutamine synthetase (GS1 ) gene in transgenic alfalfa demonstrates that GS1 may be regulated at the level of RNA stability and protein turnover. Plant Physiol 126:109–121 Ortega JL, Temple S, Bagga S, Ghoshroy S, Sengupta-Gopalan C (2004) Biochemical and molecular characterization of transgenic Lotus japonicus plants constitutively over-expressing a cytosolic glutamine synthetase gene. Planta 219:807–818 Ortega JL, Moguel-Esponda S, Potenza C, Conklin CF, Quintana A, Sengupta-Gopalan C (2006) The 3 untranslated region of a soybean cytosolic glutamine synthetase (GS1 ) affects transcript stability and protein accumulation in transgenic alfalfa. Plant J 45:832–846 Papadopoulos YA, Mckersie BD (2005) A comparison of protein degradation during wilting and ensiling of six forage species. Can J Plant Sci 63:903–912 Potenza C, Aleman L, Sengupta-Gopalan C (2004) Targeting transgene expression in research, agriculture and environmental applications: promoters used in plant transformation. In Vitro Cell Dev Biol Plant 40:1–22 Ray H, Yu M, Auser P, Blahut-Beatty L, McKersie B, Bowley S, Westcott N, Coulman B, Lloyd A, Gruber MY (2003) Expression of anthocyanins and proanthocyanidins after transformation of alfalfa with maize Lc. Plant Physiol 132:1448–1463 Reish B, Duke SH, Bingham ET (1981) Selection and characterization of ethionine-resistant alfalfa (Medicago sativa L.) cell lines. Theor Appl Genet 59:89–94 Riday H, Brummer EC (2002a) Forage yield heterosis in alfalfa. Crop Sci 42:716–723 Riday H, Brummer EC (2002b) Heterosis of agronomic traits in alfalfa. Crop Sci 42:1081–1087 Riday H, Brummer EC (2006) Persistence and yield stability of intersubspecific alfalfa hybrids. Crop Sci 46:1058–1063 Riday H, Brummer EC, Moore KJ (2002) Heterosis of forage quality in alfalfa. Crop Sci 42:1088– 1093 Riday H, Brummer EC, Campbell TA, Luth DE, Cazcarro PM (2003) Comparisons of genetic and morphological distance with heterosis between Medicago sativa subsp. sativa and subsp. falcate. Euphytica 131:37–45 Rishi AS, Nelson ND, Goyal A (2001) Molecular farming in plants: a current perspective. J Plant Biochem Biotechnol 10:1–12 Robbins MP, Paolocci F, Hughes JW, Turchetti V, Allison G, Archioni S, Morris P, Damiani F (2003). Sn, a maize bHLH gene, modulates anthocyanin and condensed tannins pathways in Lotus corniculatus. J Exp Bot 54:239–248 Rosellini D, Barone P, Bouton J, LaFayette P, Sledge M, Veronesi F, Parrot W (2002) Aluminum tolerance in alfalfa with the citrate synthase gene. In: NAAIC (ed) 38th report of the north american alfalfa improvement conference. Available at: www.naaic.org/Meetings/National/2002meeting/2002Abstracts/Rosellini.pdf Saalbach I, Pickardt T, Machemehl F, Saalbach G, Schieder O, Muntz K (1994) A chimeric gene encoding the methionine-rich 2S albumin of the Brazil nut (Bertholletia excelsa HBK) is stably expressed and inherited in transgenic grain legumes. Mol Gen Genet 242:226–236 Samac DA, Tesfaye M, Dornbusch M, Saruul P, Temple SJ (2004). A comparison of constitutive promoters for expression of transgenes in alfalfa (Medicago sativa). Transgenic Res 13:349– 361 Schroeder HE, Khan MRI, Knibb WR, Spencer D, Higgins TJV (1991) Expression of a chicken ovoalbumin gene in three Lucerne cultivars. Aust J Plant Physiol 18:495–505

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Seger M (2005) Modulation of cytosolic glutamine synthetase: mesophyll-specific vs constitutive overexpression. MSc thesis, New Mexico State University. Las Cruces Segovia-Lerma A, Murray LW, Townsend MS, Ray IM (2004) Population-based diallel analyses among nine historically recognized alfalfa germplasms. Theor Appl Genet 109:1568–1575 Shearer HL, Friedberg J, Bowley SR (2002) The effect of enhanced sucrose-phosphate synthase (SPS) activity on the low-temperature survival of alfalfa (Medicago sativa L.). In: NAAIC (ed) 38th report of the north american alfalfa improvement conference. Available at: www.naaic.org/Meetings/National/2002meeting/2002Abstracts/Shearer.pdf Sledge M, Ray I, Rouf Mian MA (2004) EST-SSRs for genetic mapping in alfalfa. In: Hopkins A, Wang ZY, Sledge RM, Barker RE (eds) Molecular breeding of forage and turf. Springer, Berlin Heidelberg New York, pp 239–243 Strizhov N, Keller M, Mathur J, Koncz-Kalman Z, Bosch D, Prudovsky E, Schell J, Sneh B, Koncz C, Zilberstein A (1996) A synthetic cryIC gene, encoding a Bacillus thuringiensis δ-endotoxin, confers Spodoptera resistance in alfalfa and tobacco. Proc Natl Acad Sci USA 93:15012–15017 Sullivan ML, Hatfield RD, Thoma SL, Samac DA (2004) Cloning and characterization of red clover polyphenol oxidase cDNAs and expression of active protein in Escherichia coli and transgenic alfalfa. Plant Physiol 136:3234–3244 Tesfaye M, Temple SJ, Allan DL, Vance CP, Samac DA (2001) Overexpression of malate dehydrogenase gene expression in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum. Plant Physiol 127:1836–1844 Tesfaye M, Denton MD, Samac DA, Vance CP (2005) Transgenic alfalfa secretes a fungal endochitinase protein to the rhizosphere. Plant Soil 269:233–243 Tesfaye M, Silverstein KAT, Bucciarelli B, Samac D, Vance CP (2006) The affymetrics Medicago genechip array is applicable for transcript analysis of alfalfa (Medicago sativa). Funct Plant Biol 33:783–788 Tuboly T, Yu W, Baily A, Erickson L, Nagy E (2000) Development of oral vaccine in plants against transmissible gastroenteritis virus of swine. In: Toutant JP, Balazs E (eds) Molecular farming. La Grande Motte, Paris, pp 239–248 Verdaguer B, Kochko AD, Fux CI, Beachy RN, Fauquet C (1998) Functional organization of the cassava vein mosaic virus (CsVMV) promoter. Plant Mol Biol 37:1055–1067 Wang L, Samac DA, Shapir N, Wackett LP, Vance CP, Olszewski NE, Sadowsky MJ (2005) Biodegradation of atrazine in transgenic plants expressing a modified bacterial atrazine chlorohydrolase (atzA) gene. Plant Biotechnol J 3:475–486 Wehrmann A, Vliet AV, Opsomer C, Botterman J, Schulz A (1996) The similarities of bar and pat gene products make them equally applicable for plant engineers. Nat Biotechnol 14:1274–1278 Winicov I (2000) Alfin1 transcription factor overexpression enhances plant root growth under normal and saline conditions and improves salt tolerance in alfalfa. Planta 210:416–422 Winicov I, Bastola DR (1999) Transgenic overexpression of the transcription factor ALfin1 enhances expression of the endogenous MsPRP2 gene in alfalfa and improves salinity tolerance of the plants. Plant Physiol 120:473–480 Winicov I, Valliyodan B, Xue L, Hoober JK (2004) The MsPRP2 promoter enables strong heterologous gene expression in a root-specific manner and is enhanced by overexpression of Alfin 1. Planta 219:925–935

IV.2 Clover A. Mouradov, S. Panter, M. Emmerling, M. Labandera, E. Ludlow, J. Simmonds, and G. Spangenberg1

1 Introduction The Leguminosae is one of the largest families of flowering plants, comprising more than 650 genera and 18,000 species (Polhill and Raven 1981). Legumes are second to cereal crops in agricultural importance, based on area harvested and total production. One of the most important attributes of legumes is their unique capacity for symbiotic nitrogen fixation, underlying their importance as a source of nitrogen in both natural and agricultural ecosystems. Seeds of legumes provide about one-third of all dietary nitrogen derived from protein and one-third of processed vegetable oil for human consumption (Graham and Vance 2003). Legumes are also sources for many natural products (secondary metabolites), such as lignins, flavones, flavonols, methoxyflavonols, coumestans, isoflavans, anthocyanins and proanthocyanidins (condensed tannins). Most of these metabolites play important biological roles in plants, including the attraction of pollinators and agents of seed dispersal to flowers and fruit by visual cues, pollen development, signalling associated with plant– microbe interactions, and protection from ultraviolet light, herbivores and pathogens. In addition to their importance as sources of dietary protein for humans and animals, legumes provide health-related natural products including dietary fibre, hormone analogues and anti-oxidants (Dixon and Sumner 2003). White clover (Trifolium repens L.) is the major legume species found in temperate dairy pastures in Australia and is one of the key pasture legumes in temperate climates with an annual rainfall in excess of 750 mm throughout the world. Its valuable features, including symbiotic nitrogen fixation, a high protein content and the accumulation of many natural products, make white clover an ideal component of temperate pastures and an attractive target for molecular breeding.

1 Primary

Industries Research Victoria, Victorian Agribiosciences Centre, La Trobe University, Bundoora, Victoria 3086, Australia, e-mail: [email protected]

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2 Improvement of Forage Quality by Modification of Secondary Metabolism 2.1 Proanthocyanidins The presence of proanthocyanidins (PAs) in edible plant tissues is a focus of research because of their potential benefits for human health and ruminant nutrition (Dixon and Steele 1999; Bagchi et al. 2000; Winkel-Shirley 2001; Lin et al. 2002; Dixon and Sumner 2003). PAs are common components of seed coats in flowering plants. They also accumulate in the vegetative tissue and floral organs of many legumes. The ability of PAs to bind proteins via their hydroxyl groups, and to chelate heavy metal ions such as iron and zinc with their o-diphenol groups (Scalbert 1991; House 1999), could form the basis for a role in plant defence against microbial pathogens, insects and herbivores (Kesley et al. 1984; Aziz et al. 2004; Dixon et al. 2005). Furthermore, the ability of PAs to chelate metal ions is thought to account, at least partly, for the aluminium tolerance of Lotus pedunculatus, a PA-rich legume (Stoutjesdijk et al. 2001). The most important agronomic trait attributed to PAs is their ability to suppress bloat-inducing characteristics of some forage legumes, such as white clover and alfalfa. An abundance of protein in the leaves of these plants leads to their rapid fermentation by rumen micro-organisms, causing gas generation and the potential development of bloat, a disease that costs the Australian pastoral industries over AUD $ 100 × 106 year−1 . PAs can bind to dietary proteins under mild acidic conditions (pH 3.5–7.0), slowing down their rate of degradation in the rumen. This increases crude protein flow to the abomasum and intestine where PA–protein complexes dissociate under more acidic conditions (pH < 3.5), releasing the plant proteins (Waghorn et al. 1987; Wang et al. 1994; Aerts et al. 1999a; Barry and McNabb 1999; Douglas et al. 1999). The presence of a low concentration of PA polymers (2–4% of dry weight) in forage can improve the efficiency of protein uptake by ruminants and prevent bloat, leading to increased milk, meat and wool production (Wang et al. 1996). A low concentration of dietary PAs can also counter greenhouse gas generation by reducing methane emissions from ruminants, and reduce eutrophication of soil and water by suppressing the excretion of soluble nitrogen, as well as providing some protection against gastrointestinal parasites. A higher concentration of PAs (6–12% of dry weight) decreases the nutritional value of forage by inhibiting bacterial enzymes needed for fermentation and by forming complexes with cell wall polysaccharides in fodder. In addition to concentration per se, the subunit composition of PAs in forages may be important for ruminant health (Foo et al. 1996, 1997; Aerts et al. 1999b). White clover plants produce an insignificant level of PAs in vegetative tissues; and this compromises their value as fodder (Aerts et al. 1999a). Staining with 4-dimethylaminocinnemaldehyde (DMACA) showed that PAs and/or their monomers are present only in the glandular trichomes of white clover

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foliage. In contrast, a substantial amount of PA accumulates in the floral organs of white clover plants (Foo et al. 2000; Fig. 1). Since flowering is seasonal, the generation of ‘bloat-safe’ transgenic white clover cultivars with a desirable concentration and composition of PAs constitutively accumulated in foliage is a major opportunity and challenge for white clover biotechnology. Conceivably, cells in white clover plants could be modified to produce PAs by re-programming the existing flavonoid pathway, diverting intermediates to PA biosynthesis. Two key genes (LAR, encoding leucoanthocyanidin reductase; BANYULS, encoding anthocyanidin reductase) were cloned from Desmodium unicinatum and Arabidopsis thaliana, respectively (Devic et al. 1999; Tanner et al. 2003). The LAR and BAN genes encode enzymes in PA-specific steps of the flavonoid pathway, converting flavan-3,4-diols generated by the general flavonoid pathway to the corresponding 2,3-trans- and 2,3-cis-flavan-3-ols, (+)catechins and (–)-epicatechins, respectively (Tanner et al. 2003; Xie et al. 2003). Ectopic expression of MtBAN, a BAN orthologue from Medicago truncatula, in tobacco plants that have no PAs in aerial organs, results in accumulation of PAs in flowers, but not in leaves. Co-transformation of tobacco plants with MtBAN and PAP1, a Myb transcription factor that regulates anthocyanin production in Arabidopsis, leads to accumulation of epicatechin monomers and PA in the leaves (Dixon et al. 2005). This indicates that additional PAP1-regulated factors

Fig. 1. Accumulation of proanthocyanidins (PAs) in different organs of white clover and Lotus corniculatus plants. DMACA staining of different tissues was visualised by light microscopy. Blue staining, indicating the presence of PAs, can be seen in floral organs (A, B) and glandular trichomes of a peduncle (C), stolon (D) and leaflet (E) of a white clover plant. In contrast, PAs were detected in mesophyll cells of L. corniculatus leaves (F). The arrow in D indicates a leaf primordium encircled by a dense layer of trichomes. Bar 1 mm

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are required for successful foliar accumulation of PA in transgenic tobacco plants. In alfalfa leaf tissue, stress-induced PA accumulation was enhanced by expressing the maize Lc gene, a Myc-family transcription factor (Ray et al. 2003). Constitutive expression of Sn, another Myc-family transcription factor from maize, in L. corniculatus increased PA accumulation in roots, but not in leaves (Damiani et al. 1999). Cloning of the BAN and LAR genes, as well as genes potentially involved in transport of PA monomers into the ER and vacuole (TT12, AHA10), will enable more precise modification of the PA pathway, opening up opportunities for metabolic engineering of white clover and other forage legumes for ‘bloat safety’. 2.2 Isoflavones Although flavonoids are found throughout the plant kingdom, isoflavones are restricted to the Leguminosae, where they function as inducers of the nodulation genes of symbiotic Rhizobia (Dixon 1999). Besides their role in symbiosis, some isoflavones have antimicrobial and insect-inhibiting properties. Isoflavones are also potential nutraceuticals with specific health benefits, namely estrogenic, anti-angiogenic, anti-oxidant and anti-cancer activities (Dixon 1999; Setchell and Cassidy 1999; Lamartiniere 2000; Dixon and Ferreira 2002). Isoflavones may have other beneficial properties, including the prevention of osteoporosis and other postmenopausal disorders, as well as cardiovascular disease (Alekel et al. 2000; Merz-Demlow et al. 2000; Uesugi et al. 2001). Major sources of isoflavones for humans are soybean products (containing daidzein and genistein) and chickpea. Transgenic approaches have been used to increase the level of certain isoflavones by blocking the competing flavonol/anthocyanin branch of flavonoid biosynthesis and over-expressing the isoflavone synthase gene, IFS (Steele et al. 1999; Jung et al. 2000; Yu et al. 2000). Analogous approaches aiming to modify isoflavone levels in a targeted manner in clovers such as red clover (T. pratense) are conceivable. Metabolic reprogramming of the flavonoid pathway to allow the production of isoflavonoids in non-legumes could also provide new dietary sources of these compounds (Jung et al. 2000; Yu et al. 2000). 2.3 Lignins Not all secondary metabolites of legumes are beneficial for forage quality. Lignins, the second most abundant plant polymers after cellulose, are a major structural component of cell walls in higher land plants. Lignins have at least three main functions in plants, namely: (a) providing mechanical strength and structural support, (b) generating a physical barrier in response to pathogens and (c) functioning as a hydrophobic component of vascular tissues to allow water transport (Lawton and Lamb 1987; Jaeck et al. 1992; Baldridge et al. 1998; Hatfield and Vermerris 2001; Peter and Neale 2004).

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Lignins adversely affect the forage quality of many legumes by reducing the digestibility of cell walls in legume-derived fodder, limiting the amount of energy available to livestock and preventing the complete utilisation of cellulose and hemi-cellulose by ruminants (Casler et al. 2002). Lignin concentrations increase progressively with maturity in the stems of many forage legumes (Jung et al. 1997). Feeding and grazing studies have shown that small changes in forage digestibility can have a significant impact on animal performance (Gressel and Zilberstein 2003). In addition to the amount of lignin per se, its monomeric composition has profound consequences for industry, agriculture and the environment. Lignin produced by dicotyledons, including legumes such as clovers, is composed of two main monomer species, termed guaiacyl (G) and syringyl (S) units, which differ in the number of methoxyl groups on the aromatic ring. A high concentration of S subunits can reduce digestibility because of cross-linking between highly methoxylated S subunits and arabinoxylans, another major cell wall component (Pond et al. 1987). Some studies link low digestibility of alfalfa and some forage grasses to an increased S/G ratio as a consequence of increasing plant maturity (Pond et al. 1987; Dixon et al. 1996; Baucher et al. 1998). Improved cell wall digestibility in forage plants is an important goal of many plant–ruminant research programmes and the molecular genetics, functional biology and chemistry of monolignol and lignin biosynthesis receives considerable attention (Morrison and Stewart 1998). As a result, biosynthesis of monolignols is one of the best understood pathways of plant secondary metabolism. Lignin biosynthesis has been successfully modified by targeting various genes in the pathway using co-suppression, antisense RNA and dsRNAi strategies (Baucher et al. 1999; Grima-Pettenati and Goffner 1999; Guo et al. 2001; Humphreys and Chapple 2002; Piquemal et al. 2002; Chen et al. 2003; Gressel and Zilberstein 2003). Silencing of a single gene can provide sufficient down-regulation of lignin production and modification of lignin structure to enhance the digestibility of maize (Halpin et al. 1998), sorghum and pearl millet (Lam et al. 1996). Some transgenic plants show improved digestibility as a result of a decreased S/G ratio (Guo et al. 2001). Metabolic profiling is a useful approach for monitoring the broader biochemical phenotypes of transgenic plants with altered expression of enzymes in the lignin biosynthesis pathway (Chen et al. 2002). 2.4 Cyanogenic Glucosides Cyanogenic glucosides (CG) are widely distributed in the plant kingdom. They are present in more than 2,650 plant species derived from about 550 genera and more than 130 families (Seigler and Brinker 1993). CGs protect certain plants from herbivores by releasing a toxin, hydrogen cyanide (HCN). The biosynthetic pathway for cyanogenic glucosides has been extensively studied in sorghum (Sorghum bicolor), cassava (Manihot esculenta), seaside arrow grass (Triglochin maritimum) and barley (Hordeum vulgare; Koch et al. 1995;

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Jones et al. 1999; Nielsen and Møller 1999; Andersen et al. 2000; Nielsen et al. 2002). Some forage legumes contain a detectable level of CGs. L. japonicus has been selected as a genetic model system for studying CGs in higher plants. The cyanogenic and nitrile glucoside content in L. japonicus varies according to the tissue type and the developmental age of plants. The cyanide potential is highest in young seedlings and in the apical leaves of mature plants. A high concentration of CGs in some plants has consequences for human health. Hence, cyanogen-free transgenic cassava was produced by down-regulating two cytochrome P450 enzymes (Siritunga and Sayre 2003). 2.5 Triterpene Saponins Triterpene saponins are a group of natural products that have both positive and negative effects on animal health. Some saponins have anti-microbial and insecticidal properties. In terms of human health, some saponins are anti-inflammatory and suppress ulcers, allergic reactions and carcinogenesis. However, saponins can be toxic to monogastric animals and reduce palatability. Saponins also reduce the digestibility of forage by ruminants (Klita et al. 1996; Oleszek 1996; Small 1996; Oleszek et al. 1999), making the development of saponin-free clovers and alfalfa an important agronomic target. Reduction of the saponin concentration in forage legumes is hindered by the fact that most genes involved in the biosynthesis of triterpene saponins have not been characterised. A genomics approach, aiming to characterise triterpene saponin biosynthesis in M. truncatula, identified sequences of genes encoding enzymes implicated in early stages of triterpene aglycone formation, namely, squalene synthase, squalene epoxidase and beta-amyrin synthase (Suzuki et al. 2002).

3 Improvement of Tolerance to Abiotic and Biotic Stresses 3.1 Aluminium Tolerance Aluminium toxicity represents one of the most important limitations to agricultural production worldwide (Von Uexkull and Mutert 1995). It is estimated that approximately 40% of all arable land is acidic (Ma et al. 2001). Soil with a pH < 5 allows the solubilisation of aluminium (Al3+) ions. Aluminium toxicity inhibits root cell elongation and division, resulting in compromised root systems with limited potential for water and nutrient uptake (Delhaize et al. 1993; Lazof et al. 1994; Delhaize and Ryan 1995). Additionally, organic phosphorus is bound in a complex with Al3+ ions in acidic soils, resulting in greatly reduced availability of phosphorus from fertilisers. Australian farmers currently spend AUD $ 600 × 106 year−1 on phosphorus fertilisers, and it is estimated that phosphorus to the value of more than AUD $ 10 × 109 is bound in Australian soils in a form unavailable to plants. White clover is very sensitive to

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aluminium toxicity and plants with elevated aluminium tolerance could allow the extension of pasture to land that is currently unsuitable because of soil acidity. Plants have complex mechanisms that enable them to tolerate toxic levels of Al3+ ions in acidic soils. Over the past decade, research efforts focused on identifying and characterising such mechanisms, of which two distinct classes have been described. The first class of mechanism involves internal detoxification of symplastic Al3+ , which is chelated by organic ligands in roots and shoots. The second class inhibits assimilation of Al3+ ions by the root apex. Although evidence for internal detoxification and sequestration of Al3+ is starting to emerge, the second mechanism, based on the secretion of organic acids leading to chelation and exclusion of extracellular Al3+ , is a focus for metabolic engineering (Ma 2000; Ma et al. 2001; Ryan et al. 2001; Kochian et al. 2004). There is strong evidence that in some plants, such as snapbean (Miyasaka and Hawes 2001) and wheat (Delhaize et al. 1993), Al3+ -tolerant genotypes exhibit Al3+ -inducible exudation of organic acids that is absent in sensitive genotypes. The organic acids malate, citrate and oxalate are intermediates of metabolic processes, notably the tricarboxylic acid/citric acid cycle, and are found in the cytoplasm, mitochondrion and glyoxysome. Key enzymes involved in the biosynthesis of these organic acids are citrate synthase (CS), malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC). The genes encoding these enzymes have been cloned in a number of plants (Kochian et al. 2004). Development of Al3+ tolerance by breeding without assistance of molecular technologies or by environmental manipulation has been shown to be limited in its potential. To date, conventional breeding efforts in white clover have failed to develop cultivars with increased aluminium tolerance under field conditions (Caradus et al. 2001). The application of calcium carbonate (liming) has long been used, but it has serious limitations, particularly when assessed for largescale use (Kochian 2000). The modification of genes encoding different steps in organic acid biosynthesis resulted in monocotyledonous and dicotyledonous crop plants with more tolerance to acidic soils than the wild-type. Transgenic alfalfa plants, constitutively expressing a nodule-enhanced malate dehydrogenase gene (neMDH), displayed greater aluminium tolerance under controlled and field conditions than wild-type plants (Tesfaye et al. 2001). Aluminium tolerance in these plants correlated with elevated accumulation and efflux of malate, and changes in the secretion of other organic acids, relative to wild-type plants (Tesfaye et al. 2001). By contrast, ectopic expression of PEPC in transgenic alfalfa plants resulted in much lower aluminium tolerance, relative to that of wild-type plants (Tesfaye et al. 2001). Transgenic canola and Arabidopsis plants overexpressing mitochondrial CS showed enhanced Al3+ -inducible accumulation and efflux of citrate from roots, correlating with elevated aluminium tolerance, as measured by root growth in Al3+ -rich conditions (Anoop et al. 2003). Ectopic expression of a carrot (Daucus carota) mitochondrial CS in

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transgenic Arabidopsis plants resulted in enhanced CS activity and secretion of citrate, and better performance compared with control plants when grown in an acidic soil with high Al3+ and low phosphate availabilities (Koyama et al. 2000). Recently, the isolation of a malate transporter (ALMT1, aluminiumactivated malate transporter), the basis for Al3+ -activated efflux of malate in wheat, provided insight into a novel molecular mechanism for natural aluminium tolerance (Sasaki et al. 2004). Transgenic wheat and barley plants overexpressing ALMT1 displayed Al3+ -inducible exudation of malate that correlated with increased aluminium tolerance. The expression of an ALMT1 transgene might also improve aluminium tolerance in clovers and other forage legumes. The experimental strategy of our research for the generation of Al3+ -tolerant and P-efficient transgenic white clover plants involved the isolation and characterisation of clover homologues of enzymes involved in the biosynthesis of key organic acids, namely, TrneMDH, TrPEPC and TrCS. White clover plants were generated expressing combinations of these genes under the control of a constitutive promoter and a root-tip-specific promoter from a gene encoding a phosphate transporter (TrPT1). Transgenic white clover plants expressing organic acid biosynthesis genes and displaying faster root growth in vitro than the wild type in the presence of a high concentration of Al3+ were produced (Labandera et al., unpublished data). 3.2 Virus Resistance Field-grown white clover is often infected by a number of plant viruses, including clover yellow vein virus (CYVV), white clover mosaic virus (WCMV) and alfalfa mosaic virus (AMV). All of these viruses reduce forage yield and quality, nitrogen-fixing capacity and vegetative persistence under conditions of biotic and abiotic stress (Gibson et al. 1981; Campbell and Moyer 1984; Latch and Skipp 1987). In Australia, AMV costs farmers approximately AUD $110 × 106 and infects up to 35% of white clover plants in dairy pastures (Norton and Johnstone 1998). Potential virus resistance or tolerance can be found in some legumes (Crill et al. 1971; Barnett and Gibson 1975). However, introgression of resistance or tolerance to viruses such as AMV into white clover by conventional breeding was unsuccessful. Biotechnology has great potential in the development of virus-resistant clovers with economic benefits for the pastoral sector. Transgenic white clover plants expressing a transgene encoding the AMV coat protein and displaying immunity to AMV under glasshouse and field conditions have been generated (Emmerling et al. 2004; Fig. 2). Following transgenic germplasm development and the establishment of a breeding nursery with 1,300 transgenic white clover plants homozygous for the transgene, a final selection of elite transgenic white clover syn0 parent plants was made for cultivar development. These plants were chosen on the basis of transgene-mediated AMV immunity, non-transgenic CYVV resistance and a range of agronomic characteristics such as plant height,

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Fig. 2. Field evaluation of transgenic white clover plants expressing the coat protein of the alfalfa mosaic virus (AMV-CP) under the control of the enhanced CaMV 35S promoter. Bars represent the percentage of plants that showed AMV infection symptoms in a planned field release carried out in Hamilton, Victoria, Australia in 1998. The first two bars represent untransformed controls, cvs. Haifa and Irrigation. The second two bars represent transgenic plants from two transformation events in white clover cv. ‘Haifa’ (D4, D6). The last 2 bars represent transgenic plants from two transformation events in white clover cv. ‘Irrigation’ (H1, H6)

stolon density, internode length, leaf length, flower number, summer growth and survival, autumn vigour and spring vigour (Emmerling et al. 2004), and they represent the basis for the first AMV-resistant transgenic white clover cultivar.

4 Functional Genomics and Metabolomics as Key Technologies for Characterisation and Modification of Natural Product Biosynthesis Over the past decade, genetic modification of forage legumes aimed to improve their forage quality and tolerance to environmental stresses. This approach integrated transgenic technologies with recent developments in functional genomics, computational and structural biology (Van den Bosch and Stacey 2003; Lee et al. 2003; Lin et al. 2003; George et al. 2004; Roy et al. 2004; Xiao et al. 2005; Zhang et al. 2005). Legumes are also the focus of efforts to provide a range of extended genomic resources (Godfree et al. 2004; Cannon et al. 2005; Gonzales et al. 2005). These include annotated sequences of bacterial artificial chromosome (BAC) ends, molecular markers based on BAC end sequencing,

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anchored physical and linkage genetic maps, libraries of expressed sequence tags (ESTs) from different organs, microarray and DNA chip resources and, ultimately, complete genome sequences (Lamblin et al. 2003; Sawbridge et al. 2003; Asamizu et al. 2004; Choi et al. 2004; Wu et al. 2004). Comprehensive EST resources and 15K unigene microarrays have been established for white clover (Sawbridge et al. 2003). Complete sequences of the genomes of Medicago truncatula and Lotus japonicus, two model forage legumes, are expected by the end of 2006 (Young et al. 2005). Functional genomic research programmes that encompass three tiers of biological function, the transcriptome, the proteome and the metabolome, are already underway in M. truncatula and L. japonicus. (Sumner et al. 2003; Watson et al. 2003, 2004; Colebatch et al. 2004). Analysis of metabolic profiles related to the expression of individual genes is a rapidly growing area of functional genomics (Trethewey et al. 1999). This integration of metabolomic and transcriptomic data is likely to provide precise information on gene-to-metabolite networks and should help to characterise novel genes involved in different biosynthetic pathways. Integration of proteomics data will improve understanding of the role of protein modifications and protein–protein interactions in biosynthesis of metabolites. A range of biochemical approaches, such as gas chromatography–mass spectrometry (GC-MS), as well as matrix-assisted laser desorption ionisation time of flight (MALDI-TOF) mass spectrometry and 13 C nuclear magnetic resonance spectroscopy (NMR), are used to analyse bioactive molecules in forage legumes and the effects of genetic and environmental manipulations on plant biochemistry (Roessner et al. 2001; Taylor et al. 2002; Behrens et al. 2003; Fiehn and Weckwerth 2003; Weckwerth et al. 2004). A metabolomic study of M. truncatula cell cultures stimulated by methyl jasmonate, a yeast elicitor, or ultraviolet light, revealed large changes in the levels of several amino acids, organic acids and carbohydrates (Suzuki et al. 2005). The generation of large numbers of transgenic white clover and M. truncatula plants is essential for functional genomic studies in these species and requires robust and efficient methods for genetic transformation and plant regeneration that are largely genotype-independent. Two rapid and simple in planta transformation methods were described for M. truncatula (Trieu and Harrison 1996; Trieu et al. 2000). The first approach was based on vacuuminfiltration of flowering plants. This procedure was used previously for the transformation of a range of Arabidopsis ecotypes. A second approach involved vacuum-infiltration of young seedlings with Agrobacterium. Although transformation efficiencies were reported, ranging over 4.7–76.0% and 2.9–27.6% for flower infiltration and seedling infiltration, respectively, both protocols are difficult to reproduce and not widely applied for forage legume transformation. A highly reproducible, robust and genotype-independent protocol for genetic transformation and regeneration of different Trifolium species was developed (Ding et al. 2003). It was successfully applied to different Trifolium species and cultivars including white clover (T. repens cvs. Haifa, Huia, Irrigation, Mink), red clover (T. pratense cvs. Astred, Colenso, Cherokee, Quinequeli,

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Redquin, Renegade), subterranean clover (T. subterraneum ssp. brachycalycinum cv. Clare, ssp. subterraneum cvs. Denmark, Woogenellup, ssp. yanninicum cvs. Larisa, Trikkala), T. michelianum and T. isthmocarpum. This methodology also proved applicable to plant regeneration in different Medicago species including alfalfa (M. sativa), M. polymorpha, M. truncatula, M. litoralis and M. tonata. A further development of this methododology, the ‘isogenic transformation’ approach, provides untransformed control plants with the same genetic background as the transgenic test plant (Ding et al. 2003).

5 Conclusions, Challenges and Future Developments The valuable features of clovers and other forage legumes, including their capacity for symbiotic nitrogen fixation and accumulation of many secondary metabolites, make them an attractive target for multi-disciplinary research approaches. This soon has led to substantial progress in our understanding of complex pathways that protect clovers from a wide spectrum of biotic and abiotic stresses. However, it is still challenging to apply this knowledge to the improvement of clovers. Molecular strategies for the re-programming of secondary metabolism need to deal not only with the complexity of these pathways, but also with gaps in our existing knowledge. For example, mechanisms for the transport of PA monomers into the endoplasmic reticulum and the vacuole, and their condensation, are poorly understood. Additional complexity is caused by the representation of some enzymes in large families of isoforms (Dixon et al. 2002; Dixon and Reddy 2003). This necessitates the identification of specific isoforms that are involved in the biosynthesis of particular metabolites by targeted gene-silencing approaches. Metabolic channelling, which potentially allows the assembly of highly specific multi-enzyme complexes to be spatially and temporally regulated, may explain why enzymes in shared steps of metabolic pathways often have multiple isoforms in plants (Winkel 2004). In the channelling model, intermediates are transferred directly between the active centres of consecutive enzymes in a biochemical pathway within a multi-enzyme complex (Srere 1987). This process increases the local concentration of intermediates and prevents unstable intermediates from reacting with other components of the cytosol. Spatial and temporal separation of ‘metabolons’ in plant cells could allow the biosynthesis of different natural products, including PAs, anthocyanins and isoflavonoids, from common intermediates in the phenylpropanoid pathway (Jorgensen et al. 2005). It is difficult to predict whether an isoform that is produced outside of its normal expression domain will interact appropriately with other enzymes in its new environment to yield the desired metabolite without proper information about the structure of metabolons and protein–protein interactions between enzymes in the pathway. In support of this model, there is already

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evidence that some enzymes in the phenylpropanoid pathway are involved in metabolic channelling (Czichi and Kindl 1975; Hrazdina and Wagner 1985; Hrazdina and Jensen 1992; Rasmussen and Dixon 1999; Achnine et al. 2004). Further understanding of specific multi-enzyme complexes, or ‘metabolons’, may improve our ability to predict the effect of genetic changes on metabolic re-programming and to modify the spatial and temporal accumulation of particular metabolites. In many cases, the annotation of metabolism-related genes is based purely on sequence similarity and not on biochemical characterisation of the encoded enzymes. As a result, some genes described as encoding particular enzymes may encode other enzymes with related functions (Jez et al. 2000a; Noel et al. 2005). Several plant polyketide synthases related to CHS by a significant level of primary sequence homology, including stilbene synthase (STS), bibenzyl synthase (BBS), acridone synthase (ACS) and pyrone synthase (PS), share a common chemical mechanism with CHS but differ from CHS in their substrate specificity (Austin and Noel 2003; Noel et al. 2005). Recent progress in structural biology provided us with a better understanding of the relationship between amino acid sequence data and catalytic activity and improved our ability to predict enzymatic function. Significant progress was made in analysing the phenylpropanoid pathway, where high-resolution structures were solved for CHS (Ferrer et al. 1999), CHI (Jez et al. 2000b), caffeic acid/5hydroxyferulic acid 3/5-O-methyltransferase (Zubieta et al. 2002), chalcone and isoflavone O-methyltransferases (Zubieta et al. 2001), anthocyanidin synthase (ANS; Wilmouth et al. 2002) and 4-CL (Schneider et al. 2003), over the past few years. These advances, together with recent progress in the understanding of metabolic channelling and progress in structural and computational biology, should enable us to modify catalytic activity, re-programme metabolic pathways and custom-design a new generation of natural products for biosynthesis in agronomically important legumes such as clovers. Bulk tissue sampling gives a loss of spatial resolution, and recent efforts were directed toward improving access to specialised cell types within plants. In recent years, a variety of technologies for obtaining data at the level of individual cells were developed. New technologies, such as laser-capture micro-dissection (Kehr 2003) and single-cell sap sampling (Karrer et al. 1995), were recently modified for successful collection of material from plant sections and are complementary to relatively old technologies, such as immuno-localisation and in situ hybridisation. Integration of single-cell sampling technology with global transcriptomic, proteomic and metabolomic approaches will open a new era of functional genomics at the nano-level, allowing the identification of cell type-specific promoters, transcription factors and metabolites. The use of state-of-the-art instrumentation for high-throughput metabolic profiling and structural characterisation of individual molecules is another key to successful metabolic engineering in clovers and other forage legumes, because amplification methods similar to those commonly used for DNA and RNA do not yet exist for proteins and metabolites. This emerging technology should allow us

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to determine the content and composition of monomers and polymers and to observe their spatial and temporal patterns of accumulation, both during development and in response to a spectrum of biotic and abiotic stresses. Technological advances in analytical chemistry, in particular in the development of high-field NMR spectroscopy and Fourier transform–ion cyclotron mass spectrometry, allow the structures of secondary metabolites to be determined, including those that are produced at low concentrations in plants. An improved understanding of the sub-cellular organisation of multi-enzyme complexes and intra- and intercellular translocation of metabolites by highly ordered, protein-mediated processes could help us to explain how metabolic channelling occurs and how highly branched pathways are regulated. An increasing variety of molecular tools are available for this type of investigation, including in situ hybridisation, sub-cellular localisation of proteins by immuno-fluorescence, yeast two-hybrid assays, improved visualisation of fluorescent reporters by confocal microscopy and fluorescent resonance energy transfer (FRET). These methods should allow multiple enzymes from the same metabolic pathway to be co-localised and should provide evidence for protein–protein interactions within multi-enzyme complexes (Hink et al. 2002). A non-invasive FRET procedure for monitoring close associations of protein molecules labelled with fluorophores (Day et al. 2001) was used in an attempt to demonstrate the intracellular co-localisation and potential interactions between l-phenylalanine ammonia lyase (PAL) and cinnamate 4-hydroxylase (C4H; Achnine et al. 2004). Co-localisation of PAL1-eGFP and C4H-eGFP constructs was observed at the surface of the endomembrane. There was also evidence for weak interactions between these enzymes. Interactions between chalcone synthase (CHS), chalcone isomerase (CHI) and dihydroflavonol 4-reductase (DFR) in Arabidopsis seedlings was shown using the yeast two-hybrid system, an assay for direct protein–protein interactions. This association appeared to be directional with CHS interacting with DFR, CHI with CHS and DFR with CHI (Burbulis and Winkel-Shirley 1999). The integration of technologies in molecular plant breeding, plant gene technology, plant functional and structural genomics, metabolomics and computational biology in a multi-disciplinary approach should greatly enhance our ability to convert genomics discoveries into genetic solutions in clovers and other forage legumes, leading to improvements in productivity, profitability and environmental sustainability of pasture-based agriculture.

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IV.3 Tall Fescue Z-Y. Wang1 and G. Spangenberg2

1 Introduction The genus Festuca includes about 80 species that are adapted to temperate or cool zones. Taxonomically, the genus can be classified into broad-leafed types (sections Bovinae and Scariosae) and fine-leafed types (section Festuca [Ovinae]; Sleper and Buckner 1995). The species within the genus vary greatly in morphology, cytology and growth habbit. Tall fescue (F. arundinacea Schreb.), the most important forage species worldwide of the genus Festuca (Sleper and West 1996), belongs to the Bovinae section of Festuca and is classified as belonging to the tribe Festuceae (Buckner et al. 1979). It was not until 1950 that tall fescue was given the name Festuca arundinacea. Prior to that date, a taxonomic debate ensued over whether meadow fescue and tall fescue constituted separate species or one broad species (Barnes 1990). Tall fescue is a wind-pollinated, highly self-infertile perennial, cool-season grass. It is an allohexaploid with 2n = 6x = 42 chromosomes. The genetic formula is considered to be PPG1G1G2G2. The P genome is from the diploid (2n = 2x = 14) meadow fescue (F. pratensis Huds.); G1 and G2 genomes are from the tetraploid (2n = 4x = 28) F. arundinacea var. glaucescens Boiss. (Sleper and West 1996). Tall fescue is indigenous to Europe, also naturally occurring on the Baltic coasts throughout the Caucasus, in western Siberia and extending into China. Introductions have been made into North and South America, Australia, New Zealand, Japan and both South and East Asia (Barnes 1990). Tall fescue was introduced to North America in the early to mid-1800s and became the predominant cool-season pasture grass in the United States (Buckner et al. 1979; Barnes 1990). Although early performance tests during the late 1980s showed tall fescue to be taller, more drought- and cold-tolerant, to form denser stands, to be more competitive with weeds and to thrive on a wider range of soils than other Festuca species, tall fescue did not attain prominence until the release of the Kentucky-31 and Alta cultivars during the early 1940s by the Kentucky and Oregon Agricultural Experiment Stations, respectively (Buckner et al. 1979; Sleper and Buckner 1995). 1 Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway,

Ardmore, OK 73401, USA, e-mail: [email protected] 2 Plant Biotechnology Centre, Primary Industries Research Victoria and Molecular Plant Breeding

CRC, La Trobe University, Bundoora, Victoria 3086, Australia Biotechnology in Agriculture and Forestry, Vol. 61 Transgenic Crops VI (ed. by E.C. Pua and M.R. Davey) © Springer-Verlag Berlin Heidelberg 2007

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Since forage production is generally a low-cash-input system, the most economical way to deliver advanced technology to farmers and ranchers is through the genetic improvement of cultivars (Spangenberg et al. 2001; Wang et al. 2001a). The high degree of self-incompatibility of tall fescue makes breeding management difficult and selection schemes complex, resulting in slow breeding progress, especially for traits with low heritability (Barnes 1990; Stadelmann et al. 1999; Spangenberg and Wang 2004). Biotechnological approaches have the potential to complement or accelerate conventional breeding by extending the range of sources from which genetic variation may be accessed, thus offering new opportunities for molecular breeding (Spangenberg et al. 1998; Wang et al. 2001a; Basu et al. 2004). There has been considerable interest in modifying tall fescue by genetic transformation in the past decade, with the aim of improving its agronomic traits. Significant progress has been made in developing transformation methods and in generating transgenic plants with improved agronomic traits.

2 Economic Importance Approximately 16,000 ha of tall fescue were grown in 1940 in the United States, since when it has become the predominant cool-season perennial grass species and is now grown on an estimated 14 × 106 ha. Some of this acreage resulted from natural seeding, but much is the result of introduced seedlings (Buckner et al. 1979; Barnes 1990). Its widespread use is due to its adaptation to a range of soil conditions, tolerance of continuous grazing, high yields of forage and seed, persistence, long grazing season, compatibility with varied management practices and low incidence of pest problems (Sleper and West 1996; Hanson 1979). Tall fescue forms the forage basis for beef cow-calf production in the eastcentral and southeast United States, supporting over 8.5× 106 beef cows, and is used for sheep and horse production (Sleper and West 1996). It is grown from northern Florida to southern Canada and is extensively used as a grass component of mixtures in irrigated pasture in the western intermountain regions of the United States (Sleper and Buckner 1995). Tall fescue is particularly adapted to the area referred to as the transition zone, defined as the area between the successful zone of cultivation of cool- and warm-season grasses (Sleper and West 1996). The plant is also cultivated in most parts of Europe. Its northernmost range occurs in Scandinavia and it proceeds south to northern Italy, with its easternmost range reaching into parts of Turkey. Other countries where tall fescue has been cultivated include Japan, southern Canada, Australia, New Zealand, Mexico, Columbia, Argentina and parts of Africa (Sleper and West 1996). For conservation purposes, the adaptability of tall fescue to a wide range of soils and climate, the coarse, deep root system and its ability to grow on low-

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lying sites on moist, heavy soils and withstand waterlogging make the species particularly valuable. Tall fescue provides excellent waterway protection and is ideal for cover in low wetlands and on steep, drought slopes. It has stabilized eroded and disturbed land areas, and helped restore fertility to such land. Millions of hectares that were gullied as a consequence of the plow have been healed and restored to lush, green productive pastures by the establishment of tall fescue (Buckner et al. 1979).

3 Current Research and Development 3.1 Genetic Transformation Methods Direct gene transfer to protoplasts was the first method used to generate transgenic tall fescue plants (Wang et al. 1992). Genetic transformation by direct gene transfer to protoplasts is based on the efficient uptake of plasmid DNA into protoplasts from the surrounding solution (Potrykus 1990; Saul and Potrykus 1990). The generation of transgenic forage- and turf-type tall fescue was reported in several laboratories in the 1990s (Ha et al. 1992; Wang et al. 1992; Dalton et al. 1995; Kuai et al. 1999). However, because protoplast culture and transformation is a difficult system with which to work, the system was later replaced by independent techniques, namely biolistic and Agrobacterium-mediated transformation. Nevertheless, direct gene transfer to protoplasts remains a valuable tool for transient gene expression studies. Biolistics, or microprojectile bombardment, employs high-velocity metal particles to deliver DNA into living cells for stable transformation (Sanford 1988; Christou 1992). Embryogenic cultures or immature embryos are ideal targets for biolistic transformation (Spangenberg and Wang 1998). Gene delivery to plant cells and tissues by microprojectiles is now a routine method for the production of transgenic tall fescue plants (Spangenberg et al. 1995a; Cho et al. 2000; Wang et al. 2001b, 2003a; Chen et al. 2003b, 2004). Additionally, the stable meiotic transmission of transgenes following Mendelian rules has been demonstrated for transgenic tall fescue plants obtained from biolistic transformation (Wang et al. 2003a). Although single-copy integration of transgenes has been demonstrated in transgenic grasses obtained by microprojectile bombardment, the majority of the transformants had complex transgene integration patterns (Spangenberg et al. 1995a, b; Ye et al. 1997; Dalton et al. 1999; Richards et al. 2001; Wang et al. 2001b, 2003a). Biolistic transformation is a reproducible procedure that can be easily adapted from one laboratory to another. However, the main disadvantage of this methodology is that it leads to the frequent recovery of transformation events showing integration of multiple, rearranged transgene copies.

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Agrobacterium-mediated transformation has the advantage of integrating a defined DNA segment into the plant genome; and it generally results in a lower copy number, fewer rearrangements and a more stable gene expression over generations than free DNA delivery methods (Dai et al. 2001; Hu et al. 2003). Spurred by successes in cereal crops (Hiei et al. 1994, 1997; Aldemita and Hodges 1996; Ishida et al. 1996; Cheng et al. 1997, 2004; Tingay et al. 1997; Zhang et al. 1997; Zhao et al. 2000; Frame et al. 2002; Hu et al. 2003), the potential has been explored of using Agrobacterium tumefaciens as a vector for producing transgenic plants of tall fescue. Using a tissue culture-responsive line and a super binary vector, Agrobacterium-mediated transformation of tall fescue was first reported by Bettany et al. (2003), although only two transgenic plants were generated; and they failed to produce seeds. Recent reports showed that larger numbers of transgenic tall fescue plants can be obtained by Agrobacterium-mediated transformation (Lee et al. 2004; Dong and Qu 2005; Wang and Ge 2005). An efficient procedure for Agrobacterium-mediated transformation of tall fescue (Wang and Ge 2005) is described here and illustrated in Fig. 1. Embryogenic calli induced from seeds/caryopsis of tall fescue cvs. Jesup and Kentucky31 (Fig. 1A), were fragmented into small pieces and used for A. tumefaciensmediated transformation. Agrobacterium strains LBA4404 and EHA105, harboring pCAMBIA vectors or the super binary vector pTOK233, were used to infect the embryogenic callus pieces. A chimeric hygromycin phosphotransferase gene (hph) was employed as selectable marker and hygromycin (Hm) used as selection agent. Hm-resistant calli were obtained after 4–6 weeks of selection (Fig. 1B). Shoots and rooted plants were obtained after transferring the Hm-resistant calli onto regeneration medium (Fig. 1C–E). Plants in soil could be obtained 4–5 months after Agrobacterium-mediated transformation (Fig. 1G, H). The number of Hm-resistant calli obtained per dish each containing 20 callus pieces subjected to Agrobacterium-transformation was in the range 2.0–5.8, while the number of transgenic plants recovered per dish was in the range 0.4–1.7. When transformation efficiency was calculated based on the number of transgenic plants recovered and the number of original intact calli used, the transformation frequency was in the range 1.9–8.7%. The use of pCAMBIA vectors lead to equivalent results as those obtained with the super binary vector pTOK233. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis. Transgene expression was confirmed by Northern hybridization as well as histochemical β-glucuronidase (GUS) enzyme assays and green fluorescent protein (GFP) fluorescence detection for transferred gusA and gfp reporter genes. Fertile transgenic plants were obtained, offspring was recovered following vernalization and crossings were undertaken under glasshouse conditions. Analysis of progenies obtained from transgenic tall fescue plants confirmed Mendelian inheritance of transgenes (Wang and Ge 2005).

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Fig. 1. Transgenic tall fescue (Festuca arundinacea) plants obtained after Agrobacterium tumefaciens-mediated transformation of embryogenic calli. A Embryogenic callus induced from seeds/caryopses. B Hygromycin-resistant calli obtained 5 weeks after Agrobacterium-mediated transformation and selection of infected callus pieces on medium containing hygromycin. C, D Shoot differentiation of hygromycin resistant calli 4 weeks after transfer onto regeneration medium. E Rooted transgenic plants 4 weeks after transfer the differentiated shoots to rooting medium. F GUS staining of leaves of transgenic plants. G, H Glasshouse-grown transgenic plants after Agrobacterium-mediated transformation

3.2 Field Evaluation, Pollen Viability and Gene Flow Agronomic performance and pollen viability of transgenic tall fescue have been studied under field conditions (Wang et al. 2003b, 2004a, b). In order to compare the agronomic performance of transgenic and non-transgenic tall fescue plants, T0 primary transgenic plants from two genotypes, the corresponding untransformed tissue culture-derived R0 regenerants from the same

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genotypes and control seed-derived plants were transferred to the field and evaluated for 2 years. Progenies of these three classes of plants were obtained and evaluated, together with seed-derived plants in a second field experiment (Wang et al. 2003b). The experimental design was a randomized complete block with three replications. The agronomic characteristics evaluated were heading and anthesis dates, height, growth habit, number of reproductive tillers, seed yield and biomass. Factor analysis showed that plants from the same genotype were more uniform than plants from seeds. This is not surprising, since seedderived plants are expected to differ at a number of loci. Genotype differences were observed in this study, with both transgenic and regenerated plants from genotype 1 performing better than plants from genotype 2. The addition of a selectable marker gene in the plant genome seemed to have little effect on the agronomic performance of the regenerated plants, since performance of the transgenic plants was comparable to the corresponding regenerants from the same genotype. Progenies of the transgenic plants performed similarly to progenies of the R0 regenerants (Wang et al. 2003b). The agronomic performance of the T0 plants and R0 regenerants was generally inferior to that of the seed-derived plants, with T0 plants having fewer tillers and lower seed yield. However, no major differences between the progenies of transgenic plants and the progenies of seed-derived plants were found for the agronomic traits evaluated (Wang et al. 2003b). In a separate study, no significant difference in pollen viability was detected between the progeny of transgenic plants and that of seed-derived plants (Wang et al. 2004a). The results obtained indicate that progenies from primary transgenic T0 plants can be expected to show normal pollen viability and agronomic performance. No indication of weediness was observed in the transgenic tall fescue plants. The field study provided evidence that out-crossing grass plants generated through transgenic approaches represent suitable material that could be incorporated into forage breeding programs. Pollen is an important vector of gene flow in plants, particularly for outcrossing species like tall fescue. To effectively assess in vitro pollen viability and longevity in tall fescue, an optimized germination medium (0.8 M sucrose, 1.28 mM boric acid, 1.27 mM calcium nitrate) was developed (Wang et al. 2004a). Treatment with relatively high temperatures (36 ◦ C, 40 ◦ C) and high doses of UV-B irradiation (900−1,500 μW/cm2 ) reduced pollen viability, while relative humidity did not significantly influence pollen viability. Progenies of primary transgenic plants (T1 , T2 ) and regenerants (R1 , R2 ) showed similar pollen viability when compared with that of seed-derived plants, although T0 and R0 plants had various levels of pollen viability in individual plants (Wang et al. 2004a). Hand pollination of T0 and R0 plants revealed that seed set could not be obtained when pollen viability was less than 5%. Pollen from transgenic progenies and non-transgenic control plants could survive up to 22 h under controlled conditions in a growth chamber. However, under sunny conditions, the viability of transgenic and non-transgenic pollen declined to 5% in 30 min,

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Fig. 2. Longevity of pollen of transgenic and non-transgenic tall fescue under ambient atmospheric conditions. Pollen was collected from seed-derived plants and transgenic progenies of tall fescue, and germination was evaluated under sunny (Sunny/nontransgenic, Sunny/transgenic) and cloudy (Cloudy/nontransgenic, Cloudy/transgenic) conditions

with a complete loss of viability in 90 min (Fig. 2). Under cloudy atmospheric conditions, some pollen remained viable up to 240 min, with about 5% viability after 150 min (Fig. 2). Since wind-pollinated grass species have a high potential to pass their genes to adjacent plants, information regarding gene flow is extremely important for the deregulation and release of transgenic cultivars. Evaluation of the biosafety of transgenic out-crossing grasses will likely focus on their environmental or ecological impacts. Transgenic plants provide unique material for studying pollen dispersal and gene transfer into related species. The detection of transgenes by molecular techniques offers distinct advantages over the use of morphological or biochemical markers by providing clear-cut information on the presence or absence of transgenes without subjective judgment or being influenced by the levels of gene expression. A small-scale experiment on pollen dispersal was carried out using transgenic tall fescue in a central plot, surrounded by exclosures containing recipient plants up to a distance of 200 m from the central source plants in eight directions (Wang et al. 2004c). Seeds were collected from the recipient plants and more than 21,000 germinated seedlings were analyzed by PCR. Transgenes were detected in recipient plants at up to 150 m from the central transgenic plot. The highest transgene frequencies, 5% at 50 m, 4.12% at 100 m and 0.96% at 150 m, were observed in the prevailing wind direction. A supplementary experiment demonstrated that transgene flow can be controlled by placing transgenic plantings downwind and long distances from non-transgenic seed increases, thus allowing tall fescue

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breeding and transgene development programs to be conducted concurrently at the same research station (Wang et al. 2004c). Obviously, larger-scale gene flow experiments are still required to assess how far grass pollen can disperse and still remain viable. Tall fescue is closely related to ryegrasses, as intergeneric crosses have been reported (Terrell 1979; Sleper and West 1996). Gene transfer by crossing among these taxa may occur and chromosome structural differentiation among them is slight (Sleper and West 1996). Thus, gene flow studies will also determine the probability of transgene escape by crossing with related grass species under natural conditions. Gene flow is a natural process that has occurred in the past and will continue in the future. Therefore, a primary focus of research on risk assessment for the release of out-crossing grasses should be placed on the consequences of transgene flow.

4 Practical Applications of Transgenic Plants 4.1 Improvement of Forage Quality by Genetic Modification of Lignin Biosynthesis In vitro dry matter digestibility is one of the most important characteristics of forages. Lignification of grass cell walls during plant development has been identified as a major factor limiting forage digestibility and, concomitantly, animal productivity. Feeding and grazing studies have shown that small changes in forage digestibility can have a significant impact on animal performance (Vogel and Sleper 1994; Casler and Vogel 1999). Improvement in forage grass cell wall digestibility is now an important goal of many plant–ruminant animal research programs. Breeding by phenotypic recurrent selection resulted in the release of grass cultivars with improved dry matter digestibility. However, continued selection for increased dry matter digestibility might affect plant fitness (Casler et al. 2002). Gene technology allows the targeted modification of genes encoding specific enzymes in the lignin biosynthetic pathway, thus offering a complementary and effective approach for improving forage digestibility of grasses. Lignin in forage grasses comprises guaiacyl (G) units derived from coniferyl alcohol, syringyl (S) units derived from sinapyl alcohol and p-hydroxyphenyl (H) units derived from p-coumaryl alcohol. In addition to lignin content (or concentration), the composition of lignin is an important factor that influences the cell wall degradability of forages (Jung and Vogel 1986; Buxton and Russell 1988; Vogel and Jung 2001). Most of the work reported on transgenic modification of lignin biosynthesis has been mainly on dicotyledonous species, such as tobacco, Arabidopsis, alfalfa and poplar (Dwivedi et al. 1994; Halpin et al. 1994; Ni et al. 1994; Atanassova et al. 1995; Baucher et al. 1996, 1998, 1999; Bernard-Vailhe et al. 1996, 1998; Sewalt et al. 1997; Lee et al. 1998; Piquemal et al. 1998; Boudet 2000; Chabannes et al. 2001; Dixon et al. 2001; Guo et al.

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Fig. 3. Maule staining of cross-sections of transgenic tall fescue plants with modified expression of COMT. A Control plant. B, C Transgenic plants showing reduced lignin

2001a, b; Anterola and Lewis 2002; Humphreys and Chapple 2002; Chen et al. 2003a; Li et al. 2003). In monocotyledonous species, there have been only few reports on transgenic modification of lignin biosynthesis; specifically, downregulation of cinnamyl alcohol dehydrogenase (CAD) in tall fescue (Chen et al. 2003b) and down-regulation of caffeic acid O-methyltransferase (COMT) in maize (Piquemal et al. 2002) and tall fescue (Chen et al. 2004). Lignin biosynthesis is comprised of a set of coordinated and regulated metabolic events involving many enzymes. CAD and COMT have been shown to play important roles in lignin biosynthesis (Halpin et al. 1998; Vailhe et al. 1998; Guo et al. 2001a). CAD catalyzes the last step in the biosynthesis of lignin precursors, which is the reduction of cinnamaldehydes to cinnamyl alcohols (Baucher et al. 1998). COMT is a multi-specific enzyme that not only methylates caffeic acid to ferulic acid and 5-hydroxyferulic acid to sinapic acid, but is also involved in the 3-O-methylation of monolignol precursors at the aldehyde or alcohol levels (Dixon et al. 2001; Humphreys and Chapple 2002). CAD and COMT cDNA sequences were cloned from tall fescue (Chen et al. 2002, 2003b, 2004). Transgenic tall fescue plants carrying either chimeric sense or antisense CAD and COMT transgenes were produced by microprojectile bombardment of single genotype-derived embryogenic suspension cells. Severely reduced mRNA levels and significantly decreased enzymatic activities were found in selected transformation events. These CAD and COMT down-regulated tall fescue plants had a reduced total lignin content and altered lignin composition. Histochemical characterization by Maule staining of the cross-sections further confirmed the alteration of lignin in the transgenics (Fig. 3). No significant changes in cellulose, hemicellulose, neutral sugar composition, p-coumaric acid and ferulic acid levels were observed in the transgenic plants. In vitro dry matter digestibility increased by 7.2–10.5% in these transgenic tall fescue plants, thus providing novel germplasm for cultivar development (Chen et al. 2003b, 2004).

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4.2 Expression and Accumulation of Rumen-Stable Sulfur-Rich Proteins Approaches to improve the nutritive value in forage-type fescues based on gene technology might contribute significantly to generate materials designed for a better supply of limiting essential amino acids for ruminant nutrition. Since wool growth in sheep is limited by the supply of sulfur-containing amino acids (S-amino acids), such as methionine and cysteine (Reis and Schinckel 1963; Ørskov and Chen 1990), it is expected that the ingestion of forage containing relatively rumen-resistant, S-amino acid-rich proteins will lead to increased wool growth (Rogers 1990). This is based on the fact that direct infusion into the abomasum (by-passing the rumen) of cysteine alone or its precursor, methionine, leads to up to 100% increase in wool growth (Reis 1989). Transgenic tall fescue plants that express foreign genes encoding a rumenstable protein rich in sulfur-containing amino acids were generated with the aim of improving protein quality of forage grass for ruminant nutrition (Wang et al. 2001b). Since sunflower seed albumin 8 (SFA8) contains high concentrations of methionine and cysteine and is resistant to rumen degradation in vitro (Kortt et al. 1991; McNabb et al. 1994), chimeric sfa8 genes including a sequence coding for the endoplasmic reticulum retention signal KDEL were constructed under the control of constitutive (cauliflower mosaic virus 35S) and light-regulated (wheat cab) promoters. These chimeric gene constructs were introduced into the tall fescue genome by microprojectile bombardment of embryogenic suspension cells. The transgenes were stably integrated in the plant genome and produced the expected transcript. The corresponding sulfurrich SFA8 protein accumulated at concentrations of up to 0.2% of total soluble protein in the leaves of individual transgenic plants (Wang et al. 2001b). In order to achieve nutritional levels useful enough to make a significant impact on ruminant diets, it would be necessary to achieve an expression level of the SFA8 to about 2–4% of total leaf protein. Strategies for increasing the accumulation levels of foreign proteins in the leaves of forage plants may include the use of stronger promoters or the development of a chloroplast transformation system to produce transplastomic lines. 4.3 Abiotic Stress Tolerance Abiotic stresses, notably drought and low temperatures, often limit the growth and productivity of perennial grasses. It has been shown that drought or cold stresses induce the expression of a number of genes. The products of these genes can be classified into two groups: those that directly counteract the detrimental conditions (such as enzymes involved in the biosynthesis of osmoprotectants) and those that regulate gene expression and signal transduction in the stress response (such as transcription factors and protein kinases). Although the effect of each individual gene in the first group is rather small, the simultaneous activation of a set of these genes by the second group can confer much greater stress tolerance.

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The transfer of a single gene, such as the dehydration-responsive element binding protein (DREB) or the C-repeat binding factor (CBF) gene, into Arabidopsis activated many stress tolerance genes under normal growth conditions and resulted in improved tolerance to drought, salt and freezing (Jaglo-Ottosen et al. 1998; Liu et al. 1998; Kasuga et al. 1999; Haake et al. 2002). Over-expression of ERF transcription factor genes, WXP1 and WXP2, activated cuticular wax production and enhanced drought tolerance in alfalfa and Arabidopsis (Zhang et al. 2005, 2007). Similar strategies could be used for improving abiotic stress tolerance in tall fescue. The expression of certain genes may have an impact on stress tolerance via an indirect effect. Isopentenyl transferase (IPT) is involved in cytokinin biosynthesis. The ipt gene from A. tumefaciens has been transferred into plants to inhibit apical dominance, stimulate axillary bud development, delay senescence and increase chlorophyll levels and secondary metabolite production (Hu et al. 2005). In turf-type tall fescue, expression of the Agrobacterium ipt gene resulted in enhanced tillering ability, increased levels of chlorophyll a and b and improved cold tolerance. The transgenic tall fescue plants were more vigorous and stayed green longer under lower temperatures conditions (Hu et al. 2005).

5 Conclusions and Future Challenges Forage and turf grasses form the foundation for grassland agriculture and play important roles in environmental protection and outdoor recreation. Although many improved forage- and turf-type fescue varieties have been developed through conventional breeding efforts, progress on improvement concerning particular traits has slowed. Gene technology and the production of transgenic plants offer the opportunity to generate unique genetic variation, when the required variation is either absent or has very low heritability. Forage quality improvement, tolerance to abiotic stresses, disease and pest resistance, manipulation of growth and development and the production of foreign proteins of industrial relevance represent current targets for the molecular breeding of tall fescue. In recent years, the first transgenic tall fescue with simple “engineered” traits have been produced and evaluated. The challenge now is how to apply the technology to generate novel genetic variability that satisfies regulatory requirements and then efficiently incorporate the new germplasm into tall fescue improvement programs for cultivar development. Risk assessment of defined “engineered” traits is extremely important for the deregulation and the future release of transgenic tall fescue cultivars.

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Wang Z-Y, Ye XD, Nagel J, Potrykus I, Spangenberg G (2001b) Expression of a sulphur-rich sunflower albumin gene in transgenic tall fescue (Festuca arundinacea Schreb.) plants. Plant Cell Rep 20:213–219 Wang Z-Y, Bell J, Ge YX, Lehmann D (2003a) Inheritance of transgenes in transgenic tall fescue (Festuca arundinacea Schreb.). In Vitro Cell Dev Biol Plant 39:277–282 Wang Z-Y, Scott M, Bell J, Hopkins A, Lehmann D (2003b) Field performance of transgenic tall fescue (Festuca arundinacea Schreb.) plants and their progenies. Theor Appl Genet 107:406– 412 Wang Z-Y, Ge YX, Scott M, Spangenberg G (2004a) Viability and longevity of pollen from transgenic and non-transgenic tall fescue (Festuca arundinacea) (Poaceae) plants. Am J Bot 91:523–530 Wang Z-Y, Hopkins A, Lawrence R, Bell J, Scott M (2004b) Field evaluation and risk assessment of transgenic tall fescue (Festuca arundinacea) plants. In: Hopkins A, Wang ZY, Mian R, Sledge M, Barker RE (eds) Molecular breeding of forage and turf. Kluwer Academic, Dordrecht, pp 367–379 Wang Z-Y, Lawrence R, Hopkins A, Bell J, Scott M (2004c) Pollen-mediated transgene flow in the wind-pollinated grass species tall fescue (Festuca arundinacea Schreb.). Mol Breed 14:47–60 Ye X, Wang Z-Y, Wu X, Potrykus I, Spangenberg G (1997) Transgenic Italian ryegrass (Lolium multiflorum) plants from microprojectile bombardment of embryogenic suspension cells. Plant Cell Rep 16:379–384 Zhang J, Xu RJ, Elliott MC, Chen DF (1997) Agrobacterium-mediated transformation of elite Indica and Japonica rice cultivars. Mol Biotechnol 8:223–231 Zhang J-Y, Broeckling CD, Blancaflor EB, Sledge M, Sumner LW, Wang Z-Y (2005) Overexpression of WXP1, a putative Medicago truncatula AP2 domain-containing transcription factor gene, increases cuticular wax accumulation and enhances drought tolerance in transgenic alfalfa (Medicago sativa). Plant J 42:689–707 Zhang J-Y, Broeckling CD, Sumner LW, Wang Z-Y (2007) Heterologous expression of two Medicago truncatula putative ERF transcription factor genes, WXP1 and WXP2, in Arabidopsis led to increased leaf wax accumulation and improved drought tolerance, but differential response in freezing tolerance. Plant Mol Biol 64:265-278 Zhao ZY, Cai TS, Tagliani L, Miller M, Wang N, Pang H, Rudert M, Schroeder S, Hondred D, Seltzer J, Pierce D (2000) Agrobacterium-mediated sorghum transformation. Plant Mol Biol 44:789–798

IV.4 Ryegrasses Y. Ran, C. Ramage, S. Felitti, M. Emmerling, J. Chalmers, N. Cummings , N. Petrovska, A. Mouradov, and G. Spangenberg1

1 Introduction Ryegrasses (Lolium spp) are among the most important pasture, forage and turf grasses in the world. Together with the closely related fescues (Festuca spp), ryegrasses are one of the major temperate grassland species of agriculturally cultivated land and a key forage species in countries with intensive livestock production systems (Forster and Spangenberg 1999). Ryegrasses are widely distributed throughout temperate regions on all continents and extensively used as a forage crop throughout mainland Europe, the United Kingdom, New Zealand, Australia, USA and Japan. Since the 1950s breeding efforts through selection, hybridization and artificial tetraploid induction have contributed to the genetic improvement of ryegrass through traits such as above-ground biomass, yield and disease resistance (Hunt and Easton 1989; Cunningham et al. 1994). The demand for high-quality ryegrass varieties for grassland agriculture continues to grow. However, genetic improvement, based exclusively on conventional breeding approaches, is becoming increasingly difficult due to the complicated genetic control of desirable traits coupled with self-incompatibility. This makes the progress of varietal development slow and demanding. The significant potential for ryegrass improvement by combining pest and disease resistance, drought and cold tolerance, increased digestibility, higher biomass production and improved nutrient content are beyond the scope of conventional breeding alone. Thus, a multi-disciplinary approach, exploiting recent advances in biotechnology, is required to allow the generation of novel variability as well as maximize efficient use of existing genetic resources.

2 Economic Importance Commercially, the most important ryegrasses are Italian or annual ryegrass (L. multiflorum Lam.) and perennial ryegrass (L. perenne L.). Italian ryegrass is indigenous to Italy where it has been grown under irrigation as a cut forage crop since the thirteenth century (Beddows 1953). Italian ryegrass is biennial (var. italicum Beck) or annual (var. westerwoldicum Mansh), with forage attributes such as rapid establishment from seed, high biomass production 1 Primary

Industries Research Victoria, Victorian Agribiosciences Centre, La Trobe University, Bundoora, Victoria 3086, Australia, e-mail: [email protected]

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during the seeding year, rapid recovery following defoliation, high palatability and nutrition (Jung et al. 1996). Italian ryegrass is an important short duration grass, highly valued for forage and livestock systems and used in many environments when a fast cover or quick feed is required. There are nearly 3 × 106 acres (approx. 1.2 × 106 ha) of Italian ryegrass in the United States, with about 90% used for winter pasture in the south. Italian ryegrass is also grown for silage and hay on poorly drained waterlogged soil where small grains are poorly adapted. In the Northeast and Pacific Northwest, Italian ryegrass is sown between maize and other row crops to absorb excess nitrogen, reduce erosion after crop harvest and provide a source of winter feed. In the northern United States and Canada, it is also grown as a summer annual, typically as a quick-cover lawn grass. Italian ryegrass is also used as fish feed in parts of China in the aquaculture of grasseating species of carp (Tilapia spp) and forms the basis of many wildlife feeds (Hannaway et al. 1999). Perennial ryegrass is indigenous to parts of Europe, Asia and North Africa, with the first records of cultivation from England about 1677 (Terrell 1968). Perennial ryegrass has a number of attributes that led to the species becoming a dominant forage crop. These include high palatability, increased persistence with high density tillering, resistance to treading and a good response to increased nitrogen input (Jung et al. 1996). Perennial ryegrass has adapted well to variation in winter severity from the harsh continental climates of North America and central Europe to the mild maritime climates of the United Kingdom and New Zealand (Easton 1989; Jung et al. 1996). Perennial ryegrass is highly valued for forage and livestock systems and grown extensively for pasture and silage. High digestibility, high yield potential and fast establishment are desirable attributes for dairy and sheep forage systems. It is also suitable for reduced-tillage rotation and is used on heavy and waterlogged soil. As a result, it is often the preferred forage in temperate regions of the world such as New Zealand and southern Australia. Perennial ryegrass is grown for hay in the Pacific Northwest in the United States, but typically provides one hay cutting per annum and little regrowth (Hannaway et al. 1999). In New Zealand, over 7 × 106 ha of perennial ryegrass are grown, providing high quality forage to support over 60 × 106 sheep and cattle (Siegel et al. 1985). In Australia, it is estimated that more than 6 × 106 ha of perennial ryegrass are cultivated. It is well adapted to regions with over 550 mm annual rainfall and a growing season of more than 7 months (Cunningham et al. 1994). In the state of Victoria, perennial ryegrass accounts for over 4 × 106 ha of agricultural land, supporting the wool, meat and dairy industries valued at over AUD $ 10 × 109 year−1 in 2003/2004 (ABARE 2004).

3 Current Research and Development Enabling methodologies for the application of molecular technologies to the improvement of ryegrasses have been developed and reviewed (Spangenberg

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et al. 2000; Forster et al. 2005). They include the establishment of efficient and robust plant regeneration systems from cells competent for genetic transformation, the production of transgenic forage plants mainly by biolistic and Agrobacterium-mediated transformation methods, the establishment of highly informative co-dominant molecular marker systems and their use in the development of framework genetic maps, the development of genomics resources for gene discovery, and genomics approaches to understanding the symbiosis between ryegrasses and their fungal endophytes. 3.1 In Vitro Plant Regeneration Systems The practical application of transgenesis for ryegrass improvement depends primarily on the development of reliable and reproducible protocols for the regeneration of a large number of fertile plants from cell cultures. Efficient plant regeneration systems are well established for ryegrasses (Lolium spp) and fescues (Festuca spp) based on embryogenic cell cultures (e.g., Ha 2000; Spangenberg et al. 2000). Embryogenic callus can be induced from cultured meristematic tissues such as shoot tips (Dale et al. 1981), immature embryos (Dale 1980), mature caryopses (Torello and Symington 1984), immature inflorescences (Dale and Dalton 1983), and anthers for the production of homozygous diploids (Olesen et al. 1988). More recently, regeneration and transient gene expression have been reported from embryogenic callus of L. perenne and L. multiflorum induced from small leaf-base sections excised from in vitro grown plantlets (Newell and Gray 2005). The majority of ryegrasses are outcrossing, require vernalization to flower and are often polyploid. The outcrossing nature of ryegrass is a major impediment to improvement through transgenesis. In particular, variation of embryogenic response due to genotype is particularly problematic because each seed-derived embryo represents a different genotype and each genotype may require optimization of culture conditions (Dale 1980; Dalton 1988; Van Heeswijck et al. 1994; Spangenberg et al. 2000; Bradley and Qu 2001; Takahashi et al. 2004; Salehi and Khosh-Khui 2005). Genotype screening for embryogenic callus response indicates that the frequency of embryogenic callus formation depends on the species, genotype, choice of explant, and development stage. For example, 5–8% of seeds from Italian ryegrass varieties and 2–5% from perennial ryegrass can be induced to form embryogenic callus (Wang et al. 1993). In contrast, the frequency of embryogenic callus formation from immature inflorescences of Italian ryegrass is generally much greater (4–17%). However, this is dependent on the developmental stage of the inflorescence (Dale et al. 1981). Darnel ryegrass (L. temulentum L.) recently emerged as a potential candidate ‘model’ system for genetic modification studies in forage and turf grasses (Dalton et al. 1999; Wang et al. 2005). L. temulentum is a self-fertile diploid grass with a short life cycle primarily studied for its flowering response to a single long day (Gocal et al. 2001). Large-scale screening was recently conducted

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of L. temulentum lines for efficient regeneration and transformation capacity (Wang et al. 2002). Crosses amongst the most responsive lines and subsequent anther culture resulted in the establishment of a few highly embryogenic doubled haploid cell suspension lines (Wang et al. 2005). Importantly, numerous fertile plants can be obtained without the requirement of vernalization (Wang et al. 2005). 3.2 Transgenesis There are many reports on the generation of transgenic plants in perennial ryegrass (e.g., Spangenberg et al. 1995; Dalton et al. 1998; Altpeter et al. 2000) and Italian ryegrass (e.g., Wang et al. 1997; Ye et al. 1997; Bettany et al. 2003). Gene transfer to ryegrasses has been achieved through a variety of transformation methods, including direct DNA transfer to protoplasts, surrogate transformation with transformed endophytes, biolistic and whiskers-mediated transformation of embryogenic and meristematic cells (Spangenberg et al. 1998, 2000). Currently, the most widely used method of transformation of ryegrasses is based on direct transfer of DNA by biolistics (Spangenberg and Wang 2004), with recent advances in Agrobacterium-mediated transformation of ryegrasses also being reported (Posselt et al. 1998; Bettany et al. 2003; Lee et al. 2004; Wu et al. 2005). Posselt et al. (1998) described transfer of the coat protein gene from ryegrass mosaic virus (RGMV) via a binary vector, also containing a chimeric β-glucuronidase (GUS) reporter gene (gusA) and a chimeric hygromycin phosphotransferase gene (hph). Cell suspension cultures from three ryegrasses (L. perenne, L. multiflorum, L. westerwoldicum) were inoculated with the super-virulent Agrobacterium strain EHA105. A total of 81 transgenic plants were recovered. However, unequivocal molecular data was not presented and meiotic stability of transgene transmission was not examined. Bettany et al. (2003) reported Agrobacterium-mediated transformation of L. multiflorum and F. arundinacea. Embryogenic suspension cells were cocultured with A. tumefaciens strain LBA4404 carrying the super-binary vector pTOK233. Colonies were grown under hygromycin selection, followed by plating of hygromycin-resistant colonies onto regeneration media. Six independent ryegrass transformation events were recovered and established in soil. All plants were hygromycin resistant, but only a few plants co-expressed GUS. More efficient methods for the Agrobacterium-mediated transformation of perennial ryegrass, including data on molecular analysis confirming the transgenic nature of the regenerated plants, were reported recently (Wu et al. 2005). 3.3 Molecular Marker Technology Both L. multiflorum (2n = 2x = 14) and L. perenne (2n = 2x = 14) are selfincompatible obligate outcrossing diploid species with relatively large genome

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sizes, estimated at 2.3 Gbp (Terrell 1968; Hutchinson et al. 1979). Species within the genus are inter-fertile, leading to short-rotation hybrid ryegrasses, and can also be crossed with Festuca species (Jung et al. 1996). Since the first report on the use of well defined allelic isozymes to identify ryegrass cultivars (Hayward and McAdam 1977), many molecular markers, including restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), and sequence tagged sites (STS) have been developed in ryegrasses (reviewed by Spangenberg et al. 1998; Forster et al. 2001, 2005). Molecular markers have been used to analyse genetic relationships between cultivars, phylogenetic relationships of related species (Jones et al. 2002a), and to establish genetic maps for ryegrasses (Jones et al. 2002b; Inoue et al. 2004; Warnke et al. 2004). The outcrossing nature of ryegrasses leads to individual genotypes that are heterozygous at a high proportion of genetic loci. From a breeding perspective, this means that pair-crosses between individuals may involve the complex segregation of up to four different alleles at a single locus. Multi-allelic codominant genetic markers are desirable in order to monitor the inheritance of multiple gene alleles (Forster et al. 2005). To this end, SSRs provide a reliable high-throughput method for large-scale characterization of perennial ryegrass germplasm collections, with a high potential value for populationbased marker trait linkage detection (Forster et al. 2005). A set of ca. 400 unique SSR markers has been developed for L. perenne and used in concert with RFLP and AFLP marker systems for reference genetic map construction (Jones et al. 2002a). Detection of quantitative and qualitative regions of genetic effect for agronomic traits allow the improvement of yield, quality and adaptation in perennial ryegrass breeding. Progress in QTL analysis and trait dissection in ryegrass was reviewed recently (Yamada and Forster 2005). A single major gene locus conferring resistance to the crown rust pathogen was detected on linkage group 2 (LG2) using an SSR-based genetic map (Dumsday et al. 2003). In contrast, five regions of genetic effect were detected for vernalization response using quantitative trait locus (QTL) analysis, measured as days to heading (Jensen et al. 2005). Cogan et al. (2005) used a molecular marker-based reference map of L. perenne to assess the genetic control of herbage quality variation. A framework marker set based on RFLP, AFLP and genomic DNA-derived SSRs was enhanced with RFLP loci corresponding to genes for key enzymes involved in lignin biosynthesis and fructan metabolism. Quality traits such as crude protein content, estimated in vivo dry matter digestibility, neutral detergent fibre content, estimated metabolizable energy, and water-soluble carbohydrate content, were measured by near infrared reflectance spectroscopy analysis of herbage harvests. A total of five QTLs for three different traits on linkage group 7 (LG7) were identified, which were coincident with a cluster of lignin biosynthesis genes. The identification of coincidence between QTLs and functionally associated genetic markers is critical for the implementation of molecular markers in ryegrass improvement

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programmes and for linkage disequilibrium studies. These strategies will form the basis of future marker-based molecular breeding in perennial ryegrass (Spangenberg et al. 2005a; Smith et al. 2005). Molecular genetic approaches to ryegrass improvement have been strengthened by a high-throughput genomics approach to establish an expressed sequence tag (EST) collection and the generation of a 15K unigene microarray (Sawbridge et al. 2003). A total of 29 cDNA libraries of L. perenne were generated, representing a range of plant organs and developmental stages. Single-pass DNA sequencing of randomly selected clones was used to establish a genomic resource of over 44,000 ESTs. A set of 14,767 genes was identified representing an estimated one-third of all expressed sequences in ryegrass. A corresponding web-based resource for Lolium EST analysis was established (Spangenberg et al. 2005b). Using this EST resource, selected perennial ryegrass cDNAs were mapped as RFLP loci (Faville et al. 2004). Clones were selected on the basis of functional annotation by sequence database searches and were classified in terms of core physiological and biochemical processes. In parallel, in silico perennial ryegrass EST analysis identified 1,175 EST-SSR loci and 9,596 single-nucleotide polymorphism (SNP) loci (Spangenberg et al. 2005b). Polymorphisms associated with EST-SSRs coupled with EST-RFLP loci were used to construct a reference genetic map of perennial ryegrass (Faville et al. 2004). Because an ideal marker system is one that is reasonably polymorphic but is in very close association or linkage disequilibrium (LD) with a target trait, these EST-derived markers greatly assist in the development of ‘perfect markers’ and the direct selection of ryegrass genotypes with superior allele content (Forster et al. 2004; Spangenberg et al. 2005a). The perennial ryegrass EST gene set is also in use for a number of functional genomic screens, including novel ryegrass gene discovery, transgenic modification and genome-wide transcriptome analysis (Spangenberg et al. 2005a). 3.4 Functional Genomics and Biotechnology of Ryegrass Endophytes Neotyphodium lolii, N. coenophialum and Epichloë festucae are common symbiotic fungal endophytes of perennial ryegrass (L. perenne), tall fescue (F. arundinacea) and red fescue (F. rubra), respectively (Christensen et al. 1993). Epichloë taxa are ascomycete fungi (family Clavicipitaceae) that are ecologically obligate symbionts of grasses (Schardl et al. 1997). They comprise both the sexual Epichloë species and their asexual Neotyphodium derivatives (Glenn et al. 1996; Schardl 1996). All establish generally asymptomatic associations with their host during the vegetative phase of growth, but the sexual species are capable of forming a stroma around the developing inflorescence that partially or completely blocks emergence of the floral meristem. Relative mutualism or antagonism of an Epichloë–grass symbiosis is largely related to the path of symbiont transmission (Schardl et al. 1997; Muller and Krauss 2005). Many of these fungi can propagate clonally in the floral

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meristems and consequently in the seed progeny of infected mother plants (vertical or matrilinear transmission). Alternatively, genotypes can transmit horizontally via sexual spores in a life cycle that also requires a third symbiont (the fly Phorbia phrenione) to mediate fungal mating (Schardl et al. 1997). The benefits of the symbiosis for the host are increased seedling vigour and persistence as well as drought tolerance in marginal environments (Hill et al. 1990; Elbersen and West 1996; Malinowski and Belesky 1999). It also provides protection against some insect pests and nematodes (Prestidge and Gallagher 1988; Breen 1993; Elmi et al. 2000). Specific metabolites produced by the endophyte, such as peramine and loline alkaloids, provide protection from insect pests (Rowan and Gaynor 1986; Wilkinson et al. 2000). However, other metabolites such as lolitrem B and ergovaline are toxic to grazing animals, causing conditions known as ryegrass staggers and fescue toxicosis, respectively (Gallagher et al. 1984; Yates et al. 1985). The most thoroughly studied compounds are alkaloids, including ergopeptine alkaloids, indole-isoprenoid lolitrems, pyrrolizidine alkaloids and pyrrolopyrazine alkaloids (Bush et al. 1997; Scott 2001). In contrast to the information on alkaloids and animal toxicosis, the beneficial physiological aspects of the endophyte/grass interactions have not been well characterized in any system. Little is known regarding the factors important in host colonization or nutrient exchange between plant and fungus. The physiological mechanisms are unknown which lead to increased plant vigour and enhanced tolerance to abiotic stresses unrelated to the reduction in pest damage to endophyte-infected grasses. Significant progress has been made in recent years in the molecular cloning and genetic analysis of Epichloë/Neotyphodium endophyte genes involved in the biosynthesis of lolines (Spiering et al. 2002, 2005; Blankenship et al. 2005), ergot alkaloids (Panaccione et al. 2001) and lolitrems (Scott 2001, 2004). The isolation and characterization of genes involved in the grass/endophyte interaction would be critical for an effective manipulation of the mutualistic associations between grasses of the Festuca–Lolium complex and Epichloë/Neotyphodium endophytes. Methodologies have been established for the genetic transformation of the perennial ryegrass endophyte N. lolii, based on direct gene transfer to protoplasts and the surrogate transformation of untransformed ryegrass host plants through inoculation with the transformed endophyte (Murray et al. 1992). More recently, a high-throughput gene-silencing approach was reported for studying the interaction between perennial ryegrass and the fungal endophyte N. lolii, based on hairpin RNA and small interfering RNAs (siRNAs; Felitti et al. 2005). Recently, an endophyte EST resource was established comprising 9507 sequences from Epichloë and Neotyphodium endophytes grown in vitro (Spangenberg et al. 2001; Felitti et al. 2004). The availability of partially sequenced endophyte cDNA clones enabled the development of specific endophyte cDNA

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microarrays, i.e., the Nchip and the EndoChip (Spangenberg et al. 2001; Felitti et al. 2004). The Nchip consists of 3,806 genes derived from four cDNA libraries from in vitro-grown Epichloë/Neotyphodium endophytes, while the EndoChip contains 18,264 features interrogating 5,325 Epichloë/Neotyphodium genes derived from six cDNA libraries, including in planta-expressed endophyte sequences. These microarrays have been applied to examine changes in Epichloë/Neotyphodium endophyte gene expression in response to growth in different culture conditions (Felitti et al. 2004). The use of the EndoChip, together with the 15K perennial ryegrass unigene microarray (Sawbridge et al. 2003), allows for the concomitant genome-wide transcriptome analysis of Epichloë/Neotyphodium endophytes and their hosts (Spangenberg et al. 2001; Felitti et al. 2004).

4 Practical Applications of Transgenic Plants In recent years, genes have been introduced into ryegrasses for lignin and fructan metabolism (Ye et al. 2001; Hisano et al. 2004c), developmental regulation of flowering (Van der Valk et al. 2004), tolerance to biotic stress (Xu et al. 2001; Takahashi et al. 2005), tolerance to abiotic stress (Li et al. 2004; Wu et al. 2005), and pollen hypo-allergenicity (Bhalla et al. 1999; Petrovska et al. 2004). 4.1 Modification of Lignin Biosynthesis Improved digestibility of grass is associated with increased animal production, through increases both in pasture intake and in energy yield from pasture. Feeding and grazing studies have shown that small changes in forage digestibility can have a significant impact on animal performance (Gressel and Zilberstein 2003). For example, increases in milk production of 90 kg ha−1 per day were observed when dairy cows grazed a ryegrass pasture with 4% units of increased digestibility (Wilman et al. 1992). A 5–6% unit increase in digestibility of perennial ryegrass was predicted to increase summer milk production in southern Australia by 27% (Smith et al. 1998). The extent and composition of lignin is an important factor affecting forage grass quality, decreasing cell wall digestibility and limiting the amount of energy available to livestock (Casler et al. 2002). For example, for every percentage decrease in the lignin content of straw, 2–4 times more cellulose is made available for digestion by ruminants (Gressel and Zilberstein 2003). Lignins from dicotyledonous plants contain two major monomer species, termed guaiacyl (G) derived from coniferyl alcohol and syringyl (S) derived from sinapyl alcohol. The (G) and (S) units differ in the number of methoxyl groups on the aromatic ring. Monocotyledonous plants contain a large proportion of an additional unit, p-hydroxyphenyl (H), derived from coumaryl alcohol. Several studies link decreased forage digestibility to an increase in

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the S/G ratio (Pond et al. 1987; Dixon et al. 1996; Baucher et al. 1998). It is postulated that elevated levels of the highly methoxylated S subunit negatively affects digestibility of ryegrass and, ultimately, the nutrient availability, because of the formation of cross-links with another major cell wall component, arabinoxylans (Pond et al. 1987). Natural variation in lignification leading to increased digestibility has been found in brown-midrib (bmr) mutants of maize (Cherney et al. 1996). This phenotype is attributed to mutations in the COMT gene (Vignols et al. 1995) or cinnamyl the alcohol dehydrogenase (CAD) gene (Halpin et al. 1998). Unfortunately, similar mutations have not been reported in wheat, barley, rice or ryegrass, probably due to redundancy within the corresponding multi-gene families in grains and grasses. Transgenesis thus offers new opportunities for the targeted modification of lignin content and composition in ryegrasses to enhance herbage quality. Considerable research effort has been made toward the cloning of genes involved in the monolignol biosynthetic pathway (Anterola and Lewis 2002; Humphreys and Chapple 2002; Boerjan et al. 2003). In perennial ryegrass, cDNAs encoding the main enzymes involved in lignin biosynthesis have been isolated and characterized, such as caffeic acid O-methyltransferase (Heath et al. 1998), cinnamyl alcohol dehydrogenase (McAlister et al. 2001; Lynch et al. 2002), cinnamoyl-CoA reductase (McInnes et al. 2002; Larsen 2004), laccase (Gavnholt et al. 2002), phenylalanine ammonia-lyase (Lidgett et al. 2005a), cinnamate-4-hydroxylase (Lidgett et al. 2005a), ferulate-5-hydroxylase (Lidgett et al. 2005a), 4-coumarate-CoA ligase (Heath et al. 2002), caffeoylCoA 3-O-methyltransferase (Lidgett et al. 2005a) and peroxidase (Lidgett et al. 2005a). These represent current targets for the genetic modification of lignin biosynthesis to improve forage quality in perennial ryegrass. Transgenic approaches to lignin modification through the partial silencing of the lignin biosynthesis genes using co-suppression, antisense RNA and dsRNAi strategies have been reported for both dicotyledonous and monocotyledonous species (reviewed by Baucher et al. 1998; Humphreys and Chapple 2002; Dixon and Reddy 2003). Importantly, these reports demonstrate that the decrease in function of a single lignin biosynthesis gene can provide sufficient down-regulation and modification of lignin structure to enhance dry matter digestibility. In transgenic tall fescue with down-regulation of cinnamyl alcohol dehydrogenase, an increase was reported in dry matter digestibility of 7.2–9.5% (Chen et al. 2003). Transgenic tall fescue plants had a significantly decreased lignin content and altered ratios of syringyl (S) to guaiacyl (G), G to p-hydroxyphenyl (H), and S to H units, with no dramatic changes in the composition of other key cell wall components, such as cellulose and hemicellulose. The down-regulation of lignin biosynthesis genes thus represents an effective strategy to alter both lignin content and composition, leading to enhanced dry matter digestibility and improved herbage quality in transgenic forage grasses. The metabolic channelling model offers a new perspective to the potential spatial and temporal assembly of multi-enzyme complexes in cells (Winkel

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2004). Applied to lignin biosynthesis, it suggests that channels differentially assembling under different developmental conditions or in response to stresses may also be differentially associated with biosynthesis of S and G lignins. This opens new opportunities for the predictable metabolic re-programming of the lignin biosynthesis pathway, precisely regulating its temporal and spatial composition and ultimately generating cultivars with improved forage quality. 4.2 Modification of Fructan Metabolism Fructans are a class of highly water-soluble polysaccharides, which consist of linear or branched fructose chains attached to sucrose. Fructans are made by approximately 15% of flowering plant species, as well as by bacteria and fungi, and represent the major non-structural carbohydrate in many plant species (Lewis 1984). Perennial ryegrass genotypes selected for high concentrations of water-soluble carbohydrates, particularly during the summer, show 2–6% increase in their in vitro dry matter digestibility (Radojevic et al. 1994). High concentrations of fructans accumulate in ryegrasses and fescues in response to environmental stresses such as drought and cold (Spollen and Nelson 1994; Amiard et al. 2003). Fructans may directly stabilize membranes under stress conditions (Ozaki and Hayashi 1996; Demel et al. 1998; Vereyken et al. 2001; Hincha et al. 2000, 2002). Plant fructans have different structures and chain lengths, ranging from three up to a few hundred fructose units. Fructan metabolism is based on the substrate sucrose and involves fructosyltransferase (FT) enzymes for fructan biosynthesis and fructan exohydrolase (FEH) enzymes for fructan degradation (reviewed by Van den Ende et al. 2004). The fructan profile of perennial ryegrass includes inulin series, inulin neoseries and levan neoseries fructans (Pavis et al. 2001a, b). The most abundant trisaccharides present in perennial ryegrass are 1-kestose and 6G-kestose, with 6-kestose being present in significantly smaller amounts (Pavis et al. 2001a, b). It is proposed that at least four enzymes, namely 1-SST, 1-FFT, 6G-FFT and 6-FFT, are required to produce this complement of fructans (Pavis et al. 2001b). Candidate cDNA sequences encoding these activities have been isolated and partly characterized in ryegrasses, including LpFT1, LpVINV, Lp1-SST, Lp1FFT, Lp6G-FFT, LpFT4, LpFEH and LpCWINV (Lidgett et al. 2002; Johnson et al. 2003; Chalmers et al. 2003, 2005a, b; Gallagher et al. 2004; Hisano et al. 2004a, b). Several fructan metabolism genes have been transformed into plants such as tobacco (Sprenger et al. 1997; Schellenbaum et al. 1999), potato (Hellwege et al. 1997), petunia (Van der Meer et al. 1998), sugarbeet (Sevenier et al. 1998), chicory (that naturally accumulates fructan; Sprenger et al. 1997; Vijn et al. 1997) and Italian ryegrass (Ye et al. 2001). The bacterial levansucrase SacB gene from Bacillus subtilis was transformed into Italian ryegrass (Ye et al. 2001). In this study, the concentration of fructan was reduced unexpectedly compared with isogenic controls, whilst levels of sucrose were unaltered and

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hexose concentrations slightly reduced. Furthermore, transgenic plants were stunted with narrower leaves and poorly developed roots (Ye et al. 2001). Although stress tolerance was observed in plants transformed with bacterial fructan synthesis genes, transgenic plants often exhibited a number of aberrant phenotypes, such as stunting, leaf bleaching, necrosis, reduction in starch accumulation and chloroplast agglutination (Cairns 2003). Hisano et al. (2004c) produced transgenic perennial ryegrass overexpressing the wheat fructosyltransferase genes, wft1 and wft2, individually and in combination. These genes encode sucrose–fructan 6-fructosyltransferase (6-SFT) and sucrose–sucrose 1-fructosyltransferase (1-SST), respectively. Significant increase in fructan content was detected in the transgenic perennial ryegrass plants expressing either chimeric wft1 or wft2 genes compared with non-transgenic control plants. However, fructan content in transgenic ryegrass plants transformed with both wft1 and wft2 chimeric genes was lower than fructan accumulation observed in transgenic ryegrass plants expressing these genes individually. Due to the high sequence homology between wft1 and wft2, the authors suggested co-supression as a possible explanation for this observation. Based on measurements of electrical conductivity (Dexter et al. 1932), transgenic ryegrass plants expressing wheat fructosyltransferase genes revealed increased tolerance to freezing at the cellular level (Hisano et al. 2004c). 4.3 Modification of Reproductive Development and Leaf Senescence Forage ryegrass varieties with an extended vegetative growth phase are of great interest since the nutritional value of ryegrass declines as plants reach maturity. Plants that are flowering (heading) contain crude protein levels, total digestible nutrients and phosphorus concentrations 45%, 25% and 33% lower than early vegetative plants, respectively (Hannaway et al. 1999). Furthermore, digestible energy, metabolizable energy and calcium levels in flowering ryegrass plants decrease by 15% (Hannaway et al. 1999). Consequently, modification of reproductive development through a delay in heading or a reversible inhibition of flowering in ryegrasses offers potential for significant improvement in highquality herbage production. To achieve a delay in flowering, Van der Valk et al. (2004) expressed the Arabidopsis light-regulated homeobox transcription factor ATH1 in perennial ryegrass. Heading was delayed significantly in ryegrass plants expressing ATH1 and, in a number of cases, plants never flowered. This phenotype was associated concomitantly with the outgrowth of normally quiescent lateral meristems increasing vegetative biomass. Plants that did eventually flower generally produced fewer inflorescences than non-transformed controls. The ABC model of floral organ specification describes the basic mechanisms of floral pattern formation in eudicots (Lohmann and Weigel 2002). Studies in monocotyledons systems such as maize, rice and, recently, ryegrass, showed

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that there is some degree of gene conservation involved at the different stages of flower initiation and development compared with dicot plants. Several monocotyledon orthologues of dicotyledon homeotic genes controlling floral organ identity have been characterized (Ambrose et al. 2000). For example, the maize silky1 and rice SUPERWOMAN1 encode apparently functional orthologues of APETALA3 (AP3), a B-class gene that regulates specification of petals and stamens in Arabidopsis (Ambrose et al. 2000). Similarly, characterization by transgenic and mutant analysis of the rice OsMADS1, demonstrated functional equivalence with the APETALA1-related A-class gene that regulates specification of sepals (Jeon et al. 2000). MADS-box transcription factors displaying homology to APETALA1, SEPALATA and AGAMOUS-like subfamilies have been isolated from ryegrass (Gocal et al. 2001; Peterson et al. 2004). Flowers in the ryegrass inflorescence are arranged in a cyme, with apical growth terminating with the production of a single terminal flower. The TERMINAL FLOWER 1 (TFL1) gene of Arabidopsis and its homologue CENTRORADIALIS (CEN) in Antirrhinum have been identified as a group of genes that specify an indeterminate identity of inflorescence meristems (Bradley et al. 1996, 1997). Mutations in these genes convert shoot apices into a single terminal flower. The TERMINAL FLOWER1-like gene, LpTFL1, was recently isolated from perennial ryegrass and shares the same function as TFL1, inhibiting the same family of flower meristem-identity genes and ultimately flowering (Jensen et al. 2001). Ectopic expression of LpTFL1 in transgenic red fescue plants prevented flowering following exposure to natural vernalization conditions over two successive years (Jensen 2001). Homologues of two orthologous flower meristem-identity genes, LEAFY/FLORICAULA from A. thaliana and A. majus were also isolated from L. temulentum (Gocal et al. 2001). A family of key flowering-time genes determining the developmental fate of plants was isolated recently from rice, maize and ryegrass. Maize plants that have mutations in the indeterminate gene (id1) are unable to undergo the normal transition to flowering; the shoot apical meristem of id1 mutants continues to initiate leaves long after normal plants have flowered. The id1 gene was found to encode a putative transcriptional regulator containing a zincfinger domain potentially involved in protein–DNA interactions (Colasanti et al. 1998). Photoperiod and vernalization are the two key environmental factors affecting floral induction in ryegrass. Transition from vegetative to reproductive growth occurs only after an extended vernalization period, followed by an increase in day length and temperature. In A. thaliana plants under a long-day photoperiod, the key flowering time gene CONSTANS initiates the transition to flowering (Mouradov et al. 2002; Valverde et al. 2004). Down-regulation of this gene causes a severe delay in flowering under both long- and short-day conditions. Interestingly, functional homologues of CONSTANS have been isolated from rice, a short-day plant (Yano et al. 2000), and perennial ryegrass (LpCO; Martin et al. 2004). LpCO is able to complement the Arabidopsis co-2 mutant, and ectopic expression in Arabidopsis wild-type plants leads to early flowering.

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The regulation of developmental genes, such as floral meristem-identity genes, to delay heading may provide a powerful approach to the improvement of forage quality in ryegrasses. Gan and Amasino (1995) developed a system for autoregulated cytokinin production in transgenic plants. A senescenceassociated SAG12 promoter from A. thaliana was linked to the isopentenyl transferase gene (ipt) from A. tumefaciens to exploit the role of cytokinins in delaying senescence. Tobacco plants transformed with the chimeric SAG12ipt gene demonstrated delayed senescence without associated developmental abnormalities (Gan and Amasino 1995). In Italian ryegrass, expression of a cysteine protease cDNA, SEE1, was also enhanced during leaf senescence and repressed by cytokinin (Li et al. 2004). The SEE1 promoter was fused to ipt and transformed into Italian ryegrass. Transgenic Italian ryegrass plants appeared greener than non-transformed control plants and exhibited a staygreen phenotype. 4.4 Increased Tolerance to Biotic and Abiotic Stresses The most important diseases of ryegrasses are caused by the fungal pathogens crown rust (Puccinia coronata f.sp. lolii) and stem rust (Puccinia graminis f.sp. lolii), the viruses ryegrass mosaic virus (RMV) and barley yellow dwarf virus (BYDV) and the bacterial wilt pathogen (Xanthomonas campestris pv. graminis). In economic terms, the most damaging foliar disease is crown rust, associated with reductions in herbage quantity, quality and palatability (Dumsday et al. 2003). The pathogenesis-related protein chitinase hydrolyses chitin, a structural component of the fungal cell wall. The rice chitinase gene (Cht-2; RCC2) was recently transformed into Italian ryegrass (Takahashi et al. 2005). A bioassay for crown rust resistance was conducted on detached leaves from transgenic Italian ryegrass plants with elevated chitinase activity. Compared with non-transgenic controls, the severity of disease symptoms was reduced and the development of uredospores was slower in leaves from transgenic Italian ryegrass plants (Takahashi et al. 2005). Ryegrass mosaic virus is also widely distributed among forage grasses (Eagling et al. 1992) and can decrease vegetative dry matter by 5–50% and reduce persistence in perennial ryegrass (A’Brook and Heard 1975). The RMV coat protein gene was transformed into perennial ryegrass via particle bombardment, with 23 independent transgenic lines recovered and examined for RMV resistance (Xu et al. 2001). The most resistant transgenic line showed no immuno-detectable RMV coat protein up to 9 months after inoculation. Molecular analysis suggests that the mechanism of RMV resistance is RNA degradation via post-transcriptional gene silencing, along with inhibition of RNA replication (Xu et al. 2001). Salinity is an emerging threat to sustainable agriculture. Increases in salinity can be attributed to a rise in underground water-tables bringing naturally

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occurring salt to the surface. Recently, the rice vacuolar membrane Na+ /H+ antiporter gene OsNHX1 was transformed into perennial ryegrass by Agrobacterium-mediated transformation (Wu et al. 2005). The Na+ /K+ antiporter prevents accumulation of toxic levels of Na+ in the cytoplasm by pumping excess Na+ into the vacuole, maintaining a higher K+ /Na+ ratio in the cytoplasm compared with the vacuole (Fukuda et al. 1999). Transgenic perennial ryegrass plants expressing a chimeric OsNHX1 gene survived more than 10 days at salt concentrations of 350 nmol l−1 , whilst non-transformed control plants perished. Levels of Na+ , K+ and the stress-related amino acid proline were elevated in the transgenic OsNHX1-expressing perennial ryegrass plants. 4.5 Down-Regulation of Main Pollen Allergens Hayfever and seasonal allergic asthma are major type I allergic diseases affecting up to 25% of the population in cool temperate climates around the world (Tamborini et al. 1995). Ryegrass is responsible for a major portion of grass pollen allergies worldwide (Smart et al. 1979; Spangenberg et al. 1998). Two proteins, designated Lol p 1 and Lol p 2, have been identified as main allergenic determinants in ryegrass pollen (King et al. 1995). These allergens exist in multiple immunologically indistinguishable forms (Johnson and Marsh 1965). Significant efforts are being made to develop diagnostic and therapeutic reagents for the design of new and more effective immuno-therapeutic treatments for hayfever and seasonal allergic asthma caused by ryegrass pollen. An alternative strategy to mitigate this environmental disease is to reduce the amount of pollen allergens produced by the source plants through targeted down-regulation of the expression of pollen allergen-encoding genes (Spangenberg et al. 1998). Bhalla et al. (1999) reported transformation of the weedy annual ryegrass (L. rigidum) with an antisense gene of the Lol p 5 protein. Transgenic plants showed significantly less Lol p 5 protein accumulation in pollen without a reduction in pollen viability. Transgenic perennial ryegrass and Italian ryegrass containing antisense Lol p 1 and Lol p 2 genes under the control of a pollen-specific promoter have been generated, showing a significant down-regulation of the respective main pollen allergens Lol p 1 and Lol p 2 (Petrovska et al. 2004). Transgenic hypoallergenic ryegrass plants with down-regulation of the Lol p 1 allergen have been evaluated under small-scale field conditions (Petrovska et al. 2005).

5 Conclusions and Future Challenges Significant progress has been made in the establishment of the methodologies required for the molecular breeding of ryegrasses. A number of biotechnological approaches currently being tested include nutritional improvements through altered biosynthesis of lignin, fructan and fatty acids, protection

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against pathogens and pests through engineered virus resistance and regulated expression of antifungal and pesticidal proteins, as well as the modification of growth and development aimed at improved persistence, improved tolerance to abiotic stresses, delayed senescence, non-flowering phenotypes and downregulation of pollen allergens. The first transgenic ryegrasses have passed field-trial evaluations, and selected transformation events are being used in the production of elite transgenic germplasm for cultivar development. Advances in functional genomics promise to close gaps in our understanding of the underlying genetics, physiology and biochemistry of many complex plant processes and thus speed-up progress in applying gene technology-based approaches to ryegrass improvement. The application of molecular methodologies and tools in ryegrass improvement will greatly enhance current empirical phenotype-based selection with more directed and predictable genotypebased approaches. However, these molecular approaches show promise only when considered as a part of plant improvement programmes. The most successful improvement programmes are expected to be those that build on multi-disciplinary teams involving plant breeders, molecular and cell biologists, biochemists, plant pathologists, agronomists and animal scientists, and that efficiently embrace appropriate new gene technology, functional genomics, bioinformatics and high-throughput genotyping tools and apply these in a sensible manner. The effective integrated effort of such teams will be critical to the competitive development of marketable forage and turf ryegrass cultivars from molecular breeding programmes. Plant genomics will play a vital role in accelerating the application of biotechnology to ryegrass improvement and grassland agriculture. Significant progress has been made in the development of genomics and transcriptomics resources and tools in ryegrasses and their fungal endophytes. They provide the basis for genome-wide gene and molecular marker discovery enabling detailed molecular genetic and functional genomic studies of both ryegrass and endophyte biology and interactions between endophytes and their grass hosts. The development of candidate gene-based SNP markers offer opportunities for the direct selection of ryegrass genotypes with superior allele content. Collectively, these technologies will accelerate and enhance ryegrass breeding efforts.

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Vereyken IJ, Chupin V, Demel RA, Smeekens SCM, De Kruijff B (2001) Fructans insert between the headgroups of phospholipids. Biochim Biophys Acta 1510:307–320 Vignols F, Rigau J, Torres MA, Capellades M, Puigdomenech P (1995) The brown midrib 3 (bm3) mutation in maize occurs in the gene encoding caffeic acid O-methyltransferase. Plant Cell 4:407–416 Vijn I, Van Dijken A, Sprenger N, Van Dun K, Weisbeek P, Wiemken A, Smeekens S (1997) Fructan of the inulin neoseries is synthesised in transgenic chicory plants (Cichorium intybus L.) harbouring onion (Allium cepa L.) fructan:fructan 6G-fructosyltransferase. Plant J 11:387– 398 Wang GR, Binding H, Posselt UK (1997) Fertile transgenic plants from direct gene transfer to protoplasts from Lolium perenne and Lolium multiflorum Lam. J Plant Physiol 151:83–90 Wang ZY, Nagel J, Potrykus I, Spangenberg G (1993) Plants from suspension cell-derived protoplasts in Lolium species. Plant Sci 94:179–193 Wang ZY, Scott M, Hopkins A (2002) Plant regeneration from embryogenic cell suspension cultures of Lolium temulentum. In Vitro Cell Dev Biol Plant 38:446–450 Wang ZY, Ge Y, Mian R, Baker J (2005) Development of highly tissue culture responsive lines of Lolium temulentum by anther culture. Plant Sci 168:203–211 Warnke SE, Barker RE, Jung G, Sim SC, Mian MAR, Saha MC, Brilman LA, Dupal MP, Forster JW (2004) Genetic linkage mapping of an annual x perennial ryegrass population. Theor Appl Genet 109:294–304 Wilkinson HH, Siegel MR, Blankenship JD, Mallory AC, Bush LP, Schardl CL (2000) Contribution of fungal loline alkaloids to protection from aphids in a grass–endophyte mutualism. Mol Plant Microbe Interact 13:1027–1033 Wilman D, Waters RJK, Baker DH, Williams SP (1992) Comparison of two varieties of Italian ryegrass (Lolium multiflorum) for milk production when fed as silage and when grazed. J Agric Sci 118:37–46 Winkel BSJ (2004) Metabolic channelling in plants. Annu Rev Plant Biol 55:85–107 Wu YY, Chen QJ, Chen M, Chen J, Wang XC (2005) Salt-tolerant transgenic perennial ryegrass (Lolium perenne L.) obtained by Agrobacterium tumefaciens-mediated transformation of the vacuolar Na+ /H+ antiporter gene. Plant Sci 169:65–73 Xu JP, Schubert J, Altpeter F (2001) Dissection of RNA-mediated ryegrass mosaic virus resistance in fertile transgenic perennial ryegrass (Lolium perenne L.). Plant J 26:265–274 Yamada T, Forster JW (2005) QTL analysis and trait dissection in ryegrasses (Lolium spp.). In: Humphreys MO (ed) Molecular breeding for the genetic improvement of forage and turf. Wageningen Academic, Wageningen, pp 43–53 Yano M, Katayose Y, Ashikari M, Yamanouchi U, Monna L, Fuse T, Baba T, Yamamoto K, Umehara Y, Nagamura Y (2000) Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS. Plant Cell 12:2473–2484 Yates SG, Plattner RD, Garner GB (1985) Detection of ergopeptine alkaloids in endophyte infected, toxic Ky-31 tall fescue by mass spectrometry/mass spectrometry. J Agric Food Chem 33:719– 722 Ye X, Wang ZY, Wu X, Potrykus I, Spangenberg G (1997) Transgenic Italian ryegrass (Lolium multiflorum) plants from microprojectile bombardment of embryogenic suspension cells. Plant Cell Rep 16:379–384 Ye XD, Wu XL, Zhao H, Frehner M, Nösberger J, Potrykus I, Spangenberg G (2001) Altered fructan accumulation in transgenic Lolium multiflorum plants expressing a Bacillus subtilis SacB gene. Plant Cell Rep 20:205–212

IV.5 Lupins L.M. Tabe and L. Molvig1

1 Introduction 1.1 Lupins in Agriculture Lupins may be most familiar as garden flowers, but the genus Lupinus (family Fabaceae) contains hundreds of species, several of which are domesticated crop plants. The majority of Lupinus species occur in the Americas, but only one of these new world taxa (L. mutabilis) has any history of human use. In contrast, all 12 species from the Mediterranean region and Africa have large seeds, which, in combination with their distribution around cultivated land, suggests that most if not all these species were utilized to some extent by humans during pre-history. The seeds of many wild lupins contain toxic concentrations of alkaloids, necessitating extensive soaking and washing before they can be consumed safely by humans or animals. For example, the large seeds of L. albus were used in Roman culture as a snack food, after boiling, leaching and cooking (Gladstones 1998). The bitter white lupin (L. albus), yellow lupin (L. luteus) and narrow leaf lupin (L. angustifolius) were used for seed, forage and green manure in Europe during the nineteenth century (Hondelmann 1984), but it was not until the early twentieth century that plant breeders in Germany selected the first “sweet” lupin mutants with low concentrations of seed alkaloids (Cowling et al. 1998). Domesticated forms of these three species were eventually produced by combining variants with low seed alkaloids, non-shattering pods and permeable seeds. Sweet lupin seeds have few anti-nutritional factors and high protein concentrations, but their carbon storage compound is predominantly non-starch polysaccharide deposited in thickened cell walls. This material is poorly digested by non-ruminant animals, so lupin seeds are best suited for ruminant animal feed, although they are also used as components of non-ruminant animal feed formulations and are considered a good source of dietary fibre for humans. Lupin seed protein is well tolerated in aquaculture feeds, where it has potential to at least partially replace fishmeal as a protein source (Booth et al. 2001; Glencross et al. 2004). Lupins are adapted to growth on poor, acidic soils and therefore they are most important in agricultural zones where other crops struggle. Being 1 CSIRO

Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia, e-mail: Linda.Tabe@ csiro.au

Biotechnology in Agriculture and Forestry, Vol. 61 Transgenic Crops VI (ed. by E.C. Pua and M.R. Davey) © Springer-Verlag Berlin Heidelberg 2007

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legumes, they can have an important role in replenishing soil nitrogen in crop rotations. Lupin (mainly L. albus) production in Europe was estimated to total approximately 150,000 t in 2001, with production mainly in France and Greece (www.nnfcc.co.uk/crops/info/lupins.htm). The major lupin-producing country in the world, and the only country to export large quantities of lupin seed, is Australia. Development of varieties of L. angustifolius suitable for cultivation in rotation with cereals on the vast sand plains of Western Australia led to lupin seed production that, in many years, exceeded around 1 × 106 t year−1 of lupin seed since the early 1990s (Pannell 1998). 1.2 Production Constraints and Targets for Improvement by Genetic Modification 1.2.1 Herbicide Tolerance As with all crops, lupin production can be limited by competition with weeds. Glyphosate-tolerant ryegrass is a particular problem in Southern Australian cropping systems, necessitating diversification in weed control measures (Schmidt and Pannell 1996). 1.2.2 Seed Protein Quality Although lupin seeds are rich in protein, the nutritional quality of the protein is limited by its sub-optimal amino acid balance. As in most grain legumes, the low proportion of the sulfur amino acid, methionine, is the first limit for lupin seed protein nutritive value, particularly in the case of L. angustifolius. Some natural variation for seed protein methionine content exists, but no current L. angustifolius variety has a methionine concentration approaching that needed for optimal animal nutrition. 1.2.3 Disease Resistance Lupins are susceptible to a number of fungal pathogens, the most important of which is Pleiochaeta setosa, the causative agent of leaf brown spot and root rot (Sweetingham et al. 1998). This pathogen causes major outbreaks in all lupin-growing areas around the world; and little natural resistance is recorded in cultivated species, with the possible exceptions of L. albus (Papineau and Huyghe 1989) and L. luteus (Yang et al. 1996). Another fungal disease, anthracnose, is currently the worst threat to the lupin industry in Australia; and resistance to this disease is an important target for the Australian breeding program (Yang et al. 2004). A wide range of other fungi cause disease in lupins. Natural resistance genes and agronomic management strategies are deployed against them with mixed success.

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The most important viral pathogen of lupins worldwide is bean yellow mosaic virus (BYMV), while cucumber mosaic virus (CMV) is a significant constraint on production of L. angustifolius and L. luteus in Australia. CMV is transmitted horizontally by aphids, and vertically through the seed, necessitating screening of seed stocks to limit virus spread. There is no effective natural resistance in lupins to BYMV, while natural resistance to CMV is limited to L. albus and possibly L. luteus (Sweetingham et al. 1998).

2 Genetic Transformation of Lupins 2.1 Lupin Regeneration in Tissue Culture Many of the targets for improvement of lupins are intractable to conventional plant breeding, due to a lack of donor germplasm with the desired traits. To overcome this problem, there was some work on inter-specific hybridization within the genus Lupinus (reviewed by Atkins et al. 1998) and good progress was made in developing genetic transformation methods for some lupin species. As reported for most grain legumes, regeneration of Lupinus species in tissue culture proves difficult. However, a number of protocols are reported, with regeneration achieved via organogenesis or somatic embryogenesis for four species (Table 1). Direct shoot organogenesis from germinating seed or seedling explants appears to be the most widely applicable method for plantlet regeneration for all four lupin species. There are also reports of plant regeneration via direct somatic embryogenesis in L. angustifolius and L. albus. In the listed regeneration protocols, the suitability of the explants for Agrobacterium infection was not always given priority. However, the regeneration protocols published by Molvig et al. (1997), Pigeaire et al. (1997), Babaoglu et al. (2000) and Li et al. (2000) are all part of successful Agrobacterium tumefaciens-mediated transformation protocols. In each of these, the explant was the wounded shoot apex of germinating seeds or seedlings, with regeneration via direct shoot organogenesis. 2.2 Lupin Transformation 2.2.1 Lupinus angustifolius In the late 1990s, two independent groups in Australia developed methods for L. angustifolius transformation (Molvig et al. 1997; Pigeaire et al. 1997). Both methods induced direct shoot organogenesis from embryonic axes, but the explants were taken from different stages of seed development (Table 1) and the target cells for transformation in the shoot apex were exposed and wounded in different ways (stabbed with a hypodermic needle by Pigeaire, versus longitudinal slices by Molvig). Both transformation methods used the bar gene, which

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Table 1. Regeneration methods for lupin species

Lupinus species Explant

Morphogenesis Plantlets

L. angustifolius Leaf, root, hypocotyl Callus Hypocotyl Shoot organogenesis from callus Immature cotyledon Direct somatic embryogenesis Immature cotyledon Direct somatic embryogenesis Embryonic axis Direct from immature seed shoot organogenesis Embryonic axis Direct shoot from organogenesis germinating seed L. luteus Leaf Shoot organogenses from callus Hypocotyl Direct shoot organogenesis Embryonic axis Direct shoot from organogenesis germinating seed L. albus Immature cotyledon Direct somatic embryogenesis Immature cotyledon Direct somatic embryogenesis Hypocotyl thin Direct shoot cell layers organogenesis L. mutabilis

Immature cotyledon Direct somatic embryogenesis Hypocotyl Organogenesis of shoot buds from callus Wounded shoot apex Direct shoot from 7-day seedling organogenesis Hypocotyl Direct shoot thin cell layers organogenesis

No Yes

Reference Sator (1985) Sroga (1987)

Yes

Molvig (unpublished data) Unknown Nadolska-Orczyk (1992) Yes Molvig et al. (1997) Yes

Pigeaire et al. (1997)

No

Sator (1985)

Yes

Daza and Chamber (1993) Li et al. (2000)

Yes

Unknown Nadolska-Orczyk (1992) Yes Rybczynski and Podyma (1993) No Mulin and Bellio-Spataru (2000) Unknown Nadolska-Orczyk (1992) No Phoplonker and Caligari (1993) Yes Yes

Babaoglu et al. (2000) Mulin and Bellio-Spataru (2000)

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confers resistance to phosphinothricin, the active constituent of the herbicide, Basta, for selection of transformed cells. The method developed by Pigeaire gave the higher transformation frequency of the two methods (0.4–2.8% of explants produced transformed plantlets) and was subsequently used by both groups to transfer a number of genes to L. angustifolius (see Section 3). In response to public concern about possible risks to the environment and human health from the use of herbicide resistance and antibiotic resistance genes as selectable markers, transformation technologies were developed for the generation of selectable marker-free genetically modified (GM) lupin plants. L. angustifolius explants were transformed with two transferred-DNAs (T-DNAs), one containing the selectable marker and the other containing the gene of interest (Molvig et al. 2002). The two T-DNAs were located adjacent to each other on a single “twin T-DNA” plasmid, which was delivered to the explant by one transformed Agrobacterium strain. Transgenic plants were screened by PCR and 85% were found to be co-transformed with both selectable marker and gene of interest. Approximately 25% of T0 plants transmitted both transgenes to the T1 generation, showing that many primary transgenic plants were chimeric. A low proportion of stably transformed events (five out of 24 events) exhibited independent segregation of transgenes in subsequent generations, indicating that the transgenes had integrated into the genome at unlinked sites in these plants. The results demonstrate the feasibility of this approach for the generation of selectable marker-free GM lupins. 2.2.2 Lupinus luteus Agrobacterium tumefaciens-mediated transformation of L. luteus was achieved via infection of wounded shoot apices from germinating seeds (Li et al. 2000). This transformation method is similar to that reported for L. angustifolius by Pigeaire et al. (1997), except that single shoots were regenerated under selection pressure for L. luteus, rather than multiple shoots, as for L. angustifolius. Fourteen transgenic events were produced from four cultivars and transmission of transgenes to the T6 generation was reported for some events. The bar gene was used for selection; and confirmation of the integration and inheritance of this transgene was shown by assay of the encoded enzyme, by PCR, and by Southern blot analysis. In addition to the bar gene, a gene for resistance to barley yellow dwarf virus was introduced. 2.2.3 Lupinus mutabilis The regeneration protocols for L. mutabilis published by Babaoglu et al. (2000) and Mulin and Bellio-Spataru (2000) appear suitable for A. tumefaciens-mediated gene transfer, but only the latter paper reported successful transformation. In this work, explants were derived from 7- to 8-day seedlings and consisted of shoot apices from which the leaf primordia and initial cell lay-

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ers of the apical meristem were removed. The neomycin phosphotransferase (nptII) gene was used as a selectable marker for transfer of the screenable marker, uidA, encoding bacterial β-glucuronidase (GUS). Ten out of 250 explants produced buds expressing GUS activity; and GUS-positive shoots were recovered from one of these explants on kanamycin selection. Multiple clones of this event were produced and rooted plantlets successfully transferred to soil. The integration of both uidA and nptII transgenes in the genome of the T0 transgenic plants was demonstrated by Southern blotting, but no data were presented regarding the fertility of these plants, or transmission of the transgenes to the next generation. L. mutabilis was also shown to be susceptible to A. rhizogenes infection. Hypocotyl, epicotyl and stem explants derived from seedlings were infected with A. rhizogenes transformed with a plasmid containing the uidA and nptII genes. Hairy roots proliferated on kanamycin selective media, and Southern blotting confirmed integration of the nptII transgene in the root cultures. However, regeneration of plants from hairy roots was not reported (Babaoglu et al. 2004).

3 Lupin Improvement Through Biotechnology 3.1 Herbicide Tolerance Several herbicides are currently used to control grass weeds in crops of L. angustifolius in Western Australia. These include the selective herbicides atrazine and simazine, and non-selective herbicides such as glyphosate. The former group has relatively high toxicity to vertebrates and persists in the environment. In all cases, development of herbicide tolerance in weed species is an inevitable consequence of continuous use of the same herbicide. The non-selective herbicide, Basta (active constituent glufosinate ammonium), is not currently approved for use on broad-acre crops in Western Australia, but is used in horticulture (Schmidt and Pannell 1996). As described in Section 2.2, the bar gene, which confers resistance to the herbicides Basta and Liberty (Hoechst), was transferred as a selectable marker in genetic transformation of L. angustifolius and L. luteus. A modelling study estimated that an integrated weed control strategy incorporating Basta-resistant, transgenic lupins would provide similar profitability to a system using current lupin varieties and paraquat for weed control (Schmidt and Pannell 1996). Basta-resistant lupins would therefore offer added diversity of options for weed control in cropping systems. 3.2 Seed Protein Nutritive Value Seeds are vital sources of protein for humans and animals. However, because of their limited diversity, seed proteins tend to have skewed amino acid com-

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position. For example, cereal seed protein is deficient in lysine, from the point of view of animal nutrition, while grain legumes are deficient in the sulfurcontaining amino acid, methionine. Both these amino acids are among the ten classified as “essential” because they cannot be synthesized by animals and must be obtained in the diet. Any amino acids in excess of the amounts defined by the most limiting amino acid are catabolized for energy production rather than supplying amino acids for protein synthesis. In order to prevent this inefficient use of dietary protein, animal feeds are formulated to combine seeds with complementary compositions and are supplemented with pure amino acids to optimize overall amino acid balance (Tabe and Higgins 1998). Like other grain legumes, lupin seeds are deficient in the sulfur-containing amino acids, methionine and cysteine. Cysteine is not classified as nutritionally essential because animals can derive it from methionine. However, dietary cysteine is said to have a “sparing” effect on methionine, and the two amino acids are generally both considered in diet formulations. Because of the limited variation available, conventional plant breeding is unable to increase methionine content in lupins (particularly L. angustifolius) to the extent necessary to meet the growth requirements of animals. A biotechnological approach was used to transfer to L. angustifolius a gene encoding an unusually methionine-rich protein normally found in sunflower seeds. A homozygous line was selected from a transgenic event that expressed the transgene at a high level, as assessed by molecular analysis of seed protein composition (Molvig et al. 1997). The sunflower seed albumin (SSA) accumulated to levels approximately equal to 5% of total seed protein and was associated with a doubling of the proportion of methionine in the seed protein of this line (Table 2). The nutritive value of the high-methionine GM lupins was evaluated in a feeding trial with broiler chickens (Ravindran et al. 2002). The trial compared the performance of chickens fed from 6 days to 20 days post-hatching on either a diet containing the GM lupin, or a diet containing lupins of the parental variety. Both lupin-containing diets and a maize–soybean control diet were formulated to contain optimal concentrations of essential amino acids, including methionine. Less pure methionine was added to the diet containing the GM lupins than to that containing the parental lupins because of the added endogenous methionine in the GM seed. As predicted, the weight gains and feed intakes were not significantly different between any of the experimental groups, although the feed to gain ratio for the GM lupin group tended to be lower (better) than that of the non-GM lupin group (Table 3). In addition, the apparent metabolizable energy of the GM lupins (10.18 ± 0.27 MJ kg−1 dry matter) was higher than that of the parental lupins (9.42 ± 0.55 MJ kg−1 dry matter). The reasons for these slight, unexpected differences between the two lupin types are not understood, but may be a consequence of slightly lower levels of the anti-nutritional, soluble non-starch polysaccharides in the GM (45.6 g kg−1 dry matter) versus parental (60.7 g kg−1 dry matter) lupin seeds.

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Table 2. Concentrations of essential amino acids, cysteine and total protein in transgenic and parental lupins. All values (g kg−1 dry matter) are the means of duplicate analyses, except crude protein which is the mean of triplicate analyses, and was derived by multiplying the nitrogen concentration by 6.25. Modified and reprinted with permission from Ravindran et al. (2002)

Seed constituent

Parental lupin

GM lupin

Essential amino acids Arginine Histidine Isoleucine Leucine Lysine Phenylalanine Threonine Tryptophan Valine Methionine Cysteine Crude protein (N × 6.25)

29.9 7.6 11.4 20.6 13.8 10.8 10.0 2.8 11.2 2.0 3.6 322

31.7 7.6 11.4 21.1 14.2 10.6 10.2 2.9 11.2 4.5 3.7 323.6

Table 3. Performance of broiler chickens fed diets containing either conventional lupins or GM lupins with increased methionine content. All values represent the mean of four replicate pens of ten birds each. Modified and reprinted with permission from Ravindran et al. (2002)

Treatment

Maize/soybean meal control 250 g kg−1 parental lupin 250 g kg−1 GM lupin Pooled SEM

Supplemental methionine (g kg−1 diet) 2.2 2.8 2.2

Weight gain (g)

Feed intake (g)

454 442 441 12.6

743 803 767 16.9

Feed/gain (g g−1 ; P = 0.09) 1.64 1.82 1.74 0.046

The results demonstrated that the extra methionine in the GM lupins was available to chickens and could reduce the need for added, pure methionine in poultry feed formulations. The dietary requirements of ruminant animals for methionine and other essential amino acids are difficult to estimate because of the intermediary step of microbial fermentation that occurs in the rumen. Ingested plant protein is first converted to microbial protein, which is subsequently digested and utilized by the animal. This process can result in the differential loss of nutrients such as sulfur amino acids, which are degraded to sulfide in the rumen (Moir et al. 1970). Some plant proteins, by virtue of their structures, are more resistant to rumen degradation than others. Such proteins, referred to as “bypass protein”, travel in a relatively intact form to the true stomach of the animal, where they are digested along with the microbial protein.

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Table 4. Wool growth and live weight gain in sheep fed diets containing either conventional lupins or GM lupins with increased content of rumen-protected methionine. Eighty merino wethers were divided into two groups and fed the experimental diets for 6 weeks. Sheep were weighed weekly, and wool samples were taken from mid-side patches at weeks –3, 0, 3 and 6 relative to the initiation of the treatment. Modified and reprinted with permission from White et al. (2001)

Diet

Wool growth (mg cm−2 day−1 )

Live weight gain (g day−1 )

65% hay + 35% parental lupin 65% hay + 35% GM lupin Significance

0.74

90

0.80

96

P < 0.001

P < 0.05

It has been established that wool growth in sheep has a particularly high demand for methionine and cysteine. Wool contains up to 150 g sulfur amino acids (SAAs) kg−1 crude protein, whereas the microbial protein supplied to the true stomach from the rumen contains approximately 35 g SAAs kg−1 crude protein (White et al. 2001). In order for dietary supplementation to be effective in correcting this deficit, SAAs must be protected from degradation in the rumen, either by chemical treatment or by incorporation in rumen-stable proteins. The sunflower albumin used to supply an additional storage sink for SAAs in GM lupin seeds was chosen for this strategy because of its stability in simulated rumen fluid in vitro (McNabb et al. 1994). The nutritive value of the high-methionine GM lupin seed for sheep was assessed in a feeding trial with merino wethers (White et al. 2001). Two groups of 40 sheep were fed hay-based diets that contained either 35% by weight of the parental type lupin seed or 35% of the GM lupin seed. After 6 weeks, the sheep fed the GM lupin diet showed 7% greater live weight gain and 8% greater wool growth than the control group (Table 4; White et al. 2001). The data were consistent with an increased supply of methionine to the animals resulting from the presence of sunflower albumin in the GM lupin seeds. The magnitude of the improvements observed was consistent with almost all the additional methionine in the GM lupins being rumen-protected. The results suggest that the use of the GM lupins as supplementary sheep feed over periods of pasture shortage would improve animal nutrition over and above the improvement afforded by conventional lupins. The sheep and poultry feeding trials were both conducted with seed of a transgenic event that also contained the bar selectable marker gene. Marker-free L. angustifolius transgenic events with the sunflower albumin gene have now been produced (Molvig et al. 2002). 3.3 Disease Resistance Resistance to disease is probably the most critical of all plant breeding objectives; however, it is often very difficult to achieve. Disease management in crops

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generally involves an integrated approach that combines improved genotypes containing natural resistance genes, along with optimized management practices and application of chemicals. Biotechnology offers an additional strategy, but its usefulness is limited by the availability of genes effective against problem diseases. In the case of lupins, the most important diseases are generally those caused by a variety of fungal pathogens. Genes with potential to confer resistance to fungal disease were recently transferred to L. angustifolius and transgenic events are now under assessment for enhanced resistance to relevant pathogens (P.M.C. Smith, University of Western Australia, and G. Thomas, Department of Agriculture Western Australia, personal communication). GM approaches using a number of modified viral genes have been employed with great success to combat plant viruses in a wide range of crops (Tepfer 2002). Major viral pathogens of lupins are being targeted by transferring a range of virus-derived gene constructs to L. angustifolius and L. luteus (S.J. Wylie, Murdoch University, personal communication). 3.4 Future Prospects The development of reliable transformation protocols for L. angustifolius and L. luteus has opened the door to the use of biotechnology for lupin improvement. L. albus has proved thus far recalcitrant to transformation, and this will be an important hurdle to overcome in the future. From the point of view of practical application of biotechnology to lupins, good progress has been made towards a number of goals, but additional tasks remain. Despite significant advances, seed sulfur amino acid content requires further optimization. Current GM events have doubled the seed methionine of the parental genotype, but this only corresponds to a 20% increase in total seed sulfur amino acids (methionine plus cysteine). A further 30% increase would be required to meet the full nutritional requirements of animals for these nutrients. This goal is being approached by pyramiding the sunflower albumin gene in existing GM lupins with genes encoding potentially limiting enzymes of sulfur amino acid biosynthesis in the maturing seed. Engineering of other aspects of seed composition, such as quantity and quality of oil is an attractive target for the future. Improvement of the harvest index of lupins is much needed, and is being attempted via the transfer of genes to manipulate hormone production in plant reproductive parts. Resistance to a broad range of fungal diseases is the most desirable GM trait for current and future development. It is to be hoped that the immediate future will bring commercial development of the existing GM lupins with tolerance to the herbicide Basta, with improved nutritive value, or with increased virus resistance. However, this will only occur in a social climate that is more accepting of GM crops in agriculture.

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References Atkins CA, Smith PMC, Gupta S, Jones MGK, Caligari PDS (1998) Genetics, cytology and biotechnology. In: Gladstones JS, Atkins CA, Hamblin J (eds) Lupins as crop plants: biology, production and utilization. CAB International, Wallingford, pp 67–92 Babaoglu M, McCabe MS, Power JB, Davey MR (2000) Agrobacterium-mediated transformation of Lupinus mutabilis L. using shoot apical explants. Acta Physiol Plant 22:111–119 Babaoglu M, Davey MR, Power JB, Sporer F, Wink M (2004) Transformed roots of Lupinus mutabilis: induction, culture and isoflavone biosynthesis. Plant Cell Tissue Organ Cult 78:29–36 Booth MA, Allan GL, Frances J, Parkinson S (2001) Replacement of fish meal in diets for Australian silver perch, Bidyanus bidyanus IV. Effects of dehulling and protein concentration on digestibility of grain legumes. Aquaculture 196:67–85 Cowling WA, Huyghe C, Swiecicki W (1998) Lupin breeding. In: Gladstones JS, Atkins CA, Hamblin J (eds) Lupins as crop plants: biology, production and utilization. CAB International, Wallingford, pp 93–120 Daza A, Chamber MA (1993) Plant regeneration from hypocotyl segments of Lupinus luteus L. cv Aurea. Plant Cell Tissue Organ Cult 34:303–305 Gladstones JS (1998) Distribution, origin, taxonomy, history and importance. In: Gladstones JS, Atkins CA, Hamblin J (eds) Lupins as crop plants: biology, production and utilization. CAB International, Wallingford, pp 1–39 Glencross B, Evans D, Hawkins W, Jones B (2004) Evaluation of dietary inclusion of yellow lupin (Lupinus luteus) kernel meal on the growth, feed utilisation and tissue histology of rainbow trout (Oncorhynchus mykiss). Aquaculture 235:411–422 Hondelmann W (1984) The lupin – ancient and modern crop plant. Theor Appl Genet 68:1–9 Li H, Wylie SJ, Jones MGK (2000) Transgenic yellow lupin (Lupinus luteus). Plant Cell Rep 19:634– 637 McNabb WC, Spencer D, Higgins TJ, Barry TN (1994) In vitro rates of rumen proteolysis of ribulose-1,5-bisphosphate carboxylase (Rubisco) from lucerne leaves, and of ovalbumin, vicilin and sunflower albumin-8 storage proteins. J Sci Food Agric 64:53–61 Moir RJ (1970) Implications of the N:S ratio and differential recycling. In: Muth OH, Oldfield JE (eds) Symposium: sulfur in nutrition. AVI, Westport, pp 165–181 Molvig L, Tabe LM, Eggum BO, Moore AE, Craig S, Spencer D, Higgins TJV (1997) Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L) expressing a sunflower seed albumin gene. Proc Natl Acad Sci USA 94:8393–8398 Molvig L, Morton RL, Tabe LM, Higgins TJV (2002) Generation of selectable marker-free transgenic lupins (Lupinus angustifolius L) using a twin T-DNA binary vector. In: IAPTCB (ed) The importance of plant tissue culture and biotechnology in plant sciences. [Proceedings of the seventh meeting of the international association for plant tissue culture and biotechnology (Australian region)] University of New England, Brisbane, pp 137–144 Mulin M, Bellio-Spataru A (2000) Organogenesis from hypocotyl thin cell layers of Lupinus mutabilis and Lupinus albus. Plant Growth Regul 30:177–183 Nadolska-Orczyk A (1992) Somatic embryogenesis of agriculturally important lupin species (Lupinus angustifolius, L albus, L mutabilis). Plant Cell Tissue Organ Cult 28:19–25 Pannell DJ (1998) Economic assessment of the role and value of lupins in the farming system. In: Gladstones JS, Atkins CA, Hamblin J (eds) Lupins as crop plants: biology, production and utilization. CAB International, Wallingford, pp 339–351 Papineau J, Huyghe C (1989) Collecting white lupin in the Azores. FAO/BPGR Plant Genetic Resour Newsl 88/89:77–78 Phoplonker MA, Caligari PDS (1993) Cultural manipulations affecting callus formation from seedling explants of the pearl lupin (Lupinus mutabilis sweet). Ann Appl Biol 123:419–432 Pigeaire A, Abernethy D, Smith PM, Simpson K, Fletcher N, Lu CY, Atkins CA, Cornish E (1997) Transformation of a grain legume (Lupinus angustifolius L.) via Agrobacterium tumefaciensmediated gene transfer to shoot apices. Mol Breed 3:341–349

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Ravindran V, Tabe LM, Molvig L, Higgins TJV, Bryden WL (2002) Nutritional evaluation of transgenic high-methionine lupins (Lupinus angustifolius L) with broiler chickens. J Sci Food Agric 82:280–285 Rybczynski JJ, Podyma E (1993) Preliminary studies of plant regeneration via somatic embryogenesis induced on immature cotyledons of white lupin (Lupinus albus L). Genet Pol 34:249–257 Sator C (1985) Studies on shoot regeneration of lupins (Lupinus L.). Plant Cell Rep 4:126–128 Schmidt CP, Pannell DJ (1996) The role and value of herbicide-resistant lupins in Western Australian agriculture. Crop Prot 15:539–548 Sroga GE (1987) Plant regeneration of two Lupinus species from callus cultures via organogenesis. Plant Sci 51:245–249 Sweetingham MW, Jones RAC, Brown AGP (1998) Diseases and pests. In: Gladstones JS, Atkins CA, Hamblin J (eds) Lupins as crop plants: biology, production and utilization. CAB International, Wallingford, pp 263–289 Tabe L, Higgins TJV (1998) Engineering plant protein composition for improved nutrition. Trends Plant Sci 3:282–286 Tepfer M (2002) Risk assessment of virus-resistant transgenic plants. Annu Rev Phytopathol 40:467–491 White CL, Tabe LM, Dove H, Hamblin J, Young P, Phillips N, Taylor R, Gulati S, Ashes J, Higgins TJV (2001) Increased efficiency of wool growth and live weight gain in merino sheep fed transgenic lupin seed containing sunflower albumin. J Sci Food Agric 81:147–154 Yang HA, Sweetingham MW, Cowling WA (1996) The leaf infection process and resistance to Pleiochaeta setosa in three lupin species. Aust J Agric Res 47:787–799 Yang H, Boersma JG, You MP, Buirchell BJ, Sweetingham MW (2004) Development and implementation of a sequence-specific PCR marker linked to a gene conferring resistance to anthracnose disease in narrow-leafed lupin (Lupinus angustifolius L.). Mol Breed 14:145–151

Section V Regulatory and Intellectual Property of GM Plants

V.1 Freedom to Commercialize Transgenic Plant Products: Regulatory and Intellectual Property Issues S. Chandler1 and J. Rosenthal2

1 Introduction If the ultimate goal of a research program is commercial release of genetically modified (GM) plants, then intellectual property (IP) and regulatory considerations are of paramount importance and must be identified and evaluated as early as possible in the research program. As the pathway to commercialization proceeds, many different options will be revealed and here too the IP and regulatory aspects of the project must be given heavy weighting. One key IP issue revolves around whether the commercial project utilizes third-party technology, and if so is it possible, economically, to access this technology. The investigation of third-party technologies that may impact on the ability to commercialize a product are generally referred to as freedom to operate (FTO) issues. In order to access this technology, there is generally a need to license it from the owner of the intellectual property (generally patents) covering the technology. Often this requires a lengthy and expensive license negotiation that can include the payment of upfront fees and/or royalties. If some of the intellectual property is unique, and protectable, a second major decision is whether the protection of the technology, and the exclusive rights this may embody, can be justified on economic grounds. Throughout the development and commercialization process, the timing and cost of IP decisions, in a background of an ever-moving technology, requires continuous assessment. The choices regarding regulatory approval are less complex. Without regulatory approval potential products simply cannot be trialed, or released commercially. Regulatory approval is generally required in order to begin the research and development (R&D) and once suitable candidate GMO plants have been identified it is necessary to go through the steps of obtaining approval for field trials and ultimately commercialization. This chapter reviews intellectual property and regulatory aspects along the road to commercialization of genetically modified plants. Attention is also drawn to reviews by Dubois (2003), Nottemburg et al. (2003), Bradford et al. (2005) and Zarrilli (2005). 1 Florigene

Pty. Ltd, 1 Park Drive, Bundoora, VIC 3083, Australia, e-mail: schandler@florigene. com.au 2 Alchemia Oncology Pty. Ltd, Pacific Towers, 737 Burwood Rd, Hawthorn, VIC 3122, Australia

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2 Intellectual Property Intellectual property is an important business asset in today’s global market. When properly managed and protected, IP is a valuable commercial asset which can provide significant competitive advantages. It is often the edge that sets successful companies apart, and as world markets become increasingly competitive, protecting IP becomes essential. Having some form of IP protection for products provides a useful legal means of protecting products from infringement by a third party. It is also a potentially valuable and marketable asset in that it can be licensed-out to a third party, bringing income to the business in the form of license payments and royalties. On the down side, key technology or plant varieties required to develop products may be owned by a third party, which can mean that a license is required. There are various different forms of IP protection available and in the area of plant biotechnology the key ones are patents, trademarks, trade secrets and plant breeder’s rights (PBRs). In addition, the regulatory process itself provides another layer of protection (see Section 3.5). However, by far the most important in terms of freedom to commercialize a GMO plant is the patent. 2.1 Forms of IP 2.1.1 Patents A patent describes an invention and must satisfy certain criteria including the key factors of: (a) being new or novel, (b) involving an inventive step, and (c) being useful. In practical terms a patent can cover products (e.g. genes, regulatory components, markers, plants, seeds, progeny), methods or processes (e.g. transformation methods, cultivation methods, gene expression technology). A patent gives the exclusive right to exploit an invention and to stop other people from utilizing it, for the life of the patent. A patent is legally enforceable and gives the owner the exclusive right to commercially exploit the invention for the life of the patent, which is 20 years for most countries. All applications for patents are examined through the relevant authorities (e.g. www.ipaustralia.gov.au, www.wipo.int, www.uspto.gov) to ensure they meet the necessary legal requirements for grant. Figure 1 provides an overview of the patent process. Patents can be filed in: (a) an individual country only, (b) several countries simultaneously (e.g. in Australia and/or Japan; see right-hand section of Fig. 1), (c) several countries through one application called a Patent Cooperation Treaty (PCT) application (see left-hand section of Fig. 1), or (d) some combination of individual countries and PCT. The decision of how and where to file is based on many factors, such as how quickly a granted patent is required, how many countries are of interest, costs, and so on.

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Fig. 1. Outline of the patent process via either Patent Cooperation Treaty or directly into national phase

Many biotechnology patents are processed via a PCT application because, by filing one international patent application, and designating any or all of the PCT Contracting States (there are presently about 182 nations which are Contracting States, including GM plant markets such as most of Europe and the United States), it is possible to: (a) simultaneously seek patent protection for an invention in each of a large number of countries, (b) gain extra time to decide which countries are of interest for national phase entry, and (c) delay

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paying substantial national/regional phase fees. The PCT application strategy provides more time to determine whether the product under development has future commercial value. PCT applications are administered through the World Intellectual Property Organization (WIPO) which is an international organization dedicated to promoting the use and protection of intellectual property (WIPO 2002, 2007). 2.1.2 Trade Secrets A trade secret is both a type of IP and a strategy for protecting ideas. It can provide effective protection for some technologies (mostly methods), proprietary knowledge (know-how), confidential information, and other forms of IP. If trade secrets are used to protect IP, then generally a confidentiality agreement is used to stop employees from revealing secret or proprietary knowledge during and after their employment or association with the business. If an agreement is breached, there will be evidence of what was agreed and protection through the law. In the GM plant area, trade secrets are mainly in some of the methodologies. Trade secrets generally apply to methods because it is often not possible to determine a method via reverse engineering of a GM plant, whereas other components of the product (e.g. genes, markers) could be determined by analysis of the GM plant. 2.1.3 Plant Breeder’s Rights Plant breeder’s rights (PBRs) are used to protect new varieties of plants by giving legally enforceable, exclusive commercial rights to market a new variety or its reproductive material. To be eligible for PBR protection, it is essential to: (a) show that the new variety is distinct, as well as being uniform and stable, and (b) be able to demonstrate, by a comparative trial, that your variety is clearly distinguishable from any other variety, the existence of which is a matter of common knowledge. Protection generally lasts for up to 25 years for trees or vines and 20 years for other species (www.ipaustralia.gov.au, www.upov.int, www.cpvo.eu.int, www.defra.gov.uk). The process for a PBR application is somewhat similar to that for patents in that there is a registration and examination process. In some countries the term plant variety rights (PVRs) is used instead of PBRs and, in the United States, plant patents. The text that follows refers to PBRs but also encompasses PVRs and plant patents. Plant IP rights are available in many countries around the world and are generally administered from within each country. An exception to this is the UPOV Convention in Europe which was first signed in Geneva in 1961 and is the basis for the plant variety protection throughout the European Union. In Europe, plant IP rights are administered by the Community Plant Variety Office (CPVO) to Member

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States (www.cpvo.eu.int). In the United States, plant patents are administered through the United States Patent and Trade Mark Office (www.uspto.gov). PBRs need to be considered at the start and end of the GM product development process. At the start, it is necessary to assess if there is protection for the plant varieties to be used in R&D, and at the end one needs to determine whether PBR protection will be taken out for the novel product. 2.1.4 Trade Marks In the commercialization phase, consideration is given to the marketing and thus the packaging and trade marking of the GM. A trade mark is a sign which distinguishes the goods or services of one trader from those of another. A trade mark can comprise or consist of a word or words, a logo, signature, letter, numeral, any aspect of packaging shapes such as those of containers and bottles, scent, color or sound. A trade mark is often identified with the product and represents the reputation, quality, and good-will of the company. Thus, it is valuable company property. The right to use a trade mark, and prevent others from using a substantially identical or deceptively similar mark, arises either by use or registration. It is not essential to register a trade mark to use it and, indeed, many plant breeders, growers, wholesalers, and retailers use unregistered marks for their different plant products. Unregistered trade marks accrue common law rights when a trade mark starts to be used. The rights are derived by virtue of the reputation which use of the mark creates. The rights are not obtained by registration and may not necessarily extend further than the region in which the products are sold. The extent of the rights is essentially limited to the area in which the reputation exists. The pros and cons of registering a mark must be weighed up against costs. Registration is often advisable because: (a) it applies to the entire country in which it is registered, (b) it prevents anyone else from using the mark as soon as the application has been submitted to the relevant authority, (c) as long as the mark is used and renewal fees are paid it has an indefinite life, and (d) it is less costly and easier to take action for infringement under the trade mark legislation than it is to exercise action under common law for an unregistered mark. 2.2 Patents and PBRs The two most important forms of IP for GM plants are patents and PBRs and a good product development strategy will take both into consideration. By far the most complicated and expensive form of IP protection is that for patents. PBRs are substantially cheaper because they are more simple and narrow in scope than patents. PBRs only protect a single product, whereas patents can

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potentially cover a whole range of products in a number of different species. Another important distinction between PBRs and patents is that PBRs are limited to GM plants. However, patents, if appropriately drafted, can cover GM plants, their progeny, products, and uses. An example of the scope of patent versus PBR coverage is illustrated by the case of Florigene’s blue GM carnation product range (www.florigene.com.au). The company’s research activity resulted in a number of patents covering genes of the anthocyanin biosynthesis pathway. One patent, the Blue Gene Patent (PCT/AU92/00334; WO 93/01290), provides coverage for all of the six products, GM carnation plants and their progeny, and cut-flower products in Europe. The patent also covers any rose, gerbera, chrysanthemum, or other product that is made using the anthocyanin gene described in the Blue Gene Patent. In contrast, if the products were protected by PBRs, six applications would be required with an additional application for each new product. While it may seem that PBRs are not so useful for GM plants (if broad patent protection is available, then why take out single PBR applications?), PBRs become useful when the patent life is coming to a close and new products are continuing to be developed. It is possible to take out PBR protection to extend the IP protection for individual new varieties for 20 years. 2.3 Freedom to Operate 2.3.1 Overview Throughout the development and commercialization process the timing and cost of intellectual property decisions, in a background of an ever-moving technology, requires continuous assessment. Figure 2 provides an overview of the development of a transgenic plant from the R&D phase to the new GM plant product and demonstrates the need to continuously assess what may potentially limit commercialization of the final GMO product. 2.3.2 R&D Phase The patterns of ownership and control of existing and forecasted technologies must be understood if the associated restrictions are to be overcome and opportunities realized. Therefore, in the planning stage it is important to consider: 1. Do PBRs exist for the plant variety to be used as a starting point for transformation experiments? Who are the PBR owners? How long will the PBR last and in which countries do they exist? 2. Do patents exist that cover any of the methods (e.g. plant transformation) or components (e.g. genes, regulatory elements, markers) to be used to develop the GM product? What is the status of the patent? How much time remains

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Fig. 2. Freedom to commercialize transgenic plants – overview of freedom to operate issues and considerations

in the patent life? Are there any court actions in progress? Do patents exist in the county where the R&D is taking place and/or in countries of interest for plant production and sales? The test for considering a license for existing IP is: would there be infringement of another party’s IP during GM product development and commercialization? At times this is difficult to answer. For example, in one scenario there could be a patent application which has broad claims that would cover the product, but during examination and prosecution the claims could be greatly limited or removed. There is therefore a need to make an assessment of the likely scope of the granted claims. This can be based on knowledge of the law and current trends, but there are no guarantees. It is at times useful to discuss outcomes with a qualified patent attorney who has experience in obtaining patent grants, and once a patent application has been identified which potentially affects FTO, it is important to monitor it.

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If there are PBRs or patents that would cover a GM product, consider: 1. Whether it is necessary to obtain a license from the IP owner. This depends on the IP laws in the county, as in some jurisdictions R&D is exempt and a license is only needed at commercialization. 2. How much is available to pay for the license in terms of up-front fees, royalties, milestone payments? 3. What type of license is needed? R&D and/or commercialization? Exclusive or non-exclusive? After consideration of the cost and benefits it may be necessary to go back to the “drawing board” and redesign the project so as to avoid the need for a license. If a license is necessary, then the next step to consider is: how much is the technology worth and is the patent or PBR granted or just in the application stage? The cost of a license will often increase once the PBR or patent is granted because it has been through examination and the scope of the IP is finalized. Other questions related to the chances of success of the project. Should one take out a license for R&D and see if there is a commercial product, and then negotiate for a commercial license? It is often cheaper to obtain a license for R&D only. However, once a marketable product is available, the owner is locked into the negotiation for a commercial license and this can weaken a negotiating position. Thus, one option is to obtain a license which includes milestone payments or royalties, such that monies are paid in proportion to the actual success of the product in the market place. 2.3.3 Commercialization When it comes to commercialization of the product, then it is important not only to revisit the patent and PBR situation, but also to consider how the product will be marketed and presented or packaged, i.e. what trade marks would be useful and in what countries? Would it be useful to take out PBR protection on the product? Is there any IP that has been developed which should be protected before being made public? What countries are of interest? While R&D may have been done in one country, plans for commercialization may include other countries, requiring analysis of the IP situation in each country of interest. 2.3.4 Licensing License fee amounts are hard to estimate and are dependent on many factors, namely: 1. Is an exclusive or non-exclusive license required? Exclusive licenses are usually “more” expensive because the IP owner cannot obtain extra revenue by issuing the IP to another party. 2. The number of plant species or varieties to be covered by the license agreement. Often more equals more expense.

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3. The status of the IP. If patents or PBRs are not yet granted the cost is often “lower” because there is some uncertainty about the IP outcome. If a patent is undergoing any legal action, such as an opposition by another party, then there is a risk that the patent will be discontinued or limited. Therefore, uncertainty drives the cost down. The types of fee components in licensing can include up-front fees, annual fees, milestone fees, and royalties. A commonly employed licensing strategy is to pay a moderate up-front fee during the R&D phase and, if a GM product comes out of the project, a royalty based on sales is payable. The art of license negotiation is very complex and there are no hard and fast rules. Each license need to be considered based on all the known facts as well as on predictions and expectations of the unknown. 2.3.5 Resources, Tools, and Vigilance It is important to regularly monitor IP databases and to investigate what applications have been filed and where, the status of each application, and the scope of the claims. As mentioned above, many patents are filed with broad claims, but by the time they have been through the examination process, the scope of patent coverage can be limited considerably. Many IP applications are filed, but do not make it to grant for a variety or reasons (e.g. expense, they do not survive examination, limited to only a few countries). There are many free IP databases available via the internet (e.g. the European Patent Office Database, the United States Patent and Trade Mark Office, IP Australia, Community Plant Varieties Office). For a fee, it is also possible to subscribe to commercial databases (e.g. Derwent, Questel–Orbit). The feepaying databases generally provide far more sophisticated search strings and are, therefore, often essential for finding all the IP of interest. Alternatively, for a fee, an IP attorney can conduct specific searches. As well as the IP databases various organizations, such as CAMBIA, provide a valuable resource in the plant biotech area (www.cambia.org.au). CAMBIA is an Australian institute that is creating new tools and technologies to foster innovation in the areas of food security, agricultural and environmental problems. These tools include the world’s largest free full-text searchable patent database, a variety of technology landscape papers and patent tutorials. CAMBIA protects its own IP through patents and trademarks in many jurisdictions, and major players in agricultural biotechnology including DuPont de Nemours, Monsanto, Novartis, Rhône-Poulenc, Dow, and Pioneer license CAMBIA’s technologies (www.cambia.org.au).

3 Regulatory Approval Since the development of genetically modified plants became an option, legislation was developed to regulate their release into the environment. Parallel

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legislation to regulate the use of genetically modified plants and plant extracts in food, food additives, and animal feed was also developed. Accordingly, the commercial release of a genetically modified plant must take into account the local, national, and international regulatory frameworks that exist around the world. Without the relevant approvals, the release of a genetically modified organism may be illegal. 3.1 The Regulatory Process The regulatory process is essentially a step-by-step process, in which laboratory results, obtained under contained conditions, are followed by trials, which are followed by applications for commercial release. Trials may be in the contained or semi-contained conditions of glasshouses, or may be large field trials. Whatever the scale, they must be of sufficient containment and competent design to provide conclusive evidence to the regulator that the GMO plant may be released into the general environment. Typically, less data is required to support an application for laboratory work than for release into the environment. The situation for an internationally traded commodity from a GM plant, such as exported grain, is that the importing country may or may not require field trials in their own country prior to allowing importation. This depends, to some extent, on whether the countries in question are signatories to the Biosafety Protocol (see below). 3.2 Legislative Frameworks Table 1 lists the agencies that legislate and/or administer the regulation of GM plants in some of the major agricultural producers and consumers of the world. Usually, multiple national agencies play a role in regulation, as some have responsibility for the environment, some for ethical and strategic issues, some for therapeutic or agricultural chemicals, and some for health and safety. A single office may play a coordination function, such as is the case in India (Indira et al. 2005) and Brazil. The internet is the first point of access to the application process in a specific country. The website addresses listed in Table 1 provide an entry point to identify the relevant contact officers and may also be used to download up-to-date legislation and application forms. Additional contacts are provided on the website of the Biosafety Clearing House (see below). Information about previous releases of GMOs are a very important resource and can be easily accessed via the internet, at the portals identified in Table 1. In the case of the United States and Canada, the sites are also a source of in-depth risk assessments directed towards certain crops. The recently established register of GM foodstuffs in the EU (europa.eu.int/comm/food/food/biotechnology/authorisation/register) provides links to detailed risk assessments for the most commonly traded GM food stuffs. The Eu-

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Table 1. Internet access points to legislation of genetically modified organisms in key countries

Country

Agency responsible Agency responsible for environmental safety for food safety

Portal details

United States

Department of The Food and Drug Agriculture (APHIS), Administration (FDA) Environmental Protection Agency (EPA) Canadian Food Canadian Food Inspection Agency Inspection Agency Office of the Food Standards Gene Technology Australia and Regulator (OGTR) New Zealand (FSANZ) Several Several community-wide community-wide directives directives Ministry of Education, Ministry of Agriculture, Culture, Sports, Forestry and Fisheries Science and Technology of Japan (NEXT), Ministry of Agriculture, Forestry and Fisheries of Japan (MAFF), Ministry of the Environment (MOE) Coordinated through Coordinated through National Technical National Technical Biosafety Committee, Biosafety Committee, Ministry of Ministry of Science and Technology Science and Technology Ministry of Agriculture, Ministry of National Environment Public Health Protection agency Ministry of Science and Ministry of Science and Technology, Technology Ministry of Environment and Forestry Ministry of Ministry of Science and Technology Agriculture and Food Agricultural Directorate Agricultural Directorate of the Secretariat of the Secretariat of Agriculture, of Agriculture, Livestock, Fisheries, Livestock, Fisheries, and Food and Food

www.aphis.usda.gov/brs

Canada Australia

European Union Japan

Brazil

China

India

Russia Argentina

www.inspection.gc.ca www.ogtr.gov.au; www.foodstandards.gov.au biotech.jrc.it; gmoinfo.jrc.it www.maff.go.jp

www.ctnbio.gov.br

www.agri.gov.cn

dbtindia.nic.in

www.aris.ru Burachik and Traynor (2002); www.sagpya.mecon.gov.ar

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ropean Community (EC) has a unique regulatory system (Christoforou 2004), in that a single application for commercial release is ultimately reviewed by, and if successful applies to, all the member countries of the EC. It can be that decisions are made on the basis of votes, and these votes are apportioned to countries as a function of their population. The approval system is therefore complex. 3.2.1 The “Cartagena Protocol” The international legislation covering genetically modified plants is the Protocol on Biosafety in Biotechnology, more commonly known as the “Cartagena Protocol”. This has been developed within the Conference of the Parties on the Convention of Biological Diversity. The protocol was approved in January 2000 and has since come into force. Details are provided on the website of the biodiversity treaty secretariat (www.biodiv.org/biosafety). In essence, the protocol commits signatory countries to ensuring that activities involving GMOs (referred to as living modified organisms, or LMOs, in the protocol) are conducted so as not to pose a risk to biodiversity or the environment. This is to be achieved through a system on notifications of transboundary movement and the provision of a Biosafety Clearing House (bch.biodiv.org). The latter is an internet-based information exchange allowing the registration of transgenic events, risk assessments, and other information by the various signatory countries. Eventually, it is expected that harmonization through the clearing house will bring conformity to legislation of genetically modified organisms and make international trade less complex. However, details such as labeling, documentation, liability, and conflict resolution are still being finalized. The United States, where the release of genetically modified plants in agriculture is well established, is not a signatory to the biodiverity treaty. Other countries are signatories, but have not adopted the Cartagena protocol. A key principle enshrined in the treaty is the precautionary principle, and the same principle is accordingly an element of the regulatory assessment process in those countries which are signatories to the protocol, such as the EC. Legislation in other countries places more emphasis on the principle of substantial equivalence (Katz 2001; Conko 2003; Daemen 2003; van den Belt 2003). 3.3 Regulatory Considerations for Researchers Before research is undertaken, there are regulatory considerations that should be included in the R&D planning. Some of the most important are outlined below. As part of the trial process, it is necessary to collect information to support subsequent applications for regulatory approval, for either subsequent trials or commercial release.

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3.3.1 The Species A key component of regulatory assessment is the potential for gene dispersal from the genetically modified plant, and this should be taken into account when considering research plans. For example, a plant species which produces copious amounts of wind-dispersed pollen, or a significant amount of seed is inevitably subject to closer scrutiny than a non-flowering crop. While this is not a fundamental barrier to commercialization, it may make the approval process longer and more involved. A species with related wild species in the release environment, particularly if these are compatible, and particularly if the compatible species are weeds, is assessed more closely than an introduced species with no relatives in the release environment. 3.3.2 The Parental Variety For the major food crops and most horticultural and ornamental species, a number of commercial varieties have been developed and there is therefore a choice of parental cultivars or varieties for transformation. Research and commercial considerations, such as ease of transformation and the commercial strength in the target market place a bearing on the choice of variety. However, the choice at an early stage of research may have a later bearing on the regulatory procedures leading to commercial release approval. For example, it may be that certain varieties have a low fertility, or are male sterile. Where the transgenic event is later to be introgressed into a number of other varieties, or “stacked” with other transgenic phenotypes, the suitability of the parent variety as a breeding line becomes important. 3.3.3 The Selectable Marker Whilst there are techniques for their removal, the selectable marker gene is normally present in the final genetically modified product. The presence of the selectable marker gene is not a fundamental barrier to commercial release, but may have implications for the regulatory process. In cases where selection uses herbicide-resistance genes and where herbicide resistance is not the intended commercial phenotype, herbicide resistance is a secondary effect of the primary phenotypic change. The regulatory process must take into account herbicide resistance as well as the “primary” genetic modification. This simply adds to the complexity of regulatory approval, as potential for spread of herbicide resistance is a key focus of regulatory review. The other commonly used selectable markers are antibiotic resistance genes. In non-food crops, these may be a more sensible choice than herbicide resistance genes. In food crops, use of antibiotic resistance genes requires more careful consideration, as there is concern in some quarters about the use of such genes in food, due to a perceived risk of horizontal gene transfer. This issue was recently reviewed by

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the Scientific Panel on Genetically Modified Organisms, in the EC (European Commission 2004b). Once the population of transgenics has been created it is usually the case that there are a number of genetic events that are suitable for further trials. The selection of events to trial can have a significant impact on the regulatory process. As the principle of substantial equivalence is still important, screening of initial transgenic populations for close similarity to the parental variety is a useful basis for early selection. If the variety is known to produce a useful, or potentially harmful, key metabolite, then analysis of the concentration of the compound is also relevant. Depending on the jurisdiction, the requirement for molecular characterization of the genetically modified plant event can be extensive: details of sites and number of transgene integrations, arrangement and sequence of inserted DNA, and sequences across the borders of the integration sites (including from the parental genome) may all be required. The collection of this information may be time-consuming and very difficult unless the integration pattern is relatively simple. 3.4 Trials and Generation of Data for Regulatory Approval Applications The trial of transgenic events is an essential part of the product development process. Trials are designed to measure the stability of gene expression, the behavior of the altered phenotype in different environmental conditions, and the performance of the GMO against that of comparator varieties. 3.4.1 Potential Effects of a Genetically Modified Plant on the Environment In both non-food and food plants, the data requirements related to potential environmental impacts of a genetically modified plant are similar, and are summarized in Table 2. 3.4.2 Food Safety Above relatively low threshold levels, it is normally not permitted to use or process a GM food or food additive, unless it has received regulatory approval. In some jurisdictions, animal feed is also subject to regulatory approval, as is material extracted from a GM plant, such as oils. It is therefore necessary to understand the precise regulations in all places the GM plant will be commercialized. Details on information required for the approval of a GM food crop are summarized in Table 3. Excellent additional information is available from the Institute of Medicine and National Research Council of the National Academies (NAS 2004). In the design of field trials in general; but particularly where the GMO will be used in the food chain, it is essential to include as a comparator

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Table 2. Summary of data and information typically required for regulatory review of the potential environmental impact of a plant GMO

Feature of the GMO

Example data sets and information

Parent organism

Biology and taxonomy, reproductive biology, weedy characteristics and survivability, geographical distribution, natural habitat Pathogenicity, natural habitat Description of the vector, description of genetic elements, sequence of the vector, the transformation process Description, morphology compared to parental organism, expected expression in a range of environments Southern analysis, Northern analysis, measures of expression of the transgene, such as Westerns, evidence for absence of vector, sequence of flanking regions at insertion sites Changes to capacity for vegetative propagation, changes in pollen viability, fertility or capacity for seed set and dispersal, hybridization experiments with potential target plants, reproductive characters compared to the parent plant Enhancement of any weedy characteristics in the GMO, potential indirect or direct effects on plant populations Potential effect on crop management, potential effects on other organisms, potential to add or subtract substances to the environment Potential selective advantages or disadvantages in the release environment or in mixed plant populations

Donor organism(s) Introduced genetic material

Altered phenotype

Genetic modification

Potential for gene dispersal from the genetically modified organism

Potential for weediness

Possible ecological effects

Possible effects on the environment

variety the parental line that was transformed, and additional iosgenic lines if they are available. This is in order to demonstrate substantial equivalence, an underlying principle in food safety studies. Whether the food product will be processed, or eaten raw, or cooked is an important factor in food safety risk assessment. 3.5 Other Regulatory Issues 3.5.1 Timeframes The timeframes for obtaining regulatory approval are normally clearly identified as part of legislation and vary depending on whether a small-scale release

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Table 3. Summary of data and information typically required for regulatory review of the potential safety in food of a plant GMO

Feature of the GMO

Example data sets and information

Parent organism Donor organism

History of safe use as a food Known pathogenicity and previous history of safe use as a food, potential allergenic properties The encoded protein and its function Description of the vector, description of genetic elements, sequence of the vector, the transformation process, sequence of flanking regions at insertion Description, morphology compared to parental organism, any intended levels of nutrient alteration, variation in concentration of nutrients in different environments, potential for modification of the modified phenotype (after processing for example) Southern analysis, Northern analysis, measures of expression of the transgene, such as Westerns, immunoblotting PCR, biochemical assay How the food will be processed and consumed Key metabolite concentrations, comparison to nutrient levels in other foods Sequence comparison to databases, similarity to know toxins, levels of exposure, heat stability of introduced protein, simulated digestion studies, acute oral toxicity studies, animal feeding studies Results of immune tests, similarity of protein to know allergens, resistance of protein to heat and digestion Assessment of interaction with gut microflora

Altered phenotype Introduced genetic material

Altered phenotype

Genetic modification

Detection technique Dietary intake Nutritional data Toxicological data

Allergenicity Horizontal gene transfer

is planned, or commercial release is needed. For release at commercial level, most legislation includes so-called stop clock provisions, under which regulators may seek further information from the applicants, hold public enquiries or seek third party advice. As these delays are not predictable, it is a high-risk strategy to base commercial plans, such as marketing strategies, on legislated timeframes for application assessment. The approval process takes several years from laboratory to marketplace. 3.5.2 Unique Identifiers Central to the regulatory process, and set up as a searchable database in the clearing house of the biosafety protocol, is the use of unique identifiers. These can be self-assigned from the field trial stage of the approval process, using the simple process adopted for unique identifier generation in the EC (European

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Commission 2004a). The unique identifier refers to a single, specific genetic event. Once fully characterized, approval for the event should allow regulatory approval for progeny carrying the event, and this is obviously critical for seed crops. The use of unique identifiers also streamlines assessments for food safety, and the future release of improved varieties from breeding programs. 3.5.3 Post-Release Monitoring In field trials, post-release monitoring may be imposed by the regulator in order for the trials to be carried out. Primarily, this is to ensure no volunteer plants grow in subsequent years as a result of seed dispersal or vegetative materials left in the soil at the conclusion of the trial. Sterilization of the field trial site at the end of the trial is an alternative approach. In either event, it is necessary to make adequate plans for monitoring. After commercial approval, post-release monitoring may or may not be required by the regulator. In Europe, post-release monitoring is mandatory. 3.5.4 Data Protection A regulatory approval is a valuable document, often acquired after extensive, and expensive, research. Unfortunately for the developer, data protection is limited, and once a GM plant is approved for commercial release, this may extend to any party. If not, then data placed in the public domain, or secured through freedom of information will certainly accelerate approvals for similar products. It should not be expected that the regulatory process be repeated each time a transgenic event is included in a commercial product by different parties, as this would be both unnecessary and a waste of administrative resources by the regulator. However, it is possible in the course of most regulatory application processes to make some parts of the information provided commercial in confidence. The degree to which this will be allowed is determined by the regulator in the jurisdiction in question. In some cases, public transparency is encouraged or legislated, perhaps to the extent of the publication of exact field trial locations. 3.5.5 Politics Though the regulation of GM plants is a science-based risk assessment process, this objective has been obscured because of the public controversy surrounding genetic modification. Accordingly ethical, religious, and economic factors must all be taken into account during the process of applying for the commercial release of a GM product, and they may be a formal part of the regulatory process. The professional lobbying skills of groups opposed to gene technology, and natural alliances to certain political parties, inevitably meant that the devel-

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opment of legislation concerning genetic modification became politicized, to some extent. From a commercial perspective, this means that scientific factors cannot be the only concerns when seeking regulatory approval for a genetically modified plant product, especially when that product is intended for the international market. At the government level of local council, city, county, and state, the prevailing policy may not necessarily be in line with that of national government policy or even legislation. For example, local governments may declare “GM free” zones or attempt to ban genetically modified plant products from the local community. Whilst these bans are essentially impossible to enforce, the underlying policy clearly makes it more difficult to begin a regulatory process where local government approval is needed, to initiate field trials for example. Most of the national governments where genetically modified plants are grown and consumed are elected, and therefore subject to change every 3–5 years. The majority of political parties have a policy towards genetic modification, sometimes as a result of the natural inclination of opposition parties to target prevailing policy. It is sensible to be aware of all policies, particularly as they might apply to any subsequent changes to legislation.

4 Conclusion The road that leads from the idea to the laboratory to commercialization of a GM product is a long one with the need for constant vigilance and activity to ensure a product is adequately protected by IP, does not infringe any other IP, and fulfills all the regulatory requirements in the jurisdiction of importance. To cover all of this requires strategic planning and a substantial budget. There are certainly many hurdles to cross before a GM product ever makes it to market and it is of concern that IP and regulatory rights and regimes could be impeding agricultural research and development, especially for small players. By the same token, these rights and regimes provide comfort in that effort is rewarded by, for example, a 20-year patent monopoly, and that the public interests are protected by regulatory authorities (Bradford et al. 2005).

References Belt H van den (2003) Debating the precautionary principle: “guilty until proven innocent” or “innocent until proven guilty”. Plant Physiol 132:1122–1126 Bradford KJ, Van Deynze A, Gutterson N, Parrott W, Strauss S (2005) Regulating transgenic crops sensibly: lessons from plant breeding, biotechnology and genomics. Nat Biotechnol 23:439–444 Burachik M, Traynor PL (2002) Analysis of a national biosafety system: regulatory policies and procedures in Argentina. (ISNAR country report 63) International Service for National Agricultural Research, The Hague

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CAMBIA (2007) Available at: www.cambia.org.au Christoforou TH (2004) The regulation of genetically modified organisms in the European Union: the interplay of science, law and politics. Common Market Law Rev 41:637–709 Community Plant Varieties Office (2007) Available at: www.cpvo.eu.int Conko G (2003) Safety, risk and the precautionary principle: rethinking precautionary approaches to the regulation of transgenic plants. Transgenic Res 12:639–647 Daemen THJ (2003) The European community’s evolving precautionary principle – comparisons with the United States and ramifications for Doha round trade negotiations. Eur Environ Law Rev 2003:6-19 DEFRA (2007) UK plant breeders’ rights handbook. (2007) Available at: www.defra.gov.uk Derwent (2007) World patents index. Available at: www.derwent.com Dubois LAM (2003) Plant patents and trademarks. In: Roberts AV (ed) Encyclopedia of rose science. Elsevier, Amsterdam, pp 473–479 European Commission (2004a) Establishing a system for the development and assignment of unique identifiers for genetically modified organisms. (Commission Regulation (EC) No 65/2004) J Eur Union 2004L:10/10 European Commission (2004b) Opinion of the scientific panel on genetically modified organisms on the use of antibiotic resistance genes as marker genes in genetically modified plants. EFSA J 48:1–18 European Patent Office Database (2007) Available at: ep.espacenet.com Florigene Limited (2007) Available at: www.florigene.com.au IP Australia (2007) Available at: www.ipaustralia.gov.au Indira A, Bhagavan MR, Virgin I (2005) Agricultural biotechnology and biosafety in India: expectations, outcomes and lessons. Stockholm Environment Institute, Stockholm Katz D (2001) The mismatch between the biosafety protocol and the precautionary principle. Georgetown Intl Environ Law Rev 2001:949–982 NAS (2004) Safety of genetically engineered foods; Approaches to assessing unintended health effects. Institute of Medicine and National Research Council of the National Academies, Washington, D.C. Nottemburg C, Binnenbaum E, Pardey PG, Wright BD, Zambrano P (2003) South–north trade, intellectual property jurisdictions, and freedom to operate in agricultural research on staple crops. Econ Dev Cult Change 51:309–335 Questel–Orbit Intellectual Property Group (2007) Available at: www.questel.orbit.com/index.htm UPOV (2007) International union for the protection of new varieties of plants. Available at: www.upov.int/index.html US Patent and Trade Mark Office (2007) Available at: www.uspto.gov WIPO (2002) Basic facts about the patent cooperation treaty (PCT): the worldwide system for simplified multiple filing of patent applications. [WIPO publication no 433 (E)] Available at: www.wipo.int/ebookshop WIPO (2007) World Intellectual Property Organization. Available at: www.wipo.int Zarrilli S (2005) International trade in GMOs and GM products: national and multilateral legal frameworks. (Policy issues in international trade and commodities study series no. 29; UNCTAD/ITCD/TAB/30) United Nations, New York

Index

abscisic acid (ABA) 68, 157, 199, 230, 326 acclimatization 158, 234, 291 Ace-AMP1 235 Agrobacterium 19, 20, 22, 70, 108, 117–119, 124, 126, 127, 255, 277, 278, 359 – A. rhizogenes 196, 234 – A. tumefaciens 8, 307 Agrobacterium-mediated transformation 9, 10, 19, 22, 41, 47, 71, 90, 92, 93, 96, 117, 118, 182, 231, 234, 235, 255, 275, 277–280, 360, 361, 375, 376, 386 agronomic performance 361 alcohol acetyltransferase (AAT) 231 alfalfa (Medicago sativa) 30, 95, 262, 321–331 Alfin1 327 alkaloids 189, 191–193, 195, 196, 198–201 – atropine 189–191, 193 – biosynthesis 179, 192–194, 196 – hyoscyamine 189, 191–193, 195, 197, 198, 200 – metabolism 178, 183 – scopolamine 189–193, 197, 198, 200 – tropane 191–193 – tropine 193, 194 aluminium 338, 342–344, 350, 354, 355 aminocyclopropane-1-carboxylate synthase 230, 245 amino acids 49, 328, 403 amplified fragment length polymorphism (AFLP) 43, 63, 64, 70, 83, 84, 86, 87, 89, 99, 184, 227, 228, 229, 230, 233, 377 androgenesis 129 annual ryegrass (Lolium multiflorum) 373, 375, 376, 386 anther culture 135, 136, 376 anthocyanins 246, 308, 312–314, 330, 337, 339, 340, 347, 416 antibiotics 9–11, 118, 136, 201, 279, 281, 282, 284

anti-cancer drug 205 – gene expression 284 – resistance 183, 281, 284, 401, 423 – selection 92, 281 antisense 108, 121, 125, 127, 329, 330 – expression 49 – RNA 72, 341, 381 – suppression 113 – technology 121 apical meristem 14, 21, 22, 66, 109, 117, 119, 132, 285, 384, 402 Arabidopsis 30, 34, 66, 70, 74, 94, 115, 119, 132, 139, 161, 230, 243, 245, 256, 257, 260, 266, 282, 285, 286, 292, 301, 329, 339, 343, 344, 346, 349, 364, 367, 383, 384 Arachis (see peanut) arginine decarboxylase (ADC) 196, 197 atrazine chlorohydrolase 325 Atropa belladonna 196–199 axillary meristem 14–17, 21 baccatin III 206, 209, 212–216, 218–220 Bacillus thuringiensis (Bt) 33, 46, 97, 120, 124, 262, 263 – Bt gene 325 – Bt toxin 262 – cry1Ab 263 – cry1Ac 46 – Cry1Ca 264, 265 – cyr1F 46 bacterial artificial chromosomes (BAC) 65, 88, 229, 323, 345 – clones 88, 162, 229 – library 161, 229 bar 19, 20, 94, 108, 111, 114, 119, 159, 276, 282, 292, 293, 295, 399, 401, 402, 405 basta 71, 73, 74, 108, 111, 118, 119, 159, 401, 402, 406 belladonna 189, 193 bialaphos 119, 121, 159, 276, 282, 292

432

biolistic 70, 90, 91, 94, 95, 99, 275, 291, 292–295, 359, 375, 376 bioreactor 151–155, 160, 198, 199, 209, 210, 215–220, 249 bipolar plantlets 233 breeding 61, 129–131, 135, 139, 322 bulked segregant analysis 229 caffeic acid 3-O-methyl transferase (COMT) 308, 329, 330, 365, 381 caffeoyl CoA 3-O-methyl transferase (CCOMT) 329, 330 calcium alginate beads 219 California poppy 183 calystegine 190, 195, 197, 200 carnation (Dianthus spp.) 241–250 – architecture 243 – breeding 242 – color 246 – flower 243, 244, 246 – structure 243 – yield 244 carotenoid 34, 259, 302, 308, 312–315 cDNA 33, 49, 63, 65, 179, 231, 257, 378, 381, 385 – clones 50, 65, 70, 83, 330–331, 379 – libraries 42, 43, 65, 162, 184, 231, 378, 380 – sequences 365, 382 cell immobilisation 214, 215, 220 cellulose 137, 329, 340, 341, 365, 380, 381 chalcone synthase 255, 257, 259, 302, 308, 312, 348, 349 chalcones 246 chitinases 95, 97, 111, 134, 135, 159, 234, 235, 249, 258, 261, 325, 385 chloroplast 21, 41, 42, 50, 114, 135, 300, 302, 383 – DNA 163, 301 – genome 42, 163 – transformation 21, 23, 42, 50, 122, 366 chrysanthemum (Chrysanthemum x morifolium) 241, 253–269 – transformation 255 chrysanthemum stem necrosis virus (CSNV) 261 chrysanthemum stunt viroid (CSVd) 257, 260, 261 chrysanthemum virus B (CVB) 260

Index

cinnamyl alcohol dehydrogenase (CAD) 137, 138, 308, 329, 365, 381 clonal fidelity 67 cocaine 190 co-cultivation 9, 11, 19, 20, 22, 32, 234, 278, 279 Coleoptera 46 color 256 – antirrhinum antisense chalcone synthase (CHS) 256 – chimeric flavonoid 3 ,5 -hydroxylase 256 – chrysanthemum chalcone synthase (CHS) 256 – chrysanthemum CmCCD4a RNAi 256 – maize flavonoid regulatory cDNA Lc 256 commercialization 411 condensed tannins 330 cotton (Gossyspium spp.) 39, 107–123 cotyledon-node explants 18 – regeneration method 15 – transformation method 17 cryopreservation 68, 254 cyanogenic glucoside 341, 342, 350, 352–355 cystathionine gamma synthase (CgS) 329 cytokinins 4, 14, 42, 68, 92, 155, 158, 245, 278, 285, 367, 385 Datura 189, 196, 198 – D. stramonium 189, 192–194 – D. metel 198 Dendrobium 274–286 diamine oxidase (DAO) 197 digestibility 327, 341, 364 dimethoxytoluene (DMT) 231 Diplocarpon rosae 228 Diptera 46 disease resistance 47 – anti-fungal protein 48 – oxalate oxidase gene (OXO) 47, 48, 50, 97, 98 – RNAi 48 – genes 43, 232 – Rdr1 232 – Rdr2 232 DNA content 161, 227 DNA markers 30, 43, 63, 242

Index

DNA methylation 69, 70, 130 dormancy 158, 292, 293, 295, 322 doubled haploids 135, 376 drought tolerance 114, 323, 367, 379 Duboisia 189, 193, 195, 198 – D. leichhardtii 189, 193 – D. myoporoides 189, 193 – hybrid 198 ectopic expression 66, 162, 312–314, 339, 343 edible oil 60, 121 edible vaccine 34, 98, 331 electroporation 21, 23, 32 elicitation 152, 153, 209, 212, 214 elicitors 151, 153, 154, 181, 199, 209, 211–213, 308 embryo – axes 6, 21, 22 – culture 131 – proliferation 9 embryo-like structures (ELS) 130–135 embryogenic – callus 5–7, 69, 117, 152, 155, 159, 232–235, 289, 360, 375 – cell suspension 67, 74, 108 – cultures 5, 12, 71, 73, 74, 90, 91, 235, 359 endophyte 375, 376, 378–380, 387 – Epichloë 378–380 – Neotyphodium 378–380 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) 110, 325 – epsps gene 19 epicotyl explants 15, 159 epigenetic 68, 69, 74, 130, 220 ethylene insensitive mutants 230 – RhETR1 230 – RhETR2 230 – RhETR3 230 – RhETR4 230 ethylene perception 230, 245, 246 expressed sequence tags (ESTs) 3, 42, 43, 65, 83, 162, 231, 246, 261, 323, 378 FAO 171 fatty acids 29, 31, 33, 39, 45, 49, 51, 59, 60, 62, 65, 71, 72, 90, 121, 123, 129, 133, 136, 138, 162, 386

433

fertility 8, 9, 12, 14, 30, 359, 402, 423, 425 Festuca (see tall fescue) fingerprinting 66, 84, 138, 308, 310 flow cytometry 59, 69, 161, 301, 305 flower 243, 244, 246 – architecture 243 – color 228, 246, 247, 253, 254, 256, 257, 259, 268, 285, 286, 305, 306, 312–316 – 2 ,4 ,6 ,4-tetrahydroxychalcone 259 – carotenoid cleavage dioxygenase CmCCD4a gene 259 – CHS gene 259 – development 59, 65, 230 – flavonoid-3 -hydroxylase (F3H) 247, 259 – flavonoid-5 ,3 -hydroxylase 246, 257, 259 – yield 230, 231, 241, 247–249, 274 flowering 6, 30, 59, 65, 68, 177, 178, 192, 193, 228, 229, 235, 241, 243, 245, 255, 257, 259, 260, 264, 265, 268, 273, 274, 285, 292, 299, 306, 337–339, 346, 375, 380, 382–384, 387 – CONSTANS 384 – indeterminate gene (id1) 384 – TERMINAL FLOWER 1 (TFL1) 384 flowering time 30, 257, 268, 274, 285, 306, 384 – Arabidopsis Leafy (LFY) 257 forage 321–323, 325, 327–331, 338, 340–342, 344–349, 357–359, 362, 364, 366, 367, 373–375, 380–383, 385, 387, 397 – quality 321, 323, 325, 327, 331, 340, 341, 345, 364, 367, 381, 382, 385 forage legume 321, 328, 338, 340–342, 344–349 Forsythia 299–302, 305–316 – biotechnology 308 – botanical origin 299 – F. europaea 299, 301, 302, 304 – F. europaea x F. suspensa 305 – F. japonica 299, 301 – var. saxatilis 301 – F. koreana 299, 301, 307, 310 – F. koreana-manshurica-saxatilis 310 – F. likiangensis 299, 301 – F. mandshurica 299, 301, 305 – F. mira 299 – F. nakaii 301

434

– – – –

F. ovata 299, 301, 305, 307 F. ovata x F. suspensa 305 F. saxatilis 299, 301 F. suspensa 299, 301, 302, 304, 305, 307, 315 – var. sieboldii 299, 305 – F. suspensa x F. viridissima hybrid 301 – F. togashii 299 – F. viridissima 299, 301, 305 – var. koreana 301 – F. x intermedia 299, 301, 305–308, 310, 312–315 – F. x kobendzae 305 – F. x variabilis 305 – mutants 305–307, 310 – plant regeneration 307 fosmidomycin 210, 214 fructan 377, 380, 382, 383, 386 – exohydrolase 382 – fructosyltransferase 382, 383 fungal resistance 32, 97 – rice chitinase gene 159, 258, 261, 262, 385 Fusarium 62, 63, 249, 261 – F. oxysporum 62, 63, 136, 289, 291 – f. sp. elaeidis 62, 63 – f. sp. dianthi 249 Ganoderma 62 gene – discovery 43, 50, 65, 74, 162, 200, 375, 378 – dispersal 423, 425 – flow 34, 50, 229, 361–364 – gun 109–111, 115, 117–119, 290–292, 295 – silencing 23, 42, 49, 96, 98, 121, 137, 139, 200, 292, 293, 296, 331, 347, 379, 330 – post-transcriptional (PTGS) 49, 96, 293, 296, 385 – virus-induced 23, 200 – stacking 324 – transfer 23, 40, 41, 90–92, 129, 136, 137, 231, 232, 274–277, 279, 280, 321, 359, 363, 364, 376, 379, 401, 423, 426 genetic engineering 31, 32, 43, 50, 71, 74, 81, 93, 107, 108, 155, 161, 227, 232, 242, 245, 254, 274, 283–286, 291, 292, 312, 315, 321, 323–325, 328, 329

Index

genetic linkage map 227, 228 genetic map 82, 88, 229, 236, 242, 323, 346, 375, 377, 378 genetic mapping 30, 64, 83, 228, 323 genetic transformation 4, 14, 70, 108, 120, 150, 158, 159, 182, 183, 185, 232–234, 236, 254, 259, 274, 291, 306, 307, 312–314, 346, 358, 359, 375, 379, 399, 402 genome 3, 30, 42, 43, 46, 50, 59, 63–65, 82, 85, 86, 88, 90, 92, 114, 118, 122, 123, 129, 134, 138, 160, 161, 163, 185, 190, 227, 229, 253, 262, 277, 278, 280, 286, 293, 310, 346, 357, 360, 362, 366, 376, 378, 380, 387, 401, 402, 424 genomics – approaches 30, 201, 227, 342, 375, 378 – resources 3, 236, 345, 375, 378 – tools 23, 232 germplasms 177, 322 ginseng (Panax ginseng) 149 – biotechnology 150, 160, 164 – adventitious root culture 153, 155, 198 – bioreactor culture 151–153 – callus culture 152 – cell culture 150 – genetic transformation 158 – hairy root culture 152 – metabolic engineering 158, 160 ginsenoside 151–154, 159–163 – biosynthesis 159, 160, 162 – dammarenediol synthase 162 – oxidosqualene cyclase 162 Gladiolus 289 – A. tumefaciens infection 291 – bean yellow mosaic virus 294 – biolistic 292 – callus 289 – cucumber mosaic virus 295 – direct regeneration 291 – phosphinotricin 292 – promoters 292 – somatic embryogenesis 290 – suspension 290 – transgene silencing 295 – ubiquitin promoters 293 globulin 68, 112 β-glucuronidase (GUS) 10, 12, 13, 21, 41, 92, 99, 109–111, 119, 158, 159, 255, 256,

Index

261, 276, 279, 292–294, 307, 360, 361, 376, 402 glucanases 95, 97, 235 glutamine synthetase 282, 326, 327 glyphosate 19, 33, 47, 325, 326 Gossypium hirsutum 107, 124–127 grasses 325, 341, 358, 359, 363, 364, 366, 367, 375, 378, 379, 381, 385 green fluorescent protein (GFP) 71, 93, 112, 117–120, 124, 235, 276, 277, 279, 349, 360 growth regulators 41, 91, 130, 151, 182, 253 – 2,3,5-triiodobenzoic acid (TIBA) 156 – 2,4-dichlorophenoxyacetic acid 4–8, 11–13, 18, 21, 67, 91, 92, 109, 121, 131, 132, 151, 152, 154, 155, 159, 212, 290 – benzylaminopurine (BAP) 4, 5, 8, 10, 15–22, 212, 214 – indole-3-acetic acid (IAA) 16, 17, 154, 245, 307, 315 – indole-3-butyric acid (IBA) 17–20, 22, 154, 158 – kinetin 10, 15, 16, 212, 215, 290 – TDZ (thidiazuron) 16, 17, 130 – zeatin 10, 17, 131, 133, 245, 290 guaiacyl 329, 341, 364, 380, 381 gynogenesis 129, 138 hairy roots 152, 153, 155, 158, 162, 182, 196, 198, 199, 402 haploid 42, 129, 135, 136, 139, 161, 229, 376 henbane (see Hyoscyamus niger) herbicides 32, 33, 44, 45, 47, 108, 110, 111, 119–121, 123, 129, 136, 159, 172, 182, 281, 282, 284, 325, 326, 398, 401, 402, 406, 423 – resistance 33, 44, 45, 47, 119, 401, 423 – selection 136, 282 – tolerance 32, 120, 325, 398, 402 heroin 169, 171, 174, 185 histodifferentiation 4, 5, 7, 8 homeobox 66, 276, 279, 285, 383 hydroxyl radicals 130, 133 hygromycin phosphotransferase (HPT) 91, 119, 276, 279, 281, 324, 360, 376 – hpt 11, 13, 19, 71, 91, 109, 114, 119, 281, 293, 324 – hph 91, 94, 95, 293, 360, 376

435

hyoscyamine-6β-hydroxylase 198 Hyoscyamus 189, 195, 196 – H. albus 193 – H. muticus (Egyptian henbane) 189, 193, 198–200 – H. niger (henbane) 189, 192, 198 immobilised cells 218 insect resistance 32–34, 44, 46, 97, 120, 256, 258, 262, 264, 268, 293 – monoterpenes 248, 266 intellectual property 411 – CAMBIA 419 – Community Plant Variety Office (CPVO) 415 – freedom to operate 416 – Patent Cooperation Treaty (PCT) 412 – patents 412 – PBRs 412 – plant variety rights 414 – trade secrets 412 – trade marks 412 International Narcotics Control Board (INCB) 172, 173 isopentenyl diphosphate (IPP) 208, 211, 213, 214 isoflavones 340, 348 kanamycin-resistance 92, 108, 114, 137 KNOX 66, 285 legumes 337, 338, 340–342, 344–349, 352, 353 – Desmodium unicinatum 339 – Lotus corniculatus 339, 340, 350–352, 355 – L. japonicus 342, 346, 350, 351, 356 – L. pedunculatus 338, 350, 352, 355 – Medicago sativa (see alfalfa) – M. truncatula 323, 339, 342, 346, 347, 350, 351, 353, 355, 356 – white clover (Trifolium repens) 337–340, 343–346, 350, 353, 354 license 411 lignans 307, 308, 310, 315, 316 lignin 329, 337, 340, 341, 350–353, 356, 364, 377, 380–382, 387, 388, 391, 392 – caffeic acid O-methyltransferase 381, 390, 396

436

– caffeoyl-CoA 3-O-methyltransferase 381 – cinnamate-4-hydroxylase 381 – cinnamoyl-CoA reductase 381 – cinnamyl alcohol dehydrogenase 381, 388, 392 – phenylalanine ammonia-lyase 381 linkage – groups 64, 83, 88, 228, 229, 377 – map 43, 64, 227, 229, 236, 346 lupins (Lupinus spp.) 397–406 – disease resistance 398, 405 – herbicide tolerance 398, 402 – seed protein 398, 402 – transformation 399 MADS box 45, 65, 70, 243, 257, 285, 384 – AGAMOUS 66, 244, 384 – AGAMOUS-like2 66 – DEFICIENS 66 – GLOBOSA 66 – SQUAMOSA 66 malate dehydrogenase (MDH) 229, 327, 343 male sterility 45, 178, 244 mandrake (Mandragora officinarum) 189 marker-assisted selection 43, 63, 64, 74, 89, 242, 323 mesocarp 59–61, 65, 71–73 metabolic engineering 122, 150, 158, 160, 164, 182, 183, 198, 312, 329, 340, 343, 348 metabolome 247, 346 methyl jasmonate 99, 151, 152, 154, 162, 199, 210–214, 216, 308, 346 methylation-sensitive amplified polymorephism (MSAP) 70 mevinolin 210, 214 microarrays 169, 184, 231, 323, 325, 346, 378, 380 microinjection 21, 22 micropropagation 63, 66, 67, 74, 233, 254, 290, 307 microsatellites 63, 64, 75, 82, 83, 162, 228 microspore cultures 135, 136 molecular markers 63, 227, 376–378, 387, 390, 391 molecular pharming 331 MsPRP2 326 muscarinic 190, 191

Index

– antagonist 190, 191 – receptors 190 mutagenesis 183, 185, 253 – induced 305 – site-directed 33 neomycin phosphotransferase II (nptII) 13, 19, 21–23, 92–95, 98, 109–116, 119, 234, 255, 281, 293, 324, 402 nightshade (Atropa belladonna) 189, 193 nucleocapsid (N) gene approach 260 nutritive value 322, 366, 398, 402, 403, 405, 406 o-diphenol 330, 331 338 O-methyltransferases (OMTs) 231, 348 oil palm (Elaeis guineensis) 59–75 – dura 62, 63 – dura x pisifera 62 – oil 59, 60, 62, 67 – pisifera 62, 63 oil quality 32, 33, 71, 129, 172 oleic acid 33, 39, 62, 65, 71–73, 112, 114, 121 opium wars 171 orchids 273–286 orcinol dimethyl ether 231 organic acids 343, 344, 346 organogenesis 4, 5, 14, 16–18, 31, 129, 133, 134, 155, 181, 182, 232, 233, 243, 290, 399, 400 ornithine decarboxylase (ODC) 196, 197 ovary cultures 135 oxycontin 174 particle bombardment 8–10, 12, 13, 18, 20–22, 41, 42, 117, 124–126, 137, 182, 275–277, 280, 282, 385 pasture bloat 330 peanut (Arachis hypogaea) 81–99 – A. cardenasii 83–86, 88, 89 – A. diogoi 88, 89 – A. duranensis 82, 84–88 – A. monticola 82, 85 – A. stenosperma 83, 84, 87, 88 – A. stenosperma x A. cardenasii 83 – allergen genes 98 – fungal resistance 97 – high oleate 90

Index

– insect resistance 97 – introgression 83 – microsatellite discovery 82 – nematode resistance 88 – origin 82 – virus resistance 93 Percocet 174 plant architecture 253, 256, 257, 259, 268, 285 pollen – allergens 386, 387 – dispersal 3643 – viability 361, 362, 386, 425 polyamines 196–198 polyhydroxybutyrate (PHB) 73, 74, 138 polymerase chain reaction (PCR) 44, 63, 71, 89, 93–95, 108, 109, 111, 113–116, 138, 234, 263, 293, 310, 312, 363, 401, 426 polyphenol oxidase (PPO) 330, 331 polyphenols 307, 308, 311 – flavonoids 308, 311–314 polyploidization 305 proanthocyanidins (PAs) 330, 337–340, 347 promoters 45, 255, 324, 325 – cassava vein mosaic virus (CsVMV) 324 – cauliflower mosaic virus (CaMV) 35S 255, 324, 326, 328, 331 – Cab 255, 366 – chalcone synthase 255, 256 – eceriferum 256 – hsp17.6G1 46 – hsp17.7G4 46 – light-inducible 324, 325 – multicystatin 256 – rubisco 256, 266 – SCP1 47 – rd29A 260 – Ubi1 44 – ubiquitin extension protein (UEP1) 255, 256 – UCP3 46 – zinc finger transcription factor (EPF2) 255 propagation 8, 149, 155, 235, 241, 245, 277, 280, 293, 306, 425 protease inhibitors 109, 262, 264, 266 protein quality 327, 366, 398

437

proteome 323, 346 protoplasts 31, 32, 40, 41, 73, 130, 134, 135, 137, 210, 233, 234, 276, 359, 376, 379 putrescine N-methyl transferase (PMT) 196, 197 – pmt gene 196, 198 quantitative trait loci (QTL) 62, 323, 327, 377, 391, 396 random amplified polymorphic DNA (RAPD) 43, 63, 83, 84–89, 138, 227, 228, 242, 244, 309, 377 recurrent selection 63, 322, 364 red clover 330, 331 regulatory approval – biosafety clearing house 420, 422 – cartagena protocol 422 – data protection 427 – European Community (EC) 422 – LMOs 422 – precautionary principle 422 – trials 424 – unique identifiers 426 regulatory elements 98 – (nopaline synthase) promoter 98 – actin2 promoter 94 – CaMV 35S promoter 71–73, 98, 112, 115, 120, 231, 263, 307, 324, 326, 328, 329, 331 – mannopine synthase promoter 94 – ubiquitin promoter 98, 293 – vegetative storage protein promoter 94 regulatory genes 200, 246, 330 reporter gene 93, 96, 119, 120, 281, 360 restriction fragment length polymorphism (RFLP) 43, 63, 64, 70, 83–86, 88, 89, 227–230, 242, 244, 377 ribosome inhibiting protein (RIP) 235 rice 5, 50, 66, 71, 74, 95, 97, 111, 159, 161, 257, 258, 261, 286, 292–294, 327, 381, 383, 386 RNAi 48, 49, 99, 108, 112, 116, 121–123, 139, 163, 183, 257, 259, 341, 382 rolA 198, 244 rolC 198, 244, 245, 257, 259 Rosa spp. 227–236 – hybrid tea 228 – R. canina 232

438

– R. chinensis 231–233 – R. damascena 231, 233 – R. hybrida 230–235 – R. multiflora 228, 233, 234 – R. persica 234 – R. persica x R. xanthina 234 – R. rugosa 229, 232 – R. wichuraian 233 – R. wichuraiana 233, 234 – R. xanthina 234 royalties 412 RT-PCR 94, 114–116, 263, 312 rumen by-pass 328 ruminant 328, 329, 338, 341, 342, 364, 366, 380, 398, 404 – nutrition 338, 366 ryegrass mosaic virus 376, 385, 387 S-adenosylmethionine synthase 329 saponin 151–153, 159–162, 164, 342, 353–355 – ginsenoside 151–154, 159–163 – triterpene 160–162 scale-up 211, 216, 218 scent 229–231, 235, 236, 247, 248, 250, 415 Sclerotinia 44 47, 48, 97 scopolamine 189–193, 195, 197–200 scopolia (Scopolia carniolica) 189, 199 secondary embryogenesis 7, 9, 12, 233, 235, 290, 292 secondary metabolites 152, 154, 162, 196, 200, 209, 212, 220, 337, 340, 347 secondary somatic embryos 7, 12, 131 seed production 61, 173, 398 selectable markers 19, 23, 91, 93, 98, 99, 119, 121, 281, 284, 324, 360, 362, 401, 402, 405, 423 – hygromycin resistant 361, 376 – kanamycin resistance 92, 114, 137, 307 selection index 63, 178 semi-synthetic opiates 173 – buprenorphine 173 – oxycodone 169, 173 sequence-specific amplified regions (SCARs) 228, 229 sequence-tagged microsatellite site (STMS) 228 shoot – apex 8, 19, 118, 399, 400

Index

– elongation 17, 20, 136, 233 – regeneration 16, 18, 92, 93, 108–111, 113, 115, 133, 245, 275, 279, 290 simple sequence repeat (SSR) 43, 82, 84, 163, 323, 377 Solanaceae 189, 190, 194, 199 somaclonal variation 68, 69, 74, 130, 136, 281, 282, 306 somatic embryogenesis 4–9, 11–14, 31, 40, 41, 45, 66, 67, 109–117, 129, 131–134, 155, 156, 158, 181, 232, 233, 235, 290, 292, 399, 400 – direct 5, 133, 155, 399, 400 – indirect 5 somatic embryos 5–13, 31, 40, 67, 68, 132–134, 156–159, 182, 232, 233, 235, 254 – conversion 4, 5, 8, 67, 157 – development 4, 5, 8, 9, 131–133, 156, 157 – induction 9, 11, 133, 155, 156, 182 – maturation 4, 7, 11, 13, 68, 69, 74, 233 stress 340, 342, 344, 345, 347, 349, 353 – abiotic 34, 44, 48, 121, 241, 257, 260, 321, 326, 344, 347, 349, 353, 366, 367, 379, 380, 385, 387 – biotic 34, 51, 342, 380 – environmental stress 48, 121, 345, 382 – oxidative 48, 133 sucrose phosphate synthase 326, 327 sunflower (Helianthus annuus) 39–51 – biodiesel 39, 49, 51 – gene 44 – harvested area 39 – oil 39, 49 – promoters 46 – seed albumin 366, 403 superoxide dismutases (SODs) 45, 112, 326 syringyl 329, 341, 380, 381 T-DNA 8–12, 18, 19, 23, 117, 137, 152, 153, 196, 198, 244, 277–279, 401 tall fescue (Festuca arundinacea) 49, 357–367 – F. arundinacea var. glaucescens 357 – F. pratensis 357 taxanes 205–207, 209, 210–216, 218, 220 taxol 205–209, 211–216, 218–220 – biosynthesis 206–208, 215

Index

thioesterase 33, 65, 72, 73 thiol compounds 19, 20 thornapple 189 threonine synthase 329 tomato spotled wilt virus (TSWV) 94–96, 257, 260, 261 tospoviruses 94, 260–262 totipotency 132–134 transcriptome 247, 346, 378, 380 transformation – efficiency 9, 12, 20, 23, 32, 41, 70, 234, 235, 255, 275, 277–279, 360 – frequency 11, 22, 41, 42, 91, 99, 235, 360, 401 – methods 9, 17, 19, 21, 23, 41, 42, 50, 92, 110–116, 119, 275, 278, 346, 358, 376, 399, 401, 412 – protocols 8–10, 12, 17, 18, 23, 92, 99, 182, 185, 235, 255, 256, 324, 399, 406 transgene – copies 90, 359 – expression 44, 96, 122, 137, 258, 263, 286, 360 – flow 42, 50, 363, 364 – integration 23, 359, 424 – silencing 292, 293, 295, 296 transgenesis 250, 375, 376, 381 transgenic plants 4, 9, 14, 19, 21, 23, 33, 34, 42, 44, 46, 48–50, 71, 92, 96, 97, 115, 155, 158, 159, 198, 201, 232, 234, 235, 244,

439

245, 247, 248, 260, 263, 264, 266, 275, 277, 294, 295, 307, 341, 345, 358, 360–367, 376, 380, 383, 385, 386, 401, 402, 417 transgenic technologies 196, 321, 345 transient expression 41, 71, 92, 137, 159, 280 transposable elements 70, 130, 162 tropane 189–200 turf grasses 367, 373, 375 two-stage culture 210, 212, 216, 217 United Nations Economic and Social Council 172 – Single Convention on Narcotic Drugs 1961 172 United Nations Office on Drugs and Crime (UNODC) 171, 173 United States Drug Enforcement Administration (DEA) 173 vase life 230, 241, 245, 246, 253, 274, 286 vegetable oils 29, 39, 49, 50, 60–62, 75, 337 virus resistance 92, 93, 257, 291, 296, 344, 387, 406 WHO 171 winter hardiness 302, 322, 323 zygotic embryos 4, 5, 40, 65, 68, 130, 131, 133, 155–158, 232