Horticultural Reviews (Volume 14)

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Horticultural Reviews, Volume 14

Edited by Jules Janick

WILEY

HORTICULTURAL REVIEWS Volume 14

Horticultural Reviews is sponsored by: American Society for Horticultural Science

Editorial Board, Volume 14 James N. Cummins Elizabeth G. Williams Naftaly Zieslin

HORTICULTURAL REVIEWS VOLUME 14

edited by

Jules Janick Purdue University

John Wiley & Sons, Inc. NEW YORK /

CHICHESTER /

BRISBANE /

TORONTO /

SINGAPORE

In recognition of the importance of preserving what has been written, it is a policy of John Wiley & Sons, Inc., to have books of enduring value published in the United States printed on acid-free paper, and we exert our best efforts to that end. Copyright © 1992 by John Wiley & Sons, Inc. All rights reserved. Published simultaneously in Canada. Reproduction or translation of any part of this work beyond that permitted by Section 107 or 108 of the 1976 United States Copyright Act without the permission of the copyright owner is unlawful. Requests for permission or further information should be addressed to the Permissions Department, John Wiley & Sons, Inc. LC card number 79-642829 ISBN 0-471-57339-6 ISSN 0163-7851

Contents Contributors Dedication Heliconia: Botany and Horticulture of a New Floral Crop

1

ix

xiii

1

Richard A. Criley and Timothy K. Broschat I. II. III. IV.

Introduction Botany Horticulture Research Needs Literature Cited

Root Physiology of Ornamental Flowering Bulbs

2

2 3

19 49 51

57

Ludwik p Kawa and A. A. De Hertogh I. II. III. IV. V. VI.

3

Introduction Root Origins Root Morphology Endogenous Factors Exogenous Factors Conclusions Literature Cited

Tuber Formation in Potato: Induction, Initiation, and Growth

57 60 67 72 72 82 84

89

E. E. Ewing and P. C. Struik

I. Introduction II. Methods of Studying Tuberization

90 94 v

III. IV. V. VI. VII.

Environmental Factors Affecting Tuberization Genetic Effects Effects of the Mother Tuber Physiological Nature of Induction to Tuberize Changes in the Stolon Tip or Bud Associated with Tuberization Patterns of Stolon and Tuber Formation Resorption and Second Growth Implications for Tuber Yield Conclusion Literature Cited

151 164 172 180 181 182

The Biology, Epidemiology, and Control of Turnip Mosaic Virus

199

VIII. IX. X. XI.

4

104 121 124 134

V. I. Shattuck

I. II. III. IV. V. VI. VII. VIII. IX. X.

5

Introduction History Characteristics of the Virus Strains and Isolates Purification Effects of Infection Detection Epidemiology Control Methods Conclusion Litera ture Cited

Thin Cell Layer Morphogenesis

199 200 201 204 208 210 215 217 221 228 229

239

Michael E. Compton and Richard E. Veilleux 1. Introduction

II. III. IV. V. vi

Flower Bud Production Vegetative Shoot Morphogenesis Somatic Embryogenesis Conclusions Literature Cited

239 240 256 258 259 260

6

Tissue and Cell Cultures of Woody Legumes

265

R. N. Trigiano, R. L. Geneve, S. A. Merkle, and]. E. Preece I. II. III. IV. V. VI.

Introduction In Vitro Propagation Crop Improvement Secondary Metabolite Production In Vitro Studies of Nitrogen Fixation Concluding Remarks Literature Cited

Polyamines in Horticulturally Important Plants

7

266 289 311 314 322 324 324

333

Miklos Faust and Shiow Y. Wang I. II. III. IV. V. VI.

Introduction Overview Polyamines and Plant Development Stress-Induced Changes in Polyamine Content Polyamines and Senescence Conclusions Literature Cited

Breeding Muscadine Grapes

8

333 334 337 344 347 349 350

357

R. G. Goldy

I. II. III. IV. V.

9

Introduction Germplasm Resources Breeding: Intraspecific Hybridization Breeding: Intersubgenetic Hybridization Future Prospects Literature Cited

Nitrogen Metabolism in Grapevine

357 359 363 383 396 398

407

K. A. Roubelakis-Angelakis and W. Mark Kliewer I. Introduction II. Uptake of Nitrogenous Compounds III. Biosynthesis of Nitrogenous Molecules

408 409 414 vii

IV. V. VI. VII. VIII.

Nitrogenous Compounds Storage and Reallocation of Nitrogen Translocation of Nitrogenous Compounds Diagnosis of Nitrogenous Status Future Research Directions Literature Cited

428 432 435 438 440 441

Subject Index

453

Cumulative Subject Index

455

Cumulative Contributor Index

470

viii

Contributors

Timothy K. Broschat, Ft. Lauderdale Research Education Center, University of Florida, Ft. Lauderdale, Florida 33314 Michael E. Compton, Central Florida Research and Education Center, Institute of Food and Agricultural Science, University of Florida, 5336 University Avenue, Leesburg, Florida 34748-8203 Richard A. Criley, Department of Horticulture, University of Hawaii, Honolulu, Hawaii 96822 A. A. De Hertogh, Department of Horticultural Science, North Carolina State University, Raleigh, North Carolina 27695-7609 E. E. Ewing, Department of Fruit and Vegetable Science, Cornell University, Ithaca, New York 14853-0327 Miklos Faust, Fruit Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, Beltsville, Maryland 20705 R. L. Geneve, Department of Horticulture and Landscape Architecture, University of Kentucky, Lexington, Kentucky 40546 R. G. Goldy, Formerly Department of Horticultural Science, North Carolina State University, Raleigh, North Carolina 27695 Present address: 86 West Albion Street, Holley, New York 14470 Ludwika Kawa, Research Institute ofPomology and Floriculture, 96100 Skierniewice, Poland w. Mark Kliewer, Department of Viticulture and Enology, University of California, Davis, California 95616 S. A. Merkle, School of Forest Resources, University of Georgia, Athens, Georgia 30602 J. E. Preece, Department of Plant and Soil Science, Southern Illinois University, Carbondale, Illinois 62901-4415 K. A. Roubelakis-Angelakis, Department of Biology, University of Crete, P.O. Box 1470, 71110 Heraklio, Greece V. I. Shattuck, Horticultural Science, University of Guelph, Guelph, Ontario, Canada N1G 2Wl P. C. Struik, Department of Field Crops and Grassland Science, Wageningen Agricultural University, Haarweg 333, 709 RZ Wageningen, The Netherlands R. N. Trigiano, Department of Ornamental Horticulture and Landscape Design, Institute of Agriculture, University of Tennessee, Knoxville, Tennessee 37901-1071 ix

x

Richard E. Veilleux, Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Shiow Y. Wang, Fruit Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, Beltsville, Maryland 20705

HORTICULTURAL REVIEWS Volume 14

James N. Moore

Dedication

James N. Moore quoted Jonathan Swift from Gulliver's Travels (1726) in his Presidential Address to the American Society for Horticultural Science (ASHS) in 1988: "Whoever could make two ears of corn or two blades of grass to grow upon a spot where only one grew before would deserve better of mankind and do more essential service to his country than the whole race of politicians put together." This aptly describes the career and philosophy of Jim Moore, needing only the exchange of a few different phrases, perhaps, "two clusters of grapes and two pints of blueberries." He has made the hills, mountains, and plains of Arkansas and far beyond flourish with new cultivars of small fruits, grapes, and tree fruits. Jim Moore was a leader in the movement in ASHS to establish a Horticultural Hall of Fame and I predict that some day he will join such horticultural greats as Liberty Hyde Bailey and Luther Burbank. Jim Moore is a "compleat" horticulturist (if I may borrow from Sir Isaac Walton): teacher, scientist, servant, author, editor; but he will be best remembered for the prolific output of fruit cultivars, some of which are now being grown worldwide. Most fruit breeders work with one or two at most. Jim has released cultivars of strawberry, blackberry, grape, peach, and apple, and is well along in developing blueberry cultivars adapted to upland mineral soils. How does he do it? He makes maximum use of his time and collaborates with many others, especially his graduate students, plant pathologists, entomologists, food scientists, engineers, and other fruit breeders around the world. Jim has visited a number of other countries on National Academy of Science Exchange Programs and as a consultant. Frequently he will collaborate with a former student from another country, such as Brazil or Mexico. Jim Moore has received much recognition and many awards. Through it all he has maintained a sense of modesty and approachability. One of Jim's former PhD students, now an associate professor at a leading land grant university, said to me after taking Jim Moore's courses in Small Fruit Production and Advanced Plant Breeding: "We were being taught by a great man but you'd never know it from his friendly, concerned attitude and open door policy to us all. He was never too busy to explain again to groups of students or individuals any concept not well underxiii

xiv

GEORGE A. BRADLEY

stood by all in the class." Jim is one of the best teachers at the University of Arkansas and receives incredibly high ratings from his students. Early in Jim's career it was evident that he was not to be a run-of-themill horticulturist, when papers from both his MS and PhD research won awards from ASHS. Jim's first award-winning paper on processing tomato yield and quality with Ahmed Kattan, his major professor, and J. W. Fleming received the Woodbury Award for Raw Products Research in 1959. Jim took an instructor's position on a fill-in basis at the University of Arkansas in 1957 and he fell in love with fruit crops. This love affair continued at Rutgers University where Jim was a Research Associate under Fred Hough. His PhD research on strawberries won the ASHS Gourley Award in Pomology in 1963. After a stint with the USDA as a small fruit breeder Jim was enticed to return to the University of Arkansas in 1964. Ten years after returning, he received the Distinguished Faculty Award for Outstanding Research. He received the Wilder Silver Medal of the American Pornological Society in 1982 for fruit breeding achievements, and in 1984 Jim received a Wilder Citation for two books on fruit breeding coedited with Jules Janick of Purdue University. In 1987 he received the USDA Distinguished Service Award, one of only three state experiment station scientists to be so honored that year. Gamma Sigma Delta, the honorary agricultural society, presented Jim with its International Award in 1988. In a typical action, Jim immediately signed over the accompanying stipend to the local chapter scholarship fund. In 1988, Jim also received the Burlington Northern Faculty Research Award in competition with all University of Arkansas faculty. In 1990 he received the Outstanding Research Scholar Award from the Arkansas Department of Higher Education in competition with all college faculty of state-supported higher education institutions in Arkansas. Jim was in the first group of five faculty members at Arkansas to receive the newly established rank of University Professor in 1985; in 1988 he was named Distinguished Professor. Jim Moore is an outstanding scholar and teacher who has also excelled in the area of service at the state, regional, national, and international levels. He has served ASHS and the Southern Region ASHS in numerous offices, including President. He is a Fellow of ASHS (1976) and the American Association for the Advancement of Science (1988). In 1973 and 1977, Jim was twice selected by the National Academy of Sciences as an Exchange Scientist with Eastern European countries. He was a consultant in Brazil and Costa Rica and hosted fruit scientists from all over the world. He guided more than 40 MS and PhD students. Jim Moore, through all of this recognition, has kept as his central focus the students at the University of Arkansas as well as the fruit growers of Arkansas and the surrounding region. The fact that many of his cultivars

1.

DEDICATION

xv

have performed well in regions far removed is a bonus, and perhaps due to Moore's philosophy of fruit breeding, in which he brings in genes for adaptation to divergent conditions into the selection pool. One of his most remarkable achievements for Arkansas was the introduction and nurturing of blueberries to a full-fledged commercial enterprise; in a few years they may well become the state's most valuable commercial fruit crop. Progressive Farmer Magazine named him Man of the Year in Arkansas Agriculture in 1990 for this and other fruit breeding achievements. A new table grape industry is under development based on Jim's grape releases, 'Venus,' 'Reliance,' 'Mars,' and 'Satern'. 'Cardinal' strawberry, Jim's first fruit release in Arkansas (1974), is still the leading cultivar grown in the region and received the ASHS Fruit Breeding Working Group Outstanding Cultivar medal. Jim is a prolific writer and an excellent editor, but he loves best to get out into the orchards, vineyards, and breeding nurseries. His efforts there have enabled Jim to reach the pinnacle of horticultural accomplishments. George A. Bradley Department of Horticulture and Forestry University of Arkansas

1 Heliconia: Botany and Horticulture of a New Floral Crop * Richard A. CrHey·· Department of Horticulture University of Hawaii Honolulu. Hawaii 96822 Timothy K. Broschat Ft. Lauderdale Research and Education Center University of Florida Ft. Lauderdale. Florida 33314

I. II.

III.

IV.

Introduction Botany A. Ecology B. Taxonomy C. Morphology D. Pollination and Compatibility E. Physiology Horticulture A. Cut-Flower Production B. Pot-Plant Production C. Interiorscape Use D. Landscape Culture Research Needs Literature Cited

• Published as Journal Series No. 3563 of the Hawaii Institute of Tropical Agriculture and Human Resources and as Florida Agricultural Experiment Station Journal Series No. R01639. "Acknowledgement is made to W. John Kress and Ray Baker for their assistance in the preparation of this review and to May Moir, Elsie Horikawa, Lillian Olviera, Ray Baker, Lisa and Ken Vinzant, Hamilton Manley, and Brian Miyamoto for their assistance in the preparation of Table 1.4. Colorseparations for Plate I were provided by the Marketing Division, Hawaii State Department of Agriculture. Funding for the color plate was provided by the College of Tropical Agriculture and Human Resources of the University of Hawaii and Jim Little provided a generous donation. Appreciation is also expressed to the authors who gave permission to reproduce illustrations from their published works. 1

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

2

I. INTRODUCTION It is hard to tell which are more exotic-the architecturally unique,

brightly colored inflorescences of heliconias or the little jewel-like hummingbirds that dart among them. The relationships between heliconias and the hummingbirds are a rich source of study for ecologists because the heliconias are an abundant nectar source. Pollen transfer is achieved when the birds feed in successive flowers. Ecologists study not only hummingbird foraging behavior and their ability to recognize color and shape but also the mechanisms which sustain or dilute species distinctiveness within Heliconia, their flowering phenology, and nectar secretion (Seifert 1975, 1982; Stiles 1975, 1978, 1979; Wolf and Stiles 1989; Kress 1983a, 1983b; Dobkin 1984, 1985, 1987; Bronstein 1988; and Wooten and Sun 1990). Heliconias occur in forest light gaps, shaded rainforest, isolated valleys, and along open roads and riverbanks from sea level to 2000 m elevation in Central and South America and to 500 m in the South Pacific Islands. Distribution of some species are very localized and uniform, while others have a wide range and exhibit polymorphism. The destruction of native habitats for agricultural purposes eliminated populations of heliconias that flourished in rich ecosystems only two or three years earlier (Woolliams 1985). Botanical and horticultural collections saved valuable germplasm, but the ecosystems will never be restored. Pacific Islanders recognized heliconias for their ornamental value and planted them in their villages. From such sites, early collectors brought them to botanic gardens in Bogor, Singapore, and Calcutta. Appreciation of heliconias for their horticultural traits extends from native Pacific Islanders who use the broad leaves to wrap portions of food for cooking, transport, or serving and the sap and solid white portion of the pseudostem as antiseptics on open wounds (Kress 1990a) to the parties of the rich and famous where heliconias in floral displays elicit amazement and fascination. In Colombia, a species tentatively identified as H. hirsuta is grown for its rhizome as a starchy vegetable (C. Clement, personal communication). In the early 1980s heliconias were such an insignificant part of the cut flower market that they were grouped together under one name and did not merit separate statistics for production and value. In the gigantic Dutch auctions small supplies have been available from the early 1970s, but heliconias remain a minor flower crop in the early 1990s when compared with the overall volume of floral produce. Nonetheless, it is appropriate to mark the progress which has been made in understanding this genus, both botanically and horticulturally, because of its increasing importance in floriculture.

1.

HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

3

II. BOTANY

A. Ecology The majority of Heliconia species are found in the New World tropics, but six species and several botanical varieties occur in the Pacific island tropics. Their distribution is from the Tropic of Cancer in Mexico and the Caribbean islands to the Tropic of Capricorn in South America. Numerous floras, descriptive reports, and monographs with keys detail the 200+ species found in different countries in the tropics (Woodson and Schery 1945; Smith 1968, 1977; Green 1969; Santos 1978; Dodson and Gentry 1978; Pingitore 1978; Daniels and Stiles 1979; Stiles 1979; Kress 1981, 1984, 1986, 1990a; Andersson 1981, 1985a, 1985b; Abalo and Morales 1982, 1983a, 1983b, 1985; Wolf and Stiles 1989). The greatest numbers of species occur at middle elevations (500-1000 m). According to Andersson (1985a), closely related species growing in the same area often differ with respect to their preferences for soil types, light, or altitude. Nearly all inhabit moist or wet environments, but some occur in seasonally dry areas. Their most vigorous growth occurs in humid lowland areas at elevations below 500 m. In shaded rainforests, the plants are locally endemic and subject to extinction as destruction of the forests proceeds. The adaptable members of the genus rapidly colonize forest light gaps as well as open sites along roads, rivers, and swamps and tend to be weaker in growth and more sparsely distributed in shaded forests. During the colonization of the New World and the Pacific tropics, colorful heliconias attracted attention from European plant collectors who brought them back to botanic gardens, private gardens, and the prominent nurseries of the era. Kress (1990a) related that H. indica was listed in cultivation in the Calcutta Botanical Gardens as early as 1814. Most of the forms were named for their bold foliage color and variegation. Correct botanical identification was obscured by such fanciful names as Spectabilis, I11ustris, and Edward us-Rex.

B. Taxonomy Gilbert S. Daniels writes in the preface to Heliconias, an Identification Guide (Berry and Kress 1991), "To the natives of the regions where they occur naturally, their abundance leads them to be considered as weeds and thus not worthy of attention, and to the scientist their large size has made them difficult to collect and preserve during any general collecting expedition". Still, herbaria all over the world have specimens which were

4


annotated by taxonomists in their efforts to identify species, and elucidate, collate, and publish relationships within the genus. However, the state of taxonomy in the genus is in flux. In 1703 the plant known today as Heliconia bihai (L.) L. was known as Bihai amplissimus foUss, with three taxa assigned longer polynomials (Plumier 1703). Linnaeus (1753, 1771) combined these under Musa in his Species Plantarum, although he later separated it out as Heliconia. Even as late as 1915, the genus was still classified by some as Bihai (Griggs 1915), although HeUconia was in use at the same time. Since the turn of the century a number of taxonomists attempted revision of the genus. These were cited and summarized by Andersson (1981, 1985a, 1985bJ and Kress (1984, 1990aJ. Heliconias resist the attempts of taxonomists to define precise species types as many forms (polymorphismJ exist in some areas. As a result of the "splitters", many new species have been described, which are not sufficiently different to merit such recognition according to Andersson (1985a). The heliconias of the central Amazon basin, Essequibo River basin, northern Venezuela at low and mid-elevations, Panama, and Costa Rica tend to exhibit polymorphism, while heliconias from the montane forests of northern Venezuela, the Guiana Shield, and the Pacific side of the Andes, the northeastern Andes, and northern Central America from southern Mexico to Honduras tend to have more morphologically constant populations. Andersson (1985aJ suggests that polymorphism in the genus results from two trends, differentiation due to isolation and convergence due to secondary contact and hybridization. Cytological studies have been carried out on about 20% of known heliconia species. In all New World species examined, 2n = 24 (Bisson, et ai. 1968; Andersson 1984), which includes the principal cut-flower heliconias such as H. bihai, H. psittacorum, and H. caribaea, (Cheesman and Larter 1935; Mahanty 1970). Counts of 2n = 16, 18, 22, and 26 were reported in the Chromosome Atlas of Flowering Plants (Darlington & Wylie 1955J. Andersson (1984J dismissed these as miscounts after studying illustrations in the original reports and suggested the possible occurrence of aneuploidy. If more recent taxonomic designations are applied to H. illustris, H. aureo-striata, and H. rubra as forms of H. indica (Kress 1990a), then at least one of the Old World tropic species is also 2n = 24 (Venkatasubban 1946). The taxa within the order Zingiberales have been debated for a long time, but the heliconias have always been placed with the Musa complex. The name, Heliconia, is derived from Mt. Helicon in southern Greece, the mythical home of the Muses, hence a supposed relationship between these plants and bananas (genus MusaJ. Eight families are recognized [see Kress (1984, 1990b) for a historical account} with the

1.


5

Heliconiaceae consisting of the genus Heliconia, an arrangement first proposed by Nakai (1941). Retaining three taxa of Griggs (1903), Andersson (1981, 1985a, 1985b) subdivided the genus into four subgenera: (1) Taeniostrobus (Kuntze) Griggs, a group with broad bracts; (2) Stenochlamys Baker, with narrow bracts; (3) Heliconia (= Platychlamys Baker, which were the remaining species of uncertain relationship); and (4) Pendulae Griggs, a group with pendent inflorescences. He further defined sections within these based largely on the consistency of vegetative structures and staminode shape and style vestiture, and disagreed with Kress (1984) in the groupings, especially of pendent heliconias. Kress' 1984 monograph expresses the opinion that pendent heliconias are not necessarily monophyletic. Kress (1990a) assigns the Pacific tropical species to the subgenus Heliconiopsis, a taxon also with earlier precedence (MiqueI1859). Table 1.1 shows the assignment to subgenera of a number of species which have been brought into commercial culture and botanical collections. The recent publication of Heliconia, an Identification Guide (Berry and Kress 1991) may not end taxonomic disagreements, but it does afford the horticulturist a convenient reference for descriptions of the majority of cultivated species, botanical varieties, and some cultivars from both the New World and Pacific tropics. In this book, groupings are by inflorescence habits, for example, erect or pendent and distichous or spiral, while bypassing keys in favor of color photographs that simplify identification.

c.

Morphology

Descriptions of the genus Heliconia appear in recent major taxonomic works (Andersson 1981, 1985a, 1985b; Kress 1981, 1984, 1990a). The anatomic and morphological characteristics of these plants have been used variously to define them within the Musaceae (Andersson 1985a,b) or as a separate and equal family, the Heliconiaceae (Kress 1990b). Kress (1990a) listed 72 characters used to distinguish among different species and a briefer list of 34 characters used to separate the eight families and represent phylogenetic relationships within the Zingiberales (Kress 1990b). Among the characteristics of greatest interest to horticulturists are vegetative habit and inflorescence size, shape, and color. Heliconias are rhizomatous, perennial, herbs with an erect, aerial, and stem-like tube composed of overlapping leaf sheaths called a pseudostem. The rhizome branches sympodially from buds at the base of the pseudostem. Vegetative growth is quite vigorous, often giving rise to large monoclonal populations.


6

Table 1.1. Tentative classification of Heliconia species (Andersson 1981. 1985a,b; Kress 1984,1990; Berry and Kress 1991). Taxonomists do not agree on placement of some species in the subgenera shown in this table, and the species listed together should not necessarily be considered to be closely related. All are new world species except those in the last subgenus, Heliconiopsis.

Subgenus Taeniostrobus (Kuntze) Griggs. Musoid plants with compact, erect inflorescences; bracts much overlapping, deeply cupped, subject to shedding at fruit maturity; flowers resupinate or somewhat twisted. atropurpurea episcopaUs imbricata reticulata Stenochlamys Baker. Musoid, cannoid, or zingiberoid plant habits, often low and slender; inflorescence upright with narrow shallowly boat-shaped bracts borne spirally or distichously; flowers resupinate (twisted 180 0 on the pedicel) and exposed. Considerable polymorphism.

Section: Lanea L. Anderss. aemygdiana burleana Ungulata pseudoamygdian a schiedeana zebrina Section: Stenochlamys Baker acuminata angusta laneana psittacorum richardiana x nickeriensis x 'Golden Torch' Section: Lasia L. Anderss. lasiorachis velutina

Section: Proximochlamys L. Anderss. densiflora gracilis ignescens

1.


Table 1.1.

7

Continued

Subgenus Section: Cannastrum L. Anderss.

calatheaphylla metallica osaensis subulata vaginalis

Section: Zingiberastrum L. Anderss.

aurantiaca hirsuta longiflora schumanniana

Heliconia L. Anderss. Stout musoid species with erect inflorescences. thick. deeply cupped bracts; and greenish flowers on untwisted pedicels. Bracts borne distichously.

Section: Heliconia L. bihai bourgaeana caribaea champneiana orthotricha x rauliniana rodriguensis stricta wagneriana

Section: Tortex L. Anderss.

beckneri farinosa irrasa lankesteri latispatha lindsayana monteverdensis sampoaioana sarapiquensis spathocircinata continued

8


Table 1.1.

Continued

Subgenus Griggsia L. Anderss. '\.., Pendulae Griggs. Stout-to-intermediate musoid species with pendent inflorescences, bracts arranged spirally or distichously, flowers red, yellow, orgreen; bracts smooth-to-densely woolly. Grouped according to pollen morphology characteristics (Andersson 1985b; Kress and Stone 1983; Kress, personal communication).

Groups 10nga: excelsa, 10nga, stilesii nutans: collinsiana, marginata, nigrapraefixa. nutans, platystachys, secunda, chartacea pogonantha: danielsiana. magnifica, mariae, pogonantha, regalis, sessilis, xanthovillosa, ramonensis trichocarpa: colgantea. maculata, necrobracteata, talamanca. trichocarpa griggsiana: griggsiana. pastazae pendula: pendula obscura: riopalenquensis, sclerotricha rostrata: rostrata, standleyi Heliconiopsis (Miq.) Kress. Pacific Island tropical species of musoid growth habit with erect or pendent inflorescences, bracts distichous or spiral and green to yellow-green; flowers green. indica ssp austrocaledonica indica ssp dennisiana indica ssp indica indica ssp micholitzii indica ssp rubricarpa lanata laufao paka papuana solomonensis

Leaf arrangement is alternate and distichous, gIvmg rise to three growth habits described as musoid, cannoid, or zingiberoid (Fig. 1.lA,B,C; Kress 1984). During the flowering phase, an unbranched aerial stem bearing a terminal inflorescence elongates within the pseudostem. Plant size is usually measured to the topmost foliage rather than to the point of origin of the inflorescence. Thus, within the genus, there are species as short as 0.5 m, as well as some as tall as 5 m. Andersson (1985a) used plant robustness and shoot organization in grouping species. Individual plants tend to develop as round clumps (often with a hollow center as old pseudostems die out), but some species have a vigorous running rhizome system (H. psittacorum, forexample) and produce dense patches in which

1.


A

B

9

c

Figure 1.1. Schematic representation of the three types of shoot organization in Heliconia: A, Musa-like; B, Canna-like; C, Zingiber-like (Kress, 1984).

the original shoots cannot be distinguished. Different management practices are called for with the clumping and running types. Apseudostem is often composed of a specific and limited number (5-9) of leaves (Criley 1985) which may be influenced by cultural and environmental conditions. Additionally, there are reduced scale-like leaves or leaf bases at the base of the pseudostem. The obvious lamina-bearing leaves are furled around the midrib, which is an extension of the long petiole. The leaf apex is acute to acuminate with the base of the lamina unequal and usually obtuse to truncate, but occasionally cordate orattentuate. While the leaves are usually a solid green, occasionally with a waxy bloom, in some species variegations of red, maroon, pink, or yellow occur along the veins, margins, or whole laminar surface. The colorful inflorescence structure (Fig. 1.2) is the main attraction of heliconia for ornamental and cut flower use. The inflorescence may be erect, nodding (rare) or pendent (Fig. 1.3; Plate 1*). The peduncle may have various colors and textures and is not included when the length of an inflorescence is measured. Colorful, modified leaf-like structures called inflorecence bracts (cincinnal bracts, branch-bracts, or spathes in earlier • Selected heliconias produced for export from Hawaii,(Color plate: Courtesy of Hawaii Department of Agriculture.)

10


Figure 1.2. Structure and measurements of inflorescences of Heliconia: A, peduncle; B, rachis; C, rachis internode length; D, basal cincinnal bract (sterile, with elongated leaflike extension); E, middle cincinnal bract; F. cincinnal bract angle with axis of inflorescence (e.g .. 80°); G, floral bract (Kress. 1984).

literature) are arranged spirally or distichously (in one plane) on a straight or flexuose rachis, also varied in color and texture. The angle of the bract with the rachis varies from 0 to 180 degrees. The bract closest to the peduncle is often sterile and may bear a reduced laminal extension. Bract margins may be straight, revolute, or involute near the rachis. Within each inflorescence bract is a cincinnus (coil with successive flowers arranged alternately along the axis) of a variable number of flowers, with each flower subtended by a floral bract. These floral bracts may persist through to fruit maturation or decompose after anthesis. Selection of heliconias for cut flower use depends more upon inflorescence bract coloration than on the colors of the floral bracts and perianth, which are usually white to green to yellow or occasionally orange. The

1.


11

Figure 1.3. Inflorescence types in Heliconia: A. pendent. distichous (flat) inflorescence of H. rostrata; B. pendent. spiral inflorescence of H. collinsiana; C, upright distichous inflorescence of H. caribaea; D, upright, spiral inflorescence of H. latispatha (Watson and Smith 1974).

12


heliconias of the old world tropics have almost exclusively green bracts and white or green perianths (Kress 1990a). The flower shape maybe uniformly curved, parabolic, orsigmoid, with a bulbous nectary at the base (Fig. 1.4). In the subgenus Stenochlamys, the perianth is markedly triangular in cross-section while in other groups it is more or less elliptic in cross-section. The floral diagram is shown in Fig. 1.5. Flower presentation may be erect and exposed as in H. psittacorum or nearly hidden with only the perianth tip extending above the level of the bract margin when anthesis occurs as in many largebracted species. Flowers may have a resupinate or nonresupinate orientation. The perianth consists of two whorls (three outer sepals and three inner petals) showing varying degrees of fusion from the base distally to form an open tube, which varies in length with the species. The two abaxial sepals are connate with the adaxial sepal free for much of its length. The petals are connate except for free margins opposite the adaxial sepal. Flowers are perfect with the filaments adnate to the perianth. Of the six stamens, five are fertile and the sixth is modified as a sterile staminode. The insertion, size, and shape of this staminode are used as identifying characteristics by taxonomists. The linear anthers may extend just beyond the perianth or end just inside the apex and

Figure 1.4. Side drawing of mid-longitudinal section of flowp", ~ r ~ •• pogonantha. nc = nectar chamber, f = filament. s = style, st = staminod" '... -belly" where nectar collects after overflowing the nectar chamber (Wolf and Stiles 1989).

o '" •

SEPAL

'" PETAL

= FERTILE STAMEN

HElfCONIACEAE

Figure 1.5.

~

Floral diagram for the Heliconiaceae (Kress 1990a).

0

=STAMINOOE

1.


13

usually surround the style. They dehisce longitudinally to shed pollen; diurnal shedding characterizes the neotropical species and nocturnal in 4 of the 6 paleotropical species (Kress 1990a). Heliconia pollen has been studied in detail for its unique structure and systematic relationships (Kress et aI. 1978; Santos 1978; Stone et a1. 1979; Kress and Stone 1982, 1983; Andersson 1985a). The long style follows the curvature of the perianth and may curve back just below a 3-lobed stigma. The degree of stigmatic lobing is also a diagnostic character among species. The ovary is inferior and 3-locular. Each locule contains a single basally attached ovule. Fruits of the New World species are blue in color and tend to be small, under 2 cm in length, while those of Pacific tropical species may be up to 3 cm in size and red or orange in color. Developing seed is hidden by the bracts and protected by bract liquid and tough surrounding tissues. Upon ripening, the fruit is elevated by elongation of its pedicel for ready visibility and dispersal by birds, bats, or other mammals. The fruit is described as a 1- to 3-seeded drupe (Andersson, 1985b; Kress 1990a) or fleshy schizocarp (Smith 1977). The hard rough seed is properly called a pyrene (Kress 1990a) because the seed covering is a stony endocarp. Unlike seeds of many other Zingiberales, those of heliconia have no arH. The embryo is reported to be poorly differentiated at the time of seed maturity (Gatin 1908).

D. Pollination and Compatibility Heliconias are primarily pollinated by hummingbirds (Stiles 1975, 1978) or bats (Kress 1985, 1986, 1990a) and secondarily pollinated by insects and mites which inhabit the inflorescences and disperse via hummingbirds (Seifert 1975, 1982; Dobkin 1984, 1985). The early emphasis by ecologists on heliconias as nectar sources for the hummingbirds or hosts for insect communities largely overlooked the breeding systems and speciation processes of the heliconia. More recent studies have examined how heliconia populations could ensure successful reproduction through the attraction of pollinators and the efficient use of their resources (Dobkin 1984). Inflorescences produce flowers over a long time, with up to five months longevity reported for H. wagneriana in a Trinidad population (Dobkin 1984). The interval between opening of successive flowers, both in a bract and within an inflorescence, ranges from 1.5-7.5 days depending on age of the inflorescence, species, and environment. The hermaphroditic flowers open shortly after dawn, although the Old World species are nocturnal (Kress, 1990a). Flowers last 24 h or less before withering or abscising. The stigmatic surfaces are receptive for only 4-5 h

14


after flower opening. The stigma and anther are in close proximity and the anthers dehisce before flower opening to shed pollen directly onto the stigma (Skutch 1933). Dichogamy probably does not playa role in the breeding systems of these self-pollinated plants (Kress 1983b). Fruit development to mature seed takes 2 to 3 months. Kress (1983a, 1983b) showed that the majority of Costa Rican heliconias were self-compatible (Table 1.2) as indicated both by pollen tube growth in the style and fruit set in controlled pollinations. The number of pollen tubes per style in both selfed and cross-pollinated flowers was found to be very close to the number (3) of ovules per ovary (Kress 1983b). Where self-incompatibility occurred, Kress (1983b) suggested it was gametophytically controlled. Limited study suggested the Old World heliconias were primarily self-incompatible (Kress 1985, 1990a). The foraging behavior of hummingbirds, either in small territories (implying visits to a small number of clones) or through systematic visits to a series of inflorescences (traplining), as well as the low number of open flowers per day in an inflorescence (limiting intraplant pollen transfer) probably contribute more to outcrossing in a species than any physiological self-incompatibility in the New World heliconias (Kress 1983b). This would explain the lack of inbreeding where self-compatibility would seem to be the normal situation. Controlled pollinations between Costa Rican species were carried out Table 1.2. Self-compatible and self-incompatible Costa Rican heliconia species and cross-compatible combinations as determined by pollen germination and growth (Daniels and Stiles 1979; Kress 1983a, 1983b). Self-compatible

Self-incompatible

Cross-compatible

H. colgantea H. collinsiana H. curtispatha H. danielsiana H. imbricata H. irrasa H. latispatha H. mariae H. pogonantha H. sarapiquensis H. stilesii H. tortuosa (partial) H. trichocarpa H. umbrophila H. wagneriana H. wilsonii

H. mathiasae H. nutans hybr. of H. latispatha with H. imbricata

H. H. H. H. H. H. H. H. H.

collinsiana x H. nutans trichocarpa X H. collinsiana imbricata X H. latispatha imbricata X H. sarapiquensis irrasa X H. sarapiquensis latispatha X H. sarapiquensis latispatha X (H. latispatha X H. imbricata) imbricata X (H. latispatha X H. imbricata) sarapiquensis X H. imbricata

1.


15

by Kress (1983a). For the most part, foreign pollen did not grow to the ovules, indicating barriers at the stigmatic and stylar levels. Successful combinations occurred more frequently among species with pendent inflorescences than erect. Table 1.2 presents examples of successful controlled interspecific crosses in Costa Rica. Ecologists have assumed that interspecific crossing normally is rare, as pollinators are adapted to different species, and heliconia species also have different geographic and temporal niches. Kress (1983a) established the importance of crossability barriers in heliconia species with similar morphological characteristics, which prevent interspecific crossing in species visited by the same hummingbird. Such traits permit sharing of pollinators while limiting interspecific genetic transfer. Although natural interspecific hybrids have been identified (Table 1.3; Kress 1990c), these occur mainly where the ecosystem has undergone disturbance and the normal plantpollinator relationships have broken down. Andersson (1981) reports convergence among several species in regions where their ranges overlap, but stated he had no evidence for interspecific hybridization. Photographs (Berry 1988; Hirano 1989) of an F1 seedling population of a cross between H. bihai and H. caribaea showed a wide range of colors, but no data were reported to suggest inheritance patterns. Andersson (1981) reported that the all-yellow form of H. bihai is rare throughout its range, suggesting a recessive character in this mostly red and variegatedred species. Despite a broad range of inflorescence characteristics throughout its wide geographic distribution, there seems to be no reason to subdivide H. bihai into additional species, but it does show convergence of characters with compatible species where their ranges overlap (Andersson 1981). For the most part, collections by hobbyists and commercial growers tend to focus on the unusual color forms; and such collections are unlikely to reflect relative abundances in natural populations. Table 1.3. Natural hybrids and hybrid swarms in Heliconia (Daniels and Stiles 1979; Kress and Stone 1983; Kress 1984, 1990a, 199OC). H. pogonantha var holerythra X H. mariae H. lankesteri X H. nutans H. latispatha X H. imbricata (sterile) H. imbricata X H. sarapiquensis H. 'Richmond Red' (H. caribaea X H. bihai) H. spathocircinata x H. bihai H. X nickeriensis (H. marginata X H. psittacorum) (sterile) H. x 'Golden Torch' (H. spathocircinata x H. psittacorum) (sterile) H. rauliana (H. bihai X H. marginata) H. curtispatha xH. pogonantha var. holerythra (sterile) H. stilesii x H. danielsiana H. tortuosa X H. nutans H. solomonensis X H. lanata

16


E. Physiology As a large perennial herb found mostly in tropical areas, the heliconia has not been well-studied in terms of its physiology. The vegetative character of the plant has received attention with respect to the branching of its rhizomes (Tomlinson 1969; Lekawatana 1986) but most attention has centered on the inflorescence in an ecophysiological sense (insect communities in the bracts, pollination by hummingbirds and bats). Flowering has been a concern of those studying its relationship to nectarsipping birds, but only since the recent introduction of heliconia to the cut-flower industry are studies now undertaking specifically to examine environmental influences on flowering. Branching of the rhizome is primarily dichotomous (Bell and Tomlinson 1980), but the timing of its development with respect to formation and elongation of the erect pseudostem has not been determined. Heliconias are very responsive to light and rapidly colonize light gaps in forested areas (Stiles 1974). Shaded plants are much taller than plants grown in full sun (Stiles 1979; Andersson 1981; Lekawatana and Criley 1989). Some evidence for an active gibberellin system is the considerable responsiveness of heliconia to gibberellin-biosynthesis inhibitors such as ancymidol, flurprimidol, uniconazole, and paclobutrazol (Tjia and Jierwiriyapant 1988; Lekawatana and Criley 1989). Reports on applied gibberellin effects have been mixed. In one instance, foliar GA applications caused elongation of H. stricta 'Dwarf Jamaican' pseudostems, but had no effect on flower induction (Broschat and Donselman 1987). On the other hand, it was reported that gibberellin applied to H. anqusta (Ball 1987b) and H. psittacorum (Natans 1989) led to improved flowering, but these were not controlled experiments. Strongly seasonal flowering patterns in many species led naturalists to suggest a dry-wet cycle control of flowering (Stiles 1979). Because many species showed similar seasonal patterns of flowering in Hawaii where no marked dry-wet cycle existed, other causes were suspected (Criley 1985). The photoperiod responsiveness of several species has been reported (CriIey & Kawabata 1986; Lekawatana 1986; Geertsen 1989, 1990; CrileyandLekawatana 1990a, 1991; Sakai, etal.1990a, 1990b), but not all species show sufficient seasonality to suggest that daylength is the only stimulus for flower initiation. Phenological patterns of flowering for 13 Brazilian species show six of these flowering heavily during or after the short day periods of the year with three showing peak flowering during the longest daylength seasons and the remainder scattered throughout the year (Santos 1978). Given developmental periods of 3-4 months or longer, such as those determined by Criley and Kawabata (1986) and Lekawatana (1986), floral

H. bihai

H. stricta

H. psittacorum

H. caribaea

H. chartacea

H. angusta

Plate 1

H. col1insiana

H. wagneriana

H. x 'Golden Torch'

1.


17

initiation probably occurred during the season of opposite daylength duration. Temperature tolerance in heliconia is associated with species and with the origin of plant material within a species. In Brazil H. episcopalis is found only at J

co

Table 1.4.

Species

Continued. Keeping" quality (days) Seasonality"

CultivarsZ

Inflorescence orientation

Bract color

Flower color

Bucky

Upright

Red-orange

White

5-7

Dwarf Jamaican

Upright

Red-orange

White

Upright

Red

Peru Royal (= Tagami)

Upright Upright

Sharonii

Height (m)

Growth habit

1.3-2.5

Spreading

5-8

Sept.-Mar.• Peak in Dec.-Feb. Year-round

0.5-1.5

White

5-6

July-Mar.

1.5-2.5

Slow spreading Spreading

Red Red-orange. yellow rim

White White

6-7

1.3-2.0

Upright

Red-orange

White

4-6

July-Jan. July-Apr. Peak in Sept.-Nov. July-Jan.

Dusty Rose

Upright

July-Jan.

1.0-1.3

Upright

Orange

Upright

Orange

Orange & White

Upright

Africa

Upright

Orange. light Yellow margin Red

White with green tip White with green tip White with green tip White with green tip

4-6

Fat Stricta

Red-orange. yellow rim Orange

(= Dwf "HumiIis")

Firebird (= Red Royal)

H. subulata

Yellow with green tip

5-7

1.5-2.5

Spreading Spreading

5-6

1.0-1.8

Slow spreading Slow spreading Clumping

5-6

1.0-1.8

Clumping

1.0-1.8

4-6

Year-round

1.0-1.3

Clumping

5-7

Jan.-May

2.0-2.5

Spreading

Table 1.4.

Continued.

Species

H. wagneriana

H. sp.

~

C&:l

Keeping Y quality SeasonalityX (days)

Height (m)

Growth habit

Jan.-May Peak Mar.Apr.

1.8-2.0

Clumping

7-14

Feb.-Mar.

1.8-2.0

Clumping

5-7

Winter

1.0-1.5

Clumping

Inflorescence orientation

Bract color

Flower color

Purple Flat (Flat Purple) Rainbow (Easter)

Upright

Purple-red

Yellow

Upright

Green

7-21

Turbo

Upright

Green

Carnaval

Upright

Green rim, pale red blotch on light yellow background Yellow & green rim, redorange base Lavender

Gold-yellow

Cultivarsz


30

Table 1.5. Heliconias with colored foliage grown for cut foliage. potted plant or interior use. (Green 1969; Kress 1990a; Berry and Kress 1991). Leaf colors

Growth habit

Rabaul

Bright yellow-green

Clumping

"Sanderi" (= Bangkok, Bangkok Gold) "Spectabilis" (= "Illustris", "Rubricaulis", "Rubra") (= "EdwardusRex") (= "Roseo-striata", "Rubro-striata")

Variable patterns of cream white to rose on glossy green

Clumping

2.0-4.0 2.0-3.0

Green and copper-red to maroon; more intensively red on underside. Tends toward burgundy Green with numerous rose-pink, red, or white lateral striations (Juvenile form?) Green with yellow or "Striata" (= "Aureo-striata") white lateral striations

Clumping

2.0-5.0

Clumping

2.0-4.0

Species

Cultivar

H. indica

Height (m)

H. stricta

Sharonii, Dusty Rose

Dark green with maroon midrib and maroon underside

Clumping

0.7-1.2

H. zehrina

Tim Plowman

Alternating bands of interveinal dark green and light green over main lateral veins; underside purplish or green (2 variants).

Clumping

0.6-1.2

ZNames shown in double quotes (" ") have been used as invalid species names but are still used in commercial trade to identify some selections.

flower markets led to a large increase in the number of commercial growers in Hawaii during the 1980s (Table 1.6). Hawaiihascompiledboth production statistics and farm gate wholesale values (Table 1.6) (Hawaii Agr. Stat. Servo 1991). A market newsletter compiled by the Hawaii Department of Agriculture with the U.S. Department of Agriculture in cooperation with the San Francisco Wholesale Flower Market (Ninomiya 1990) gives some measure of the wholesale values of several kinds of heliconias (Table 1. 7), but volumes were not reported. During 2 recent years (Tsugawa 1988; Ninomiya 1990) wholesale prices for Hawaii-grown heliconias were strong, fluctuating with season, availability, and holiday demands. 3. Modeling. Coincident with the increased interest in cut-flower production of heliconia has been a demand from potential growers for

1.


31

Table 1.&. Producing farms, estimated production area, numbers of stems sold, and farm value for heliconias in Hawaii 1985-1990. (Hawaii Agr. Stat. Servo 1991)

Year

No. farms

Estimated production area (ha)

1985 1986 1987 1988 1989 1990

34 58 78 83 120 117

27.8 46.3 61.4 78.5 76.8

No. stems sold

Value of sales

(X 1000)

($ X 1000)

31 77 161 206 185 220

125 391 1427 1364 1130 1339

NA

Table 1.7. Wholesale prices for Hawaii-grown heliconia on the San Francisco Wholesale Flower Market during 1990 (Ninomiya 1990). The lower prices predominate during seasons of peak production; higher prices occur during low season of production and near holidays. Occasionally, prices will fluctuate one dollar higher or lower than these ranges for exceptional quality or demand or if too many flowers have been shipped. Wholesale price range/stem ($) Species

Small

Medium

Large

H. angusta

Red Christmas

2.00-3.00

H. bihai

Lobster Claw H. caribaea

'Purpurea'

3.50-4.00

3.50-7.50

5.00-6.50 3.50-5.50

6.50-7.50 4.50-7.50

5.50-7.00

7.00-8.00

0.75

1.00

1.00-2.50

1.00-1.25

1.00

1.00-2.00

2.50-4.50

3.50-6.00

3.50-4.50 3.00-4.50

H. chartacea 'Sexy Pink'

H. psittacorum 'Parakeet' type H. hybrid 'Parrot' Misc. Upright Hanging

4.50-7.50 7.50-8.50

data on productivity of different species. Ecologists note considerable variation in flower production from year to year and within marked clumps of wild heliconia. Flowering is believed to depend on factors, such as light availability and wet versus dry season (Seifert 1975; Stiles 1975, 1978; Dobkin 1984). H. psittacorum 'Andromeda' produced 160 flowers m-Zyr- 1 in outdoor

32


beds during the second year of production and 175 flowers m-2 yr-1 in a greenhouse, while 1.5-year-old plantings of H X 'Golden Torch' averaged 84 flowers m-2 in outdoor beds in south Florida (Broschat et a1. 1984). Commercial producers rarely keep records by clump or area allocated to a given species/cultivar, and the increased demand for heliconias has resulted in increased planted areas, which renders year to year comparisons invalid. At present, it is not possible to provide even modestly accurate productivity estimates for most large-flowered species and cultivars in commercial production. The geometry of rhizome growth and pseudostem development (Bell and Tomlinson 1980) allow a predictive capacity for shoot and flower production. Single rhizomes of the cultivar, Parrot (= 'Golden Torch'), (H. spathocircinata X H. psittocorum) showed an exponential increase in pseudostem and flower number per plant over a 19-month period (Figure 1.6) (Manarangi et a1. 1988). Flower production trailed pseudostem production by 16-18 weeks. The responsiveness of this heliconia to high light intensities was shown by the production of more shoots during summer months (Figure 1.6). H. angusta (Sakai et a1. 1990a) showed a similar growth pattern. Complicating model development, however, is a tendency of the inflorescence to abort early in its development (Criley and Kawabata 1986; Lekawatana 1986). Efforts to model growth and floral development build upon studies 70

70

60

60

~

50

50

a: w a-

40

w 40 a-

:5 a-

en a:

.....

:I:

(J)

w

3:

30

30 0

..J

u..

..J

..J

;5 0

.....

f

z

a:

(J)

0 0

.....

:5a-

« .....

20

0 20 .....

10

10

0

0

J J A SON D J F M A M J J A SON D 1985

1986

Figure 1.6. Shoot and flower production per plant for Heliconia X 'Parrot' (= 'Golden Torch') during an 18-month period following planting of single rhizome pieces (adapted from Manarangi et al. 1988).

1.


33

involving morphological and physiological understanding as well as the impacts of environmental factors, such as photoperiod, temperature, and solar integral. Ranges in floral development times for the few species studied are shown in Table 1.8. CrUey and Lekawatana (1990a,b) reported that H. chartacea growth rate from shoot emerge.nce to flowering was strongly correlated with temperature in a degree day model, at least through the first four to five emerged leaves. Aplot of leaf number (representing pseudostem elongation) versus the mean time for each leaf to unfurl was linear from the second leaf up to flowering for H. chartacea (Figure 1.7) and could provide assistance to commercial growers in predicting flowering time. 4. Propagation. Heliconias can be propagated vegetatively or by seed, but seed set is often sporadic in areas lacking suitable pollinators (Broschat and Donselman 1983a; Montgomery 1986). Desirable phenotypic characteristics may not be maintained by seed propagation, although Criley (1989a) found that seedlings of H. stricta 'Dwarf Jamaican' were uniform and similar to their parents. Also, since seed germination is often slow and poor (Lekawatana and Criley 1989), vegetative propagation is normally used for heliconias. a. Seed propagation. Heliconia fruits are blue (new world species) orred (old world species) at maturity and contain from one to three stony 5-20 mm long seeds each (Criley 1988; Carle 1989). The soft fruit is removed

8r-------------------. 7 I-

6

o

----+------1

••

•• t

•

••

5

~ 4

L1i 3

...J

2

o

10

20

30

40

50

60

TIME (WEEKS) Figure 1.7. Leaf number on H. chartacea pseudostems (N = 181 pseudostems that flowered over a 2 .5-year period; 746 data points) versus time to achieve leaf unfurling from shoot emergence. Solid triangle'" represents the mean time to flower ± 1 SD as well as the mean leaf number ± 1 SD subtending the inflorescence (Criley and Lekawatana, unpublished).


34 Table 1.8.

Development times to flower for selected heIiconias in Florida and Hawaii.

Species Cultivar

Development time (wk)

Conditions

Reference

H. psittacorum 'Andromeda'

8-9

From shoot emergence; full sun, summer, south Florida, 21-35°C

Broschat et al. 1984

H. spathocircinata X H. psittacorurn 'Golden Torch'

9-10

From shoot emergence; full sun, summer, south Florida, 21-35°C

Broschat et al. 1984

16-19

From shoot emergence; full sun, high rainfall, (minimax) of 17/26°C winter, 22/30°C summer, Hilo, Hawaii

Manarangi et al. 1988

H. stricta 'Dwarf Jamaican'

13-14

From start 0 f 4 or more wk SD, potted plants. 21/36°C (NT/DT), Shaded greenhouse, 83 klx

Criley and Kawabata 1986

'Dwarf Jamaican'

19

From start of SD: first 4 wk SD at 15-20°C, remaining development at

Lekawatana 1966

H.

X

'Parrot'

(= 'Golden Torch')

20/37°C (NT/DT);

potted plants, shaded greenhouse H. angusta

14-18

From start of 10 to 14h daylengths. 19.5-

Lekawatana 1986

23.5/29.5-36°C (NTIDT); potted

plants. shaded greenhouse

'Holiday'

14.5

From start of LD to anthesis for pseudostem with 4 leaves

CrUey and Lekawatana 1991

21-26

New shoots, outdoor beds, Hilo. Hawaii, daylength > 13.3 h, 21-25°C

Sakai et al. 1990a.b

1.


Table 1.8.

35

Continued.

Species Cultivar

Development time (wk)

Conditions

Reference

H. wagneriana 'Turbo'

14-17

From start of 8 h SO, full sun, 100 I tubs, Honolulu, Hawaii

Crileyand Lekawatana 1991

H. chartacea 'Sexy Pink'

46

From shoot emergence, year round avg, full sun, field conditions, Hawaii

Criley and Lekawatana 199oa,b

usually prior to planting; fruit that has dried in the bract or seed which floats in water should not be used (Carle 1989). Seed germination occurs sporadically over a period from 3 months to 3 years (Criley 1988). Kress and Roesel (1987) found that holding seeds of H. stricta 'Dwarf Jamaican' for 2 weeks prior to planting resulted in a greater germination speed and percentage than when planted immediately. However, in a similar experiment with H. aurantiaca, no differences in germination were observed between seeds held for 2 weeks or those planted immediately (Kress and Roesel 1987). The effects of holding seed of other species prior to planting are unknown. Embryos may not be mature at the time of fruit ripening and an after-ripening period may be needed by some species (Gatin 1908; Kress and Roesel 1987). Acid scarification of heliconia seedcoats is not effective in promoting seed germination (Kress and Roesel 1987). Light requirements for seed germination have not been studied, but seed is usually sown in fIa ts and covered to a depth equal to the seed thickness using a well-drained medium or is held in a moist vermiculitesphagnum moss medium in plastic bags until germination occurs (Criley 1988; Carle 1989). Temperature requirements for optimum seed germination are unknown, but Carle (1989) recommends 25-35°C. Seedlings may be transplanted into larger containers when they are 2-4 em tall. b. Vegetative propagation. Heliconias are usually propagated by dividing clumps into sections containing one or two pseudostems (Broschat and Donselman 1983a; Criley 1989a). Existing pseudostems are cut back from 15-30 cm from the rhizome, all dead root, stem and leaf tissue is removed, the rhizomes are dipped in a fungicide, and planted in a well-drained rooting medium such as perlite, vermiculite, or sand (Criley 1988, 1989). For rhizomes that will be shipped, or are suspected of harboring nematddes, all roots should be cut off. Soaking cleaned rhizomes

36


4-5 em in diameter in 48°C water for up to 1 h or in 50°C water for up to 30 minutes can be used to kill nematodes in the rhizomes without affecting plant survivial (Criley 1988). Exposure to higher temperatures or durations over 1 h for 50°C water resulted in death of small rhizome pieces. Surface sterilization with hypochlorite bleach (1 part bleach to 9 parts water) or formalin (1 part formaldehyde to 99 parts water) for 10-15 min can also be helpful in reducing microorganism contamination prior to shipping or planting (Criley 1988). In the 4 weeks or so required for new root development, the existing pseudostems will die back to the rhizomes, but will be replaced by new pseudostem buds in about 4-6 weeks (Criley 1988). Optimum root and shoot development occurred at 20°C for H. stricta, 'Dwarf Jamaican' (Lekawatana and Criley 1989). Rhizomes can be transplanted into the field or large containers once new pseudostems emerge from the rooting medium. For direct planting in fields or beds, rhizome clumps containing 3-5 pseudostems and intact root systems result in more rapid regrowth than single pseudostem rhizomes (Broschat and Donselman 1983b). Although related to bananas, which were micropropagated for years, heliconias were only recently successfully tissue cultured. In 1988 Criley (1988) listed a single lab producing H. psittacorum 'Andromeda' and H. X 'Golden Torch'. By 1990 at least three other laboratories were producing H. psittacorum cultivars, H. lankesteri, H. caribaea, H. stricta 'Dwarf Jamaican', H. stricta 'Sharonii', H. latispatha, and H. X 'Golden Torch', although techniques used by the various labs have not been published (Berry 1990). Tissue cultured heliconias produce many more basal shoots than rhizome-propagated plants, an advantage for pot production (Tjia and Jierwiriyapant 1988). Tissue culture also allows for the movement of disease-free stock into other countries. 5. Production environment.

a. Light. In their natural habitat, heliconias grow best in forest clearings, with the number of flowering stalks decreasing as light intensity decreases (Stiles 1979). Most heliconias are grown commercially in open field situations, although bract color for some species may be more intense under light shade (Criley 1989a). Insufficient light intensity is a primary factor limiting H. psittacorum flower production. Flower production by H. psittacorum 'Andromeda' in south Florida was 3-4 times as great for full sun beds as for beds under 63% shade in first-year beds and about twice as great in second year beds (Broschat and Donselman 1983a). Reduced light intensities within beds due to mutual shading by pseudostems eventually limits flower production in this species, even under full sun. Under optimum fertility levels (650 g N m-2 yr-l), densities of up to 700 pseudostems/m 2 were reported for second-year beds of

1.


37

H. psittacorum 'Andromeda' (Broschat et a1. 1984). Under these conditions virtually no light penetrates through the foliage, succeeding pseudostems become weak and excessively elongated, and flower production and quality decline (Broschat et a1. 1984). Annual flower production of H. psittacorum in heated greenhouses in south Florida having about 80% light transmittance was actually less than that obtained outdoors under suboptimal temperatures due to the inadequate light intensities in the greenhouse during the fall through spring months (Broschat and Donselman 1987). b. Photoperiod. Species such as H. psittacorum, H. X nickeriensis, H. episcopalis, H. hirsuta, H. X 'Golden Torch', H. chartacea, and some cultivars of H. stricta and H. bihai flower year round under suitable light intensities and are generally considered to be day-neutral or nonphotoperiodic (Broschat and Donselman 1983b; Criley 1989). However, many other species are seasonal in their flowering and photoperiod control of flowering cannot be ruled out (Criley 1989). Although H. angusta 'Holiday' flowers during winter months, Sakai et a1. (1990a) found that flowers were initiated 6 months earlier during the longest days of the year. A critical daylength of 13.3 h was determined for this cultivar (Sakai et al 1990a). On the other hand, H. stricta 'Dwarf Jamaican', H. wagneriana, and H. aurantiaca have been shown to initiate flowers under short days (Criley and Kawabata 1986; CrUey 1989; Geertsen 1990) with 4 weeks of long nights required at 15°C for flower initiation in H. stricta 'Dwarf Jamaican' (Criley 1989). A minimum of 3 leaves must be present for this species to respond to photoperiodic stimuli (Criley and Kawabata 1986). Because H. stricta 'Dwarf Jamaican' flowers throughout the year, it is considered to be a facultative, rather than an obligate short day plant (Criley and Kawabata 1986). Criley and Lekawatana (1990a) demonstrated that H. wagneriana 'Turbo' was a short day plant which flowered 100 to 120 days from the start of short day treatment. Experimental evidence for a photoperiodic response in other species of seasonal heliconias does not currently exist. c. Temperature. While no Heliconia species is reported to initiate flowers in response to temperature, increasing temperature' indirectly increases flowering rate due to an increased overall growth rate for H. aurantiaca, H. psittacorum, and H. X 'Golden Torch' (Criley, 1989; Geertsen 1989, 1990). For H. psittacorum, 'Tay', increasing temperature from 15-21°C increased flower production in Denmark from 25-60 flowers/m 2 , and flower grade and stem length were also increased (Geertsen 1989). Armbruster (1974) recommended a soil temperature of 18-23°C and venting when air temperature exceeded 28°C. Optimum temperatures for cut flower production vary, but for H. psittacorum Broschat et a1. (1984) and Van Raalte and Van Raalte-Wichers

38


(1973) suggest a 21°C minimum, with increased production up to about 35°C. H. stricta 'Dwarf Jamaican' and H. angusta 'Holiday' will grow and flower at 15°C (Criley 1989), although optimal growth undoubtedly occurs at higher temperatures. Temperature is a major limiting factor in the production of H. psittacorum in Florida (Broschat and Donselman 1983a). Growth and flower production decline as minimum temperature decreases from 2110°C and ceases altogether at 10°C. At 10°C, cold injury symptoms first appear as small black spots on the floral rachis at the point of bract attachment. At colder temperatures the entire infloresence blackens, followed by necrosis on the foliage. Freezing temperatures kill the pseudostems back to the ground, but rhizomes may survive l-ZoC colder temperatures. Flower buds of H. psittacorum exposed to temperatures below lO°C will not develop normally, if at all (Broschat and Donselman 1983a). These high temperature requirements for this species led to its abandonment by Dutch growers during the oil energy crisis of the 1970s (J. van der Krogt, personal communication). d. Growing Medium. Most species of heliconia are highly tolerant of different soil types and commercial production has been successful on soils ranging from volcanic cinders to heavy clay soils (Criley 1989). Although acid soils are preferred, slightly alkaline soils have also been successfully used for many species. H. psittacorurn and H. X 'Golden Torch', however, are highly intolerant of alkaline or poorly drained soils and in south Florida are grown in soilless container media in order to prevent Mn and Fe deficiencies from occurring (Broschat et a1. 1984). 6. Planting.

a. Beds. In field production situations, little bed preparation is required prior to planting. Steam pasteurization or chemical fumigation is recommended to eliminate nematodes, soil borne pathogens, and weed seeds (Criley 1989). For H. psittacorum production in areas such as south Florida that have alkaline soils, raised beds containing a soilless container medium are recommended (Donselman and Broschat 1986). To maximize space utilization, beds approximately 0.8 m wide, surrounded by a solid 30-cmdeep barrier are used to contain the medium and confine the aggressive rhizomes. Wider beds make more efficient use of space but make harvesting more difficult and result in reduced light penetration through the foliage and subsequent plant stretching (Broschat and Donselman 1983a). The extremely high densities of pseudostems in confined beds of H. psittacorum result in peripheral shoots being forced outward and into the aisles. In order to preserve aisle space for access, prevent the formation

1.


39

of bent peduncles, and prevent lodging of the stalks due to wind, some type of support is required. One or 2 wires supported 0.6-1.2 m over the perimeter and every 2 m across the beds will help confine and support the plants (Donselman and Broschat 1986). b. Spacing. Spacing for heliconias depends on factors, such as species size, habit (spreading vs clumping), and growth rate (Criley 1989). Spreading species rapidly fill in beds whereas clumps of clumping species may expand rather slowly and may therefore be planted more closely. Criley (1989) suggests the following within row spacing for field production of heliconias: H. psittacorum- 0.75-1 m; H. hirsuta, H. metallica, H. angusta, H. aurantiaca, H. vaginalis, and small H. stricta cultivars-1.2-1.5 m; H. rostrata, H. angusta 'Flava', H. X 'Golden Torch', H. latispatha, and larger H. stricta cultivars-1.5-2 m; and H. caribaea, H. bihai, H. chartacea, H. wagneriana, H. collinsiana, H. bourgaeana, H. champneiana, H. platystachys, and H. indica- 2-2.5 m. Rows or beds can be as little as 1.5 m apart for small species, but 2-3 m or more for larger species, depending on machinery access requirements. For H. psittacorum production in confined beds in Florida, Broschat and Donselman (1983a) recommend planting rhizomes on 30-cm centers. At this spacing, clumps containing 3-4 pseudostems each filled in 1-mwide beds in 6-8 months, but single bud rhizomes required somewhat more time. A planting depth of 10 em was recommended for this species (Broschat and Donselman 1983a). c. Renovation and Replanting. Eventually heliconia beds become overcrowded and unless they are are renovated, flower production and quality will decline. Clumping species spread primarily outward, often leaving the center of the clump devoid of shoots. Such clumps should be dug up and replanted at 2-3 year intervals (Criley 1989). New pseudostems from rapidly spreading species, such as H. psittacorum, quickly invade aisles in field plantings. In confined beds, aisle integrity is preserved, but shoot density within the beds becomes too high. In south Florida, confined beds of H. psittacorum and H. X 'Golden Torch' should be cut back to the ground after 2 years of production, but after 3 years the beds should be dug up and replanted to maintain optimum flower production and quality (Broschat and Donselman 1987). More frequent renovation of H. psittacorum beds is usually required in more tropical climates. 7. Nutrition.

a. Disorders. Nitrogen deficiency is common on most species of heliconia and appears as an overall light yellow-green coloration of the foliage and decreased growth rate (Broschat and Donselman 1983b). Potassium deficiency is very common on H. angusta and H. stricta 'Sharonii', but less so on other species. Symptoms include extensive

40


marginal necrosis on the oldest leaves. The necrosis may be accompanied by a diffuse marginal and/or interveinal chlorosis (Broschat 1989). Magnesium deficiency symptoms appear first on oldest leaves as broad yellow bands along the lateral leaf margins (Broschat and Donselman 1983a). Under alkaline soil conditions Fe and Mn deficiencies are common, particularly in species such, as H. psittacorum and H. X 'Golden Torch' (Broschat and Donselman 1983a). Iron deficiency is also induced by poor soil aeration, cold soil temperatures, or root injury caused by root rot diseases or nematodes. Symptoms of Fe deficiency occur on newest leaves as uniformly yellowish-white leaves, although the midrib may be slightly greener (Broschat and Donselman 1983a). Manganese deficiency symptoms occur first on new leaves as an interveinal chlorosis accompanied by transverse necrotic streaks (Donselman and Broschat 1986). Other nutritional disorders have not been reported on heliconias. b. Fertilization. H. psittacorum responds positively to high rates of N fertilization. In Florida, Broschat and Donselman (1983a) found that a rate of 650 g N m-2 yr-l from a 3N-1P-2K ratio controlled-release fertilizer produced more flowers than lower rates and did not decrease flower quality. Studies on N to K ratios for this species showed no differences in flower quality or quantity among beds treated with K at rates of 0-650 g K m-2 yr-l, suggesting that K is not a limiting factor in this species (Broschat and Donselman 1987). When container media are used they should be amended with dolomite and a micronutrient blend to prevent Mg and micronutrient deficiencies (Donselman and Broschat 1986). Scientific studies on the fertilization of other species of heliconias are lacking, but a typical program includes the application of about 200 g of a IN-1P-1K ratio soluble fertilizer 3 or4 times per year per plant (Criley 1989). Because of the decreased potential for leaching and reduced labor requirements, Donselman and Broschat (1986) recommend using the longest term slow release fertilizers available. Beds of H. psittacorum can thus be fertilized at planting time and annually thereafter when they are cut back or replanted. The extreme density of foliage in confined beds of H. psittacorum makes uniform application of any granular fertilizer virtually impossible at other times of the year. Alternatively, injection of fertilizer through the irrigation system is an efficient and convenient method for fertilizing heliconias, but specific rate recommendations have not yet been developed (Donselman and Broschat 1986).

8. Irrigation. Water stress is frequently a limiting factor in flower production and flower quality for H. psittacorum. Cut-flower vase life is decreased by inadequate watering during the production phase

1.


41

(Donselman and Broschat 1986). Water stress is indicated in H. psittacorum by a longitudinal rolling of the foliage (Broschat and Donselman 1983a). Although heliconias use a substantial amount of water, poor soil aeration is a major cause of root rots and nutritional disorders (Brochat and Donselman 1983a). On well-drained sandy soils or in soilless container media, overhead irrigation is the most efficient method of irrigation, since lateral movement of water from drip irrigation systems is inadequate to thoroughly moisten the soil. Also, water from irrigation heads installed at ground level seldom penetrates into the center of the beds, due to the high density of pseudostems in species', such as H. psittacorum (Brochat and Donselman 1983a). For H. psittacorum growing in well-drained container media, daily irrigation with ca. 1 em of water is essential for maintaining rapid growth and high flower quality (Donselman and Broschat 1986), but for other species growing in heavier soils, 2.5 em of water per week plus natural rainfall appears to be sufficient (Criley 1989).

9. Pest management. a. Insects and Mites. Heliconias are relatively free of serious insect problems, although aphids, mealybugs, scales, earwigs, thrips, and leafeating beetles and caterpillars are known to feed on heliconias (Broschat and Donselman 1983a; Criley 1989). Aphids are the most common pest on H. psittacorum" feeding primarily on nectar in the inflorescences (Broschat and Donselman 1983a). Ants frequently tend aphids and may themselves cause injury to the bracts of some species (Criley 1989). Mites can infest the foliage during ~ot, dry weather outdoors or in greenhouses, but are seldom a serious problem (Broschat and Donselman 1983a; Criley 1989). Because the presence of insects in the inflorescences of heliconias is cause for rejection by agricultural inspectors of shipments into the mainland United States, most heliconia inflorescences are dipped in solutions of insecticides, such as malathion or diazinon, and hand cleaned to remove debris and dead insects prior to shipment (Criley 1989). Looking to the future, Hansen et a1. (1991a) have demonstrated that fumigation with 2500 ppm HCN for 30 minutes caused no phytotoxicity to cut heliconias, although efficacy against major quarantine insect pests still must be determined. Hansen et a1. (1992) recently reported that largeflowered heliconia inflorescences exposed to vapor heat treatment (1D60-min exposure to heated (46.6°C) water-saturated air] were undamaged although the smaller H. psittacorum flowers suffered unacceptable damage when given 2-3-h exposures. Virtually all aphids, mealybugs, scales, and thrips were killed within 1 h. b. Diseases. Diseases of heliconias include root rots caused by

42


Cylindrocladium Morgan sp., Pythium splendens H. Braun, and Rhizoctonia solani Kuhn (Alfieri et a1. 1984; Broschat and Donselman 1988; Uchida et a1. 1989) and leafspots caused by Cercospora Fres. sp., Curvularia Boedijn sp., Helminthosporium Link ex Fr. sp., Phomopsis Sacco sp., Phyllosticta heliconiae F. Stevens & P. A. Young, Septoria Sacco sp., and Mycosphaerella Johanson sp. (Raabe et a1. 1981; Alfieri et a1. 1984; Criley 1989). A bacterial wilt of heliconia caused by an undescribed pathovar of Pseudomonas solanacearum has been reported from Hawaii (Ferreira 1990). Although not susceptible, heliconias are known carriers of the pathovar of P. solanacearum (E. F. Smith) E. F. Smith that causes Moko disease of bananas, and banana-growing countries often prohibit the importation of heliconias from Moko-infested areas (Ferreira 1990). Leafspot diseases on heliconias are usually rather insignificant and treatment may not be required, but root rots do cause serious damage and must be prevented by using pasteurized soil and clean stock, or treated with appropriate ,fungicides. Cucumber Mosaic Virus has been reported from H. psittacorum, but symptoms appeared only on stressed plants (Ball 1986a). Plant parasitic nematodes often infest heliconias and may result in water stress or micronutrient deficiency symptoms being expressed (Broschat and Donselman 1983a). In Hawaii, the 4 types of nematodes most commonly found on heliconias are burrowing [Radopholus similis (Cobb) Thorne], lesion [Pratylenchus coffeae (Zimmermann) Filipjev & Stekhoven], reniform (Rotylenchulus reniformis Linford & Oliveira), and root-knot (Meloidogyne spp.), with burrowing nematodes being the most widespread and damaging (Holzmann and Wong 1986). Spiral nematodes [Helicotylencus erythrinae (Zimmermann) Golden] and lesion (Pratylenchus goodeyi Sher & Allen) are reported on heliconias in California (Siddiqui et a1. 1973). Most of these types also occur on heliconias in Florida (Fla. Div. of Plant Industry, unpublished data). Treatment for nematodes include steam pasteurization or chemical fumigation of soil prior to planting, planting only hot-water-treated or nematode-free rhizomes, or post-planting chemical treatments (Holzmann and Wong 1986). c. Weeds. Weed problems can occur between rows of heliconias or in recently planted beds, but established beds of many species will shade out most weeds. Preemergent applications of oxydiazon and postemergent directed sprays with glyphosate are commonly used in Hawaii (Criley 1989). 10. Harvest and postharvest.

a. Harvesting. Heliconias flowers are usually harvested when about

1.


43

two-thirds of the bracts are open, although H. psittacorum are sometimes cut with only one or two bracts open. Bracts will not continue to open after harvest, even if sucrose-containing bud-opening solutions are used (Broschat and Donselman 1983a). Heliconias are harvested by cutting the flowering pseudostems off at ground level (Broschat and Donselman 1983b). Flowering pseudostems of H. psittacorum can also be harvested by pulling with a quick tug (Broschat and Donselman 1983a). Broschat and Donselman (1987) found that flowers of H. psittacorum 'Andromeda' harvested at 0800 had an average postharvest life of 23 days versus 16.3 days for those harvested at 1300. All leaves are generally removed from the stalks and for species other than H. psittacorum, petioles are cut just above the top of the inflorescences to protect the bracts during shipment (Hansen et a1. 1990). b. Cleaning, packing, and shipping. Since the bracts of most heliconia species contain dead floral parts, insects, and other debris, they must be cleaned prior to sale. Florets, insects and other debris are removed manually and/or with pressurized water and the inflorescences are often dipped in insecticide and sometimes fungicide solutions prior to shipping (Hansen et a1. 1990). Individual inflorescences (or bunches of inflorescences for H. psittacorum) mayor may not be sleeved or wrapped prior to packing (Inouye 1986). Heliconias are typically shipped and packed in moist shredded paper. Upon receipt, heliconia flowers are placed in water to rehydrate them and are stored in water at 13-15°C. Flowers should not be exposed to temperatures below 10°C or cold injury will result (Broschat and Donselman 1983a). c. Postharvest handling. Postharvest life of cut heliconia flowers varies considerably among species and cultivars within species. Postharvest life for good H. psittacorum cultivars is about 14-17 days, but flowers of other species often last less than one week (Table 1.5). Most studies on the postharvest handling of heliconias have been done on H. psittacorum. Results of studies evaluating various types of floral preservatives have consistently shown no significant extension of vaselife of H. psittacorum cultivars over that of control flowers (Broschat and Donselman 1983a; Tjia and Sheehan 1984; Tjia 1985; Ka-Ipo et a1. 1989). This is not surprising since uptake of water or preservative solutions by cut H. psittacorum flowers is negligible (Broschat and Donselman 1983a; Ka-Ipo et a1. 1989). Some antitranspirants increased the postharvest life of H. psittacorum 'Parakeet' (Ka-Ipo et a1. 1989) and 'Andromeda' (Broschat and Donselman 1987). Since water uptake is minimal in cut flowers of this species, conservation of existing internal water may be important in prolonging their vase-life. H. psittacorum 'Parakeet' flowers with 1-3 leaves left on the stem were

44


found to take up much more water than did leafless flowers (Ka-Ipo et al. 1989]. Since the leaves have no above-ground vascular connections with the peduncle, predictably, leaf number did not affect the postharvest life of the flowers (Ka-Ipo et al. 1989). They also found that cutting the stems at 45 cm versus 90 cm did not significantly affect cut flower postharvest life.

B. Pot-Plant Production Production of heliconias as flowering pot plants has been limited primarily by the large size of most heliconia species at maturity. For this reason, pot culture has concentrated on the smallest species such as H. psittacorum, H. X 'Golden Torch', H. angusta 'Holiday', and H. stricta 'Dwarf Jamaican', but each of these species has its own unique cultural problems. Ball (1987c) forsees perhaps 10% of heliconia species ashaving commercial pot plant potential, but this is undoubtedly optimistic. 1. Planting. Since tissue-cultured material has not been readily available, most propagation has been by division of rhizomes. Broschat and Donselman (1988) found that for direct planting of H. psittacorum 'Choconiana' in 15-cm azalea pots, rhizomes with new pseudostems less than 20 cm long (plantlets) had a 93% survival rate and produced an average of 1.4 additional new shoots within 140 days. Rhizomes with pseudostem bases that had previously flowered had only a 62% survival rate, but produced an average of 2.3 new shoots per rhizome. Since the original pseudostems from the plantlets flower much earlier than lateral shoots they are cut off at ground level after flowering to make room for development of the lateral shoots (Ball 1987a). Tissue-cultured plantlets of heliconias produce many more lateral shoots and are preferred for container production. Tjia and Jierwiriyapant (1988) found that H. X 'Golden Torch' from tissue culture produced an average of 30 lateral shoots and thata single plantlet easily filled a 15-cm pot within 200 days. Ball (1987b) suggests direct planting 4-5 rhizomes of H. psittacorum or H. X 'Golden Torch' per 25-cm pot, 3-4 in a 20-cm pot, or 3 in a 15-cm pot. A well-drained medium and shallow planting are recommended for rooting, as well as growing to prevent Pythium and Cylindrocladium root rots (Broschat and Donselman 1988]. Rooting is successful under intermittent mist or fog (Ball 1986b), but Lekawatana and Criley (1989] hold rhizome pieces in plastic bags at 20°C in the dark until rooting is evident, then plant them. For H. stricta 'Dwarf Jamaican', Lekawatana and Criley (1989) recommend planting 1-2 rhizomes per 15-cm pot for

1.


45

optimum density. Ball (1987b) recommends planting 2 rhizomes of H. angusta 'Holiday' per 25 em pot. 2. Light Intensity. H. psittacorum and H. X 'Golden Torch' are normally grown under full sunlight to keep the plants compact and improve flowering, but foliage color may be lighter as a result. Ball (1986b) suggests growing in full sun until 4 or 5 leaves have been produced and then covering the plants with 30% shade for the remainder of the production cycle. H. angusta will burn under full sun and should therefore be grown under 30-40% shade (Ball 1987b). H. stricta 'Dwarf Jamaican' should be grown under full sun or light shade to prevent stretching (Lekawatana and CrHey 1989). During the propagation phase, all species should be maintained under shade. 3. Temperature. Although H. angusta 'Holiday' is more tolerant of cool temperatures than H. psittacorum, it grows best with night temperatures above 18-21°C (Ball 1987b). Ball (1987a) suggests 21-24°C minimum night temperatures for H. psittacorum production. Rooting of H. stricta 'Dwarf Jamaican' occurs more rapidly at 20-25°C, but optimum flowering occurs when grown at 15°C night temperatures (Lekawatana and CrHey 1989). 4. Photoperiod and Scheduling. For H. stricta 'Dwarf Jamaican', long nights should be provided for 4 weeks once pseudostems have developed 3 leaves (Lekawatana and CrHey 1989). Flower development requires 1319 weeks from the start of long nights, depending on the environment. In Florida, Ball (1987b) recommends planting rhizomes of H. angusta 'Holiday' in April for natural flowering in November through February. Sakai et a1. (1990b) obtained greater flower production in this species in Hawaii during November through April by providing a 16-h photoperiod from April through December. 1'hey suggest that flowers might be produced at any season by providing long days for 5-6 months. H. psittacorum is not photoperiodic and flowers year-round, but growth rate and flowering are strongly affected by temperature and light intensity (Broschat and Donselman 1983a). Ball (1987b) suggests that a crop could be produced in 12 weeks under high light and 21°C minimum temperatures starting in May, but 24 weeks are required to produce a crop starting in December with its lower light levels and 15°C minimum temperatures. 5. Fertilization. Very little research has been published on fertilization requirements for potted heliconias. Ball (1987a) recommends using Osmocote 18-6-12 at the highest label rate, plus semiweekly drenches with 300 ppm N to promote rapid growth of H. psittacorum, but suggests that under reduced light intensities these rates may have to be reduced. Ball (1987b) did not notice a response to varying fertilizer rates for


46

H. angusta 'Holiday', indicating that its fertilizer requirements may be somewhat lower than those of H. psittacorum. CrUey and Lekawatana (unpublished data) found a ratio of 3 N0 3 to 1 NH 4 to be optimal for H. angusta when nitrogen was provided at 200 ppm in a liquid fertilization program. 6. Growth Retardants. Most heliconias grow too tall for pot plant use and growth retardants must be used to control their height. H. stricta 'Dwarf Jamaican' and H. angusta 'Holiday' can be grown without using growth retardants if they are not crowded and light intensities are adequate. If retardants are needed for height control on H. stricta 'Dwarf Jamaican', Lekawatana and Criley (1989) suggest drenching with ancymidol at 2 mg/15-cm pot after inflorescence initiation occurs. Use of growth retardants is essential for pot production of H. psittacorum and H. X 'Golden Torch'. Tjia and Jierwiriyapant (1988) produced attractive plants with drenches of paclobutrazol at 0.25 mg or ancymidol at 0.5 or 1.0 mg/15-cm pot for tissue-cultured H. X 'Golden Torch' (Table 1.9). Higher rates of paclobutrazol and ancymidol, and all tested rates of uniconazole inhibited flowering in this species. Broschat and Donselman (1988) found that paclobutrazol drenches at 0.2 mg/15-cm pot or uniconazole sprays at 105 mg/15-cm pot produced the most attractive pots of H. psittacorum 'Choconiana' without affecting flowering. Higher rates of these materials reduced plant height severely and decreased flowering rate as well. The only rates of ancymidol that effectively reduced plant height in this cultivar also reduced flowering. Table 1.9 shows the response of several heliconia cultivars to growth retardants.

C. Interiorscape Use Heliconias have been successfully maintained under interiorscape conditions. If installed in the bud stage, flowers of H. psittacorum, H. X 'Golden Torch', and H. angusta 'Holiday' will continue to develop and provide 3-4 months of flowering in the interiorscape (Ball 1986b). Ball (1986b) suggests that light levels over 3.8 klx should be adequate for flower development in H. psittacorum or H. X 'Golden Torch' having a minimum of 4 or 5 leaves. Optimum watering and fertilization regimes have not been studied in the interiorscape and undoubtedly vary with the environment. Mites appear to be the only pest of significance in the interiorscape (Ball 1986b).

D. Landscape Culture Heliconias are often included in tropical and subtropical landscapes

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47

Table 1.9. Effects of soil-applied growth retardants (mg a.U15-em-pot) on growth and flowering of H. X 'Golden Torch' (Tjia and Jierwiriyapant 1988), H. stricta 'Dwarf Jamaican' (Lekawatana and Criley 1989), and H. angusta 'Holiday' and H. psittaeorum 'Parakeet' (Lekawatana and Criley, unpublished data). Height is expressed as a percent of control. Flower production is represented as + if flowers were produced or a if no flowers were produced. 'Golden Torch'

'Dwarf Jamaican'

'Holiday'

'Parakeet'

Height Flower Height Flower Height Flower Height Flower Retardant (mg/15-cm pot) (em) production (em) production (em) production (em) production Control Ancymidol 0.5 1.0 2.0 Paclobutrazol 0.25 0.5 1.0 2.0 Flurprimidol 0.5 1.0 2.0 Uniconazole 0.05 0.10 0.25

100

+

100

+

100

+

100

+

77 52 38

+ +

71

+ + +

105 97 84

0 0 0

123

66 53

75

0 0 0

75 57

+ 53 55 31

+ + +

105 89 73

a 0 0

110 75 66

0 0 0

22 34

0 0 0

73 39 39

0 0 0

22 18 18

0

0

0

11

30 18 17

71

0

0

0 0 0

for their bold foliage or showy inflorescences (Watson 1986). Species having a clumping growth habit crable 1.1) are well suited for landscape use since their rate of spread is rather slow. Those species with spreading growth habits (e.g., H. psittacorum, H. latispatha, and H. X nickeriensis) are effective for quickly filling in larger areas, but without a physical barrier to prevent their spread, they will quickly take over large areas of the landscape. Although most species of heliconias can be effectively used as landscape plants, usage of a particular species is limited primarily by temperature requirements and secondarily, by soil requirements (Drysdale 1987; Shimonski 1990). Heliconias have been grown as landscape ornamentals in tropical areas, such as Hawaii, for decades. In 1974, 13 species were listed as being common in Hawaii (Watson and Smith 1974), but now most of the species used for cut-flower production are also utilized as landscape ornamentals. In subtropical climates such as that of southern Florida, occasional

48


frosts and frequent temperatures below 10°C during the winter months restrict the usage of heliconias in landscapes (Shimonski 1990). Minimum temperatures below 5°C appear to kill floral meristems, although not the rhizomes in most species of heliconias. Since most species flower on second-year pseudostems in subtropical areas, flowering in these species occurs only in years when yearly minimum temperatures exceed 5°C (Shimonski 1990). Slightly more cold hardy species that will usually flower following exposure to temperatures down to 1° or 2°C in south Florida include H. collinsiana, H. stricta cultivars, H. hirsuta, H. latispatha, H. rostrata, and H. caribaea (Shimonski 1990). Although rhizomes of most species may survive limited exposure to temperatures down to -2°C, old world species such as H. indica arl; exceptionally cold sensitive and seldom survive unprotected in south Florida or in southern California (Drysdale 1987). Most, if not all heliconias are native to tropical or subtropical climates and appear to be poorly adapted to Mediterranean type climates (Hodel 1985). In coastal southern California where freezing temperatures rarely occur, consistently cool nights prevent most heliconias from thriving (Hodel 1985; Drysdale 1987). H. schiedeana is best adapted to those climatic conditions, but H. collinsiana, H.latispatha, H. meredensis, and H. stricta can also be grown (Hodel 1985; Drysdale 1987). Most heliconias tolerate a wide range of soil types, but species such as H. psittacorum and H. X 'Golden Torch' are prone to Fe and Mn deficiencies on alkaline soils, such as those found in south Florida (Donselman and Broschat 1987). Soils in south Florida are also deficient in magnesium and potassium, and H. angusta and H. stricta 'Sharonii' are particularly susceptible to K deficiency (Broschat 1989). Fertilization practices in south Florida vary, but generally include 2 or more applications per year of a 1N-1P-IK ratio fertilizer with magnesium and micronutrients (Fanning and Allen 1986; Shimonski 1990). Broschat (1989) recommends supplementing these fertilizers with one or two applications per year of controlled-release K fertilizers in south Florida to prevent or correct K deficiencies. Heliconias will grow under a range of light intensities from full sun to deep shade, but optimum color and growth usually occur under very light shade. Plants grown in full sun require additional fertilization to retain their dark green foliage (Fanning and Allen 1986). In soils with good water holding capacity, most heliconias grow well with one or two waterings per week, but H. psittacorum may require daily watering on sandy soils (Fanning and Allen 1986).

1.


49

IV. RESEARCH NEEDS A substantial amount of research has been conducted on cultural aspects of H. psittacorum (Broschat and Donselman 1983a, 1983b, 1987, 1988; Donselman and Broschat 1986,1987), but there has been little work of similar scope on the larger-flowered species. The State of Hawaii through its grower advisory panels and its Industry Analysis program elaborated a set of research priorities for heliconia (Leonhardt 1991). Among the horticultural and physiological aspects in need of research, postharvest needs rank highest. A major limitation to increased use of heliconias as cut flowers is their limited postharvest life. Many last only a week or less after cutting (Table 1.3), and bract drop of H. chartacea is identified as an important problem. Sensitivity to cold limits the value of cold storage as an approach to extending postharvest life or extending the seasons of availability. Methods must be found to achieve better hydration, retard color loss, and otherwise extend the postharvest life. New introductions must be screened for their postharvest characteristics. Costa Rican heliconia grower David Carli (1989) indicates that less than 10% of more than 500 accessions have survived rigorous selection criteria to make it to the market. His qualifications for a commercial flower may be paraphrased as: (1) eye appeal, (2) productivity, (3) robust, healthy growth, (4) long vase life, and (5) suitable size and shape for packing. Last, but not least, is the acceptance of the product by consumers, and this aspect requires study by marketing specialists and education of the consumers by everyone along the production-marketing chain. A related aspect is the need for pre- and postharvest treatments to ensure pest-free shipments. Field-grown heliconias harbor a remarkable variety of insect life in their bracts. These insects are unacceptable both in the marketplace and to plant quarantine inspectors. Advances in safe and effective disinfestation treatments of cut heliconias and other bold tropical flowers have been made (Hansen, et al. 1991, 1992). Growers must also develop field procedures to reduce insect infestation before harvest. A third category of research needs is disease control. Both fungal and bacterial diseases have the potential to reduce productivity and quality. As a minor crop, there are no pesticides directly registered for heliconias, but some can be applied under certain general use labels; their efficacy and effective rates need to be established. Development of new orimproved cultivars is also an important priority to commercial growers. Although commercial growers have grown heliconias from seed collected in the wild or from cultivated plants, there has been no release of cultivars of known parentage from controlled

50


crosses. The large number of cultivars of some species, e.g., H. caribaea, H. bihai, H. stricta, and H. psittacorum (Berry 1988; Hirano 1989; Criley 1990) suggests that there is considerable potential for a breeding program. Many forms and species of heliconias remain in their native habitats, but destruction of the ecosystems will continue to reduce the diversity of germplasm. Although heliconias have not yet been placed on endangered species lists, this possibility remains. Existing germplasm in private and public collections needs to be documented and evaluated. The existence of natural hybrids suggests breeding programs can be developed, but there has been a dearth of genetic studies to support them. Sexual compatibilites need to be determined in order to exploit the potential for intraand interspecific hybrids. Pollen transfer may prove to be easy without the natural pollinators, but even this simple procedure must be refined, including the timing of pollen application, prevention of early floral abscission, pollen storage, and so on. Inheritance of color and marking patterns needs to be determined. The goals of the cut-flower markets must be considered-smaller and lighter weight flowers are desired, with ·bright colors, good longevity, and high productivity. Hybrids between H. psittacorum and H. spathocircinata would appear to be worth pursuing given the success of 'Golden Torch' and similar selections. Besides longer postharvest life, some of the desirable qualities for commercially adapted cut flowers include stronger stems, different bract coloration and color combinations, smaller and larger inflorescences, longer flowering periods, decreased sensitivity to stresses that cause flower abortion, greater cold tolerance, and a capacity to open more bracts after harvest. Selection for basal branching characteristics could lead to greater productivity. Potted types with a greater degree of dwarfness without the use of retardants are another need. Responsiveness to daylength control permits programming for potted types, but selection for day neutral flowering could also have merit. Commercial growers also seek improved winter flowering and extension of the flowering season. Some of these goals may be achieved through breeding, and others by cultural and daylength manipulations. Outside of Hawaii, the seasonal patterns of flowering (Table 1.3) need to be determined. Interestingly, Hawaii growers rank cultural information low in their list of needs, perhaps because heliconias are really very easy to grow. Nonetheless, information on effects of nutrients on growth rate, productivity, quality, pseudostem sturdiness, and disease susceptibility has yet to be developed. Plant water requirements, drought tolerance, irrigation frequency, and the effects of water quality are unknown. Planting density, plant longevity, as well as irrigation and fertilization requirements

1.


51

must be integrated, but insufficient information presently exists to develop a management plan. There are many horticultural problems. Heliconias are sensitive to cold temperatures, light intensity, and biological stresses. Information is still needed on the potential adaptability of species to protected outdoor environments in mild winter climates, such as the southern United States. Factors responsible for failure of inflorescences to develop need to be identified. There are still aspects of propagation that require attention despite ease of vegetative propagation and fast growth. The increase and distribution of new cultivars requires efficient, rapid multiplication techniques. Tissue culture approaches are being explored (Berry 1990), but results are not yet published. Improvements to seed germination are needed because germination is slow and uneven. Heliconias could find greater use as container-grown plants, both in traditional markets and for interiorscapes. Research is required on the whole range of container culture conditions and post-production environments. Screening for suitable heliconias-both species and cultivars, use of growth retardants, nutrition, media, insect control, acclimatization, cropping cycles, induction of flowering are challenges to be faced by horticultural scientists and commercial growers before heliconias can leave the jungles for more domesticated climes. LITERATURE CITED Abalo, J. E. and L. G. Morales. 1982. Veinticinco(25) heliconias nuevas de Colombia. Phytologia 51:1~1. · 1983a. Doce (12) heliconias nuevas del Ecuador. Phytologia 52:387-413. _ _ . 1983b. Diez (10) heliconias nuevas de Colombia. Phytologia 54:411-433. _ _ . 1985. Siete (7) heliconias nuevas de Colombia. Phytologia 57:42-57. Alfieri, S. A.• Jr., K. R. Langdon, C. Wehlburg, and J. W. Kimbrough. 1984. Index of plant diseases in Florida. Fla. Dept. of Agr. and Consumer Affairs, Div. of Plant Industry Bul. 11 (Rev.) Andersson. L. 1981. Revision of Heliconia sect. Heliconia (Musaceae). Nordic J. Bot. 1:759-784. . 1984. The chromosome number of Heliconia (Musaceae). Nord. J. Bot. 4:191-193. · 1985a. Revision of Heliconia subgen. Stenochlamys (Musaceae-Heliconioideae). Opera Bot. 82:1-123. · 1985b. No. 221. Musaceae. pp 1-87 In: G. Harling and B. Sparre, (eds.) Flora of Ecuador. 22. Swedish Res. Council Pub!. House. Stockholm, Sweden. Armbruster, J. 1984. Heliconia psittacorum-eine interessante schnittblume aus der familie de bananengewachse. Gartnerbauliche Versuchbericht. p. 175-178. Ball, D. 1986a. Viruses on heliconias. Bu!. Heliconia Soc. Int. 1(3):7. · 1986b. Hues of heliconia. Interior Landscape Indust. 3(8):25-29. · 1987a. Heliconia update. Nursery Dig. 21(12):36-41. · 1987b. Container culture for Heliconia augusta [sic] cv. Holiday. Bu!. Heliconia

52


Soc. Int. 2(1):2. ___ . 1987c. Heliconia brightens pot plant options. Greenhouse Manager 6(4):60-66. _ _ . 1988. In the trade. Bul. Heliconia Soc. Int. 3(2):2-3. Bar-Zvi, D. "1990. Status reports of 4 of the 6 HSI gennplasm repository gardens: Flamingo Gardens. Bul. Heliconia Soc. Int. 5(1):3. Bell, A. D. and P. B Tomlinson. 1980. Adaptive architecture in rhizomatous plants. Bot. J. Linnean Soc. 80:125-160. Berry, F. 1988. Windward Island Heliconia. Bul. Heliconia Soc. Int. 3(4):4-5, 7 (Abstr). ___ . 1990. Tissue culture of heliconias. Bul. Heliconia Soc. Int. 4(4):11. Berry, F., and W. J. Kress. 1991. Heliconia: an identification guide. Smithsonian Institution Press, Washington. Bisson, S., S. Guillmet, and I.-L. Hamel. 1968. Contribution a l'etude caryo-taxonomique des Scitaminees. Mem. Mus. Mat. d'Hist. (Paris) Nouv. Ser. B. 18:59-145. Bronstein, J. L. 1988. The origin of bract liquid in a neotropical Heliconia species. Biotropica 18:111-114. Broschat, T. K. 1989. Potassium deficiency in south Florida ornamentals. Proc. Fla. State Hort. Soc. 102:106-108. Broschat, T. K., and H. M. Donselman. 1983a. Production and post-harvest culture of Heliconia psittacorurn flowers in south Florida. Proc. Fla. State Hort. Soc. 96:272-273. ___ . 1983b. Heliconias: a promising new cut flower crop. HortScience 18:1-2. ___ . 1987. Tropical cut flower research at the University of Florida's Ft. Lauderdale research and education center. Bul. Heliconia Soc. Int. 2(3-4):5-6. _ _ . 1988. University of Florida research update #1. Bul. Heliconia Soc. Int. 3(4):4. (Abstr.) Broschat, T. K., H. M. Donselman, and A. A. Will. 1984. 'Andromeda' and 'Golden Torch' heliconias. HortScience 19;736-737. Carle, A. W. 1989. Heliconias by seed. Bul. Heliconia Soc. Int. 4(1):6. Carli, D. 1989. The world's largest heliconia fann. Bul. Heliconia Soc. Int. 4(3):9-11. Cheesman, E. E., and L. N. H. Larter. 1935. Genetical and cytological studies of Musa. III. Chromosome numbers in the Musaceae. J. Genet. 30:31-52. Clement, C. Inst. Nac. Pesguisas Amaz., Manaus, Brazil, personal communication. Criley, R. A. 1985. Heliconias. p. 125-129. In: A. H. Halevy (ed). Handbook of Flowering II. CRC Press, Inc., Boca Raton, FL. ___ . 1988. Propagation of tropical cut flowers: Strelitzia, Alpinia, and Heliconia. Acta Hort. 226:509-517. ___ . 1989. Development of Heliconia and Alpinia in Hawaii: Cultivar selection and culture. Acta Hort. 246:247-258. ___ . 1990. Production of heliconia as cut flowers and their potential as new potted plants. Hort. Dig. (Univ. Hawaii) 92:1-7. Criley, R. A. and O. Kawabata. 1986. Evidence for a short-day flowering response in Heliconia stricta 'Dwarf Jamaican'. HortScience 21:506-507. Criley, R. A. and S. Lekawatana. 1990a. Environment influences seasonality in flowering of Heliconia. XXIII Int. Hort. Congr. Abstr. 1:376 (Abstr. 1824). ___ . 1990b. Phenology of flowering in cultivated Heliconia chartacea. HortScience 25:138 (Abstr. 530.) ___ .1991. Managing seasonality in heliconia. Proc. 1990 Hawaii Cut Tropical Flower Conf. Univ. Hawaii, HITAHR Res.-Ext. Ser. 124:167-172. Daniels, G. D., and F. G. Stiles. 1979. The Heliconia taxa of Costa Rica. Keys and descriptions. Brenesia 15 (Suppl.):1-150. Darlington, C. D., and A. P. Wylie. 1955. Chromosome atlas of flowering plants. Allen & Unwin, London.

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53

Dobkin, D. S. 1984. Flowering patterns of long-lived Heliconia inflorescences: implications for visiting and resident nectarivores. Oecologia 64:245-254. ___ . 1985. Heterogeneity of tropical floral microclimates and the response of hummingbird flower mites. Ecology 66:536-543. ___ . 1987. Synchronous flower abscission in plants pollinated by hermit hummingbirds and the evolution of one-day flowers. Biotropica 19:90-93. Dodson, C. H., andA. H. Gentry. 1978. Heliconias (Musaceae) ofthe Rio Palenque science center, Ecuador. Selbyana 2:291-299. Donselman, H. M., and T. K. Broschat. 1986. Production of Heliconia psittacorum for cut flowers in south Florida. BuI. Heliconia Soc. Int. 1(4}:4-6. ___ . 1987. Commercial heliconia production in south Florida. Nurserym. Dig. 21(1):48-52. Drysdale, W. T. 1987. Heliconias in California. BuI. Heliconia Soc. Int. 3(1}:3-4. Fanning, K., and C. Allen. 1986. How to grow heliconias. Fairchild Trop. Gardens BuI. 41(1}:24. Ferreira, S. 1990. Bacterial wilt on heliconia: Hawaii's experience. BuI. Heliconia Soc. Int. 5(1}:9-11. Gatin, C. L. 1908. Recherches anatomiques sur l'embryon et la germination des Cannacees et des Musacees. Annu. Sci. Nat. Bot. 8:113-146. (cited by Kress, 1990b). Geertsen, V.1989. Effect of photoperiod and temperature on the growth and flower produclion of Heliconia psittacorum 'Tay'. Acta Hort. 252:117-122. _ _ . 1990. Influence of photoperiod and temperature on the growth and flowering of Heliconia aurantiaca. HortScience 25:646-648. Green, P. S. 1969. Notes on Melanesian plants. II. Old world heliconia (Musaceae) Kew BuI. 23:471-478. Griggs, R. E. 1903. On some species of Heliconia. BuI. Torrey Bot. Club. 30:641-664. ___ . 1915. Some new species and varieties of Bihai. BuI. Torrey Bot. Club. 42:315-330. Hansen, J. D., H. T. Chan, Jr., A. H. Hara, and V. L. Tenbrink. 1991. Phytotoxic reaction of Hawaiian cut flowers and foliage to hydrogen cyanide fumigation. HortScience 26:53-56. Hansen, J. D., A. H. Hara, and V. L. Tenbrink. 1992. Vapor heat: a potential treatment to disinfest tropical cut flowers and foliage. HortScience 27:139-143. Hansen, J. D., E. Tanoue, and R. Peckenpaugh. 1990. Flower cleaning and handling for export shipment. Bul. Heliconia Soc. Int. 5(1}:6. (Abstr.) Hawaii Agricultural Statistics Service. 1991. Statistics of Hawaiian Agriculture 1990. Hawaii Dept. Agr., Honolulu. Hirano, R. T. 1989..Some observations of Heliconia 'Richmond Red' in relation to H. caribaea and H. bihai. Bul. Heliconia Soc. Int. 4(1}:4-5, 9. Hodel, D. H. 1985. Status and potential of Heliconia in California. BuI. Heliconia Soc. Int. 1(1}8-9 (Abstr.) Holtzmann, O. V., and M. Wong. 1986. Nematodes in tropical cut flowers and their control. Hort. Dig. (Univ. Hawaii) 80:5-6. Inouye, G. 1986. Grower panel on packing and shipping cut flowers: the larger heliconias. Hort. Dig. (Univ. Hawaii) 80:8. (Abstr.) Ka-Ipo, R., W. S. Sakai, S. C. Furutani. and M. Collins. 1989. Effect of postharvest treatment with antitranspirants on the shelf-life of H. psittacorum cv. Parakeet cut flowers. Bul. Heliconia Soc. Int. 4(3}:13-14. Kepler, A. K. 1991. Exotic tropicals of Hawaii. Mutual Publ., Honolulu. Kress, W. J. 1981. New Central American taxa of Heliconia (Heliconiaceae). J. Arnold Arb. 62:243-260. ___ . 1983a. Crossability barriers in neotropical Heliconia. Ann. Bot. 52:131-147.

54


___ . 1983b. Self-incompatibility in Central America Heliconia. Evolution 37:735--744. ___ . 1984. Systematics of Central American heliconia (Heliconiaceae) with pendent inflorescences. J. Arnold Arb. 65:429-535. ___ . 1985. Bat pollination of an old world Heliconia. Biotropica 17:302-308. ___ . 1986. New heliconias (Heliconiaceae) from Panama. Selbyana 9:156-166. _ _ . 1990a. The taxonomy of old world HeliconiB (Heliconiaceae). Allertonia 6:1-58. ___ . 1990b. The phylogeny and classification of the Zingiberales. Ann. Missouri Bot. Gard. 77:698-721. ___ . 199OC. Pollination and potentials in breeding heliconias. Bul. Heliconia Soc. Int. 5(1):1-2 (Abstr.) Kress, W. J., and C. Roesel. 1987. Seed germination trials in H. stricta cv. Dwarf Jamaica. Bul. Heliconia Soc. Int. 2(2):6-7. Kress, W. J., and D. E. Stone. 1982. Nature of the sporoderm in monocotyledons, with special reference to the pollen grains of Canna and Heliconia. Grana 21:129-148. ___ . 1983. Morphology and phylogenetic significance of exine-less pollen of Heliconia (Heliconiaceae). Syst. Bot. 8:149-167. Kress, W. J., D. E. Stone, and S. C. Sellers. 1978. Ultrastructure of exineless pollen: Heliconia (Heliconiaceae). Am. J. Bot. 65:1064-1076. Leonhardt, K. W. 1991. Tropical flower and foliage industry analysis No. 1. College of Tropical Agriculture and Human Resources, Univ. Hawaii, Honolulu. Lekawatana, S. 1986. Growth and flowering of Heliconia stricta Huber. and H. angusta VeIl. MS Thesis, Univ. Hawaii, Honolulu. Lekawatana, S., and R. A. CrBey. 1989. Pot culture of Heliconia stricta'Dwarf Jamaican'. Acta Hort. 252:123-128. Linnaeus, C. 1753. Species plantarum. Vol 2. (Cited by Kress, 1984) _ _ . 1771. Mantissa plantarum. (Cited by Kress, 1984). Mahanty, H. K. 1970. A cytological study of the Zingiberales with special reference to their taxonomy. Cytologia 35:13-49. Manarangi, A., W. S. Sakai, C. Gerkin, M. Crowell, G. Nielsen, and R. Short. 1988. Growth and flowering of Heliconia psittacorum cv. Parrot in Hawaii. J. Hawaiian Pacific Agr. 1(1):1-3. Miquel, F. 1859. Heliconiopsis. Florae Indiae Batavae 3:590. (Cited by Kress, 1990a). Montgomery, R. 1986. Propagation of Heliconia from seed. Bu!. Heliconia Soc. Int. 1(2):6-7. Nakai, T. 1941. Notulae ad Plantas Asiae Orientalis (XVI). Japan. J. Bot. 17:189-203. Natans, J. 1989. Panel on research priorities and educational goals of HSI. Bul. Heliconia Soc. Int. 4(3):2. Ninomiya, D. 1990. Ornamental Crops News. 54(1-52). Market News Branch, Hawaii Dept. Agr. Honolulu. Pingitore, E. J. 1978. Lasespecies cultivadas del genero Heliconia (Musaceae) en la Republica Argentina. Rev. Inst. Municipal Bot. (Argentina) 4:77-93. Plumier, P. C. 1703. Nova plantarum Americanum genera. Joannem Bondot, Paris. (Cited by Kress, 1984). Raabe, R. D., I. L. Conners, and A. P. Martinez. 1981. Checklist of plant diseases in Hawaii. Hawaii Inst. Trop. Agr. &: Human Resources, Univ. Hawaii Info. Text Series 22. Sakai, W. S., A. Manarangi, R. Short, G. Nielson, and M. D. Crowell. 19908. Evidence for long-day flower initiation in Heliconia anqusta cv. Holiday-Relationship between time of shoot emergence and flowering. Bu!. Heliconia Soc. Int. 4(4):1-3. Sakai, W. S., G. Nielsen, S. Short, R. Ka-Ipo, A. Umemoto, and K. Inada. 1990b. Forcing off-season flower production in Heliconia angusta cv. Holiday with artificial long-days. Bu!. Heliconia Soc. Int. 4(4):10-11.

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55

Santos, E. 1978. Revisao das especies do genero HeIiconia L. (Musaceae s. 1.) espontaneas na regiao fluminense. Rodriguesia 30(45):99-221. Seifert, R. P. 1975. Clumps of Heliconia inflorescences as ecological islands. Ecology 56:1416-1422. ___ . 1982. Neotropical Heliconia insect communities. Quart. Rev. BioI. 57(1):1-28. Shimonski, J. 1990. Parrot Jungle and gardens-a special heliconia attraction. Bul. Heliconia Soc. Int. 4(4):4-5. Siddiqui, I. A., S. A. Sher, and A. M. French. 1973. Distribution of plant parasitic nematodes in California. Calif. Div. Plant Ind. Skutch, A. F. 1933. The aquatic flowers of a terrestrial plant, HeIiconia bihai L. Am. J. Bot. 20:535-544. Smith, R. R. 1968. A taxonomic revision of the genus Heliconia in middle America. Unpubl. Ph.D. dissertation, Univ. Florida, Gainesville. _ _ . 1977. HeIiconia in Nicaragua. Phytologia 36:251-261. Stiles, F. G. 1975. Ecology, flowering phenology, and hummingbird pollination of some Costa Rican Heliconia species. Ecology 56:285-301. ___ . 1978. Temporal organization of flowering among hummingbird food plants of a tropical wet forest. Biotropica 10:194-210. ___ . 1979. Notes on some natural history of HeIiconia (Musaceae) in Costa Rica. Brenesia 15 (Suppl):151-180. Stone, D. E., S. C. Sellers, and W. J. Kress. 1979. Ontogeny of exineless pollen in HeIiconia, a banana relative. Ann. Missouri Bot. Gard. 66:701-730. Tjia, B. 1985. Longevity and postharvest studies of various Heliconia psittacorum bracts. Bul. Heliconia Soc. Int. 1(1):6. Tjia, B., and U. Jierwiriyapant. 1988. Growth regulator studies on 'Golden Torch' (Heliconia psittacorum X spathocircinata). Bul. Heliconia Soc. Int. 3(3):1, 6. Tjia, B., and T. J. Sheehan. 1984. Preserving beauty and profits. Longevity, quality studies help prolong life of Heliconia. Greenhouse Manager 2(11):94-100. Tomlinson, P. B. 1969. Classification of the Zingiberales (Scitaminae) with special reference anatomical evidence. p. 295-302. In: C. R. Metcalfe (ed.) Anatomy of the Monocotyledons. Vol. 3. Clarendon Press, Oxford. Tsugawa, K. 1988. Ornamental Crops Market News. 52(1-52). Market News Branch, Hawaii Dept. Agr., Honolulu. Uchida, J. Y., M. Aragaki, and P. S. Yahata. 1989. Heliconia root rot and foliar blight caused by Cyc1indroc1adium. Hawaii Inst. Trap. Agr. Hum. Res. Brief 085. Van Raalte, D., andD. Van Raalte-Wichers. 1973. Heliconia. VakbladBloem. 28(23):12-13. VBA. 1989. Statistisch overzicht snijbloemen 1989. Verenigde Bloemen-veilingen Aalsmeer, Aalsmeer, Holland. Venkatasubban, K. R. 1946. A preliminary survey of chromosome numbers in Scitaminae of Bentham and Hooker. Proc. Indian Acad. Sci. Ser. B. 23:281-300. Watson, D. P., and R. R. Smith. 1974. Ornamental heliconias. Univ. Hawaii Coop. Ext Servo Cir. 482. Watson, J. B. 1986. Heliconias: a new challenge for landscape design. Fairchild Trap. Gardens Bul. 41(1):6-19. Wolf, L. L., and F. G. Stiles. 1989. Adaptations for the 'fail-safe' pollination of specialized ornithophilus flowers. Am. MidI. Nat. 121:1-10. Woodson, R. E., Jr., and R. W. Schery. 1945. Flora of Panama. Musaceae. Ann. Missouri Bot. Gard. 32:48-57. Woolliams, K. R. 1985. Endangeredheliconia: How serious a problem? Notes Waimea Arb. Bot. Gardn. 12(1):11-12. Wootton, J. T., and I-F. Sun. 1990. Bract liquid as a herbivore defense mechanism for Heliconia wagneriana inflorescences. Biotropica 22:155-159.

2 Root Physiology of Ornamental Flowering Bulbs Ludwika Kawa * Research Institute of Pomology and Floriculture, 96-100 Skierniewice, Poland A. A. De Hertogh * * Department of Horticultural Science, North Carolina State University, Raleigh, North Carolina 27695-7609 1.

II.

III.

IV. V.

VI.

Introduction Root Origins A. Natural Root Development from Seeds B. Natural Root Development from Storage Organs C. Artificially Induced Development Root Morphology A. Branching Habit B. Contractile Habit C. Root Hairs D. Tuberization Habit Endogenous Factors Exogenous Factors A. Temperature B. Moisture C. Soils and Artificial Planting Media D. Mycorrhiza E. Light F. Plant Growth Regulators G. Diseases H. Other Factors Conclusions Literature Cited

I. INTRODUCTION Traditionally, ornamental flowering bulbs have been classified as true bulbs, corms, tubers, tuberous roots, and rhizomes (Hartmann et a1. *We thank Ms. A. Lutman and Mrs.. G. Pemberton for their assistance in preparing the manuscript and Dr. Marcel Le Nard for reviewing the manuscript. **1 acknowledge and appreciate the support of the Dutch Bulb Exporter's Association, Hillegom, The Netherlands, who provided funding for flower-bulb research under my direction at Michigan State University and at North Carolina State University.

57

58

L. KAWA AND A. DE HERTOGH

1990). However, in 1934, Raunkiaer classified all land plants whose surviving bud or shoot apices are borne on subterranean organs as geophytes. The plants were categorized into five groups: (1) bulb geophytes, (2) stem-tuber geophytes, (3) root-tuber geophytes, (4) rhizome geophytes, and (5) root geophytes. Recently, Rees (1989J and Halevy (1990) have applied the term geophyte to all types of flower bulbs. Botanically, all geophytes are perennials and they may persist either by annual replacement (e.g., Tulipa and Gladiolus), or by perennial tissues (e.g., Muscari and NarcissusJ. In this review, we also utilize the term geophyte as synonymous with flowering bulbs. We feel that this term is physiologically more useful than denoting whether the survival organ is called a bulb, corm, tuber, rhizome, or root. The important aspect is whether the storage tissue is an enlarged leaf, stem, or root. When the growth and developmental cycle of flowering bulbs has been the subject of a review, the major emphasis has been either on flower development (Hartsema 1961J or on bulb yield (Rees 1972J. The establishment, maintenance, morphology, and anatomyofbulb roots has not been intensively studied. There have been, however, some isolated reports. In this review, we focus on four major aspects of root physiology. First, what is the precise origin of the root(s)? It can be a primary root from the seed or they can be adventitious roots from either perennial bulbs, cuttings, or from tissue culture propagules. Second, we review the general morphological characteristics of many taxa (Table 2.1). In this regard, the ornamental geophytes have unique features. Some do not branch, others have no root hairs, and many have contractile roots (eR), for example, after elongation the root reduces in length and this causes distinct wrinkling of the root surface and anatomical changes in the cortical cells (Fig. 2.1J. Third, we review the endogenous factors regulating root initiation and growth. Fourth, the effects of exogenous factors

Figure 2.1. Anatomy of root contraction in Hyacinth us. (A) Macroscopic view of contractile roots showing the conspicuous wrinkling of roots near the bulb which is indicative of the contractile zone. X %; bar = 1 em. (B) Median longitudinal section of noncontracted region of root showing large vacuolate cortical parenchyma cells. x 33; bar = 300 fJ.m. (C) Median longitudinal section of contracted region of root showing radially expanded inner cortical cells and folded collapsed outer cortex. X 33; bar = 300 fJ.m (from Cyr et al. 1988).

2.

ROOT PHYSIOLOGY OF ORNAMENTAL FLOWERING BULBS

Table 2.1.

59

General root characteristics of ornamental flowering bulbs.

Taxa

Allium schubertii Alstroemeria X hybrida Amaryllis belladonna Anemone blanda Anemone coronaria Anigozanthos spp. Begonia tuberosa Caladium bicolor Convallaria majalis Crocus flavus Crocus sativus Crocus vernus Dahlia x hybrida Eucomis spp. Freesia X hybrida Fritillaria meleagris Gladiolus X hybrida Hippeastrum X hybridum Hyacinthus orientalis Incarvillea delavayi Ipheion uniflorum Iris hollandica Iris reticulata Ixia X hybrida Leucojum aestivum Lilium longiflorum Lycoris spp. Muscari armeniacum Narcissus pseudonarcissus Nerine bowdenii Nerine sarniensis Oxalis braziliensis Polianthes tuberosa Ranunculus asiaticus Scilla tubergeniana Thlipa Y Zantedeschia X hybrida

Cultivar

Circum- Avg. no. ference basal Characteristics z bulb size roots/ (em) bulb BR. CON. R.H. TUB. No Yes

Yes

No

?

?

Yes Yes Yes Yes

? ?

Yes ? ? ? ?

Yes No ?

Remembrance

10/11

Seedling

22/23

Sun Dance Ostara

28/30 17/18

204 85

31 111

No Yes Yes Yes No Yes Yes No ? ?

Wedgwood Harmony

10/11 6/7

35 26

Yes Yes Yes ?

Early Giant

10/11

53

Explorer

24/26

190

Yes Yes No No Yes Yes Yes ?

Paul Richter Majestic Red

12/13 6.5/8

215 51

Yes No No Yes

?

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes

Yes ? ? ? ?

Yes No ?

No Yes Yes Yes Yes Yes Yes Yes

No Yes ?

No No ?

No ?

No No No No Yes No No ?

No No No

? ?

? ?

Yes Yes

No No No

? ?

?

Yes Yes No

No No No

Yes Yes No

No No No

? ?

? ?

Yes Yes No Yes

Yes No No

zBR = Branched, CON = Contractile, R.H. = Root Hairs, TUB. = Tuberized, ? = Information Not Reported. With the exception of the contractile habit that was extensively studied by Rimbach (1929), most of the characteristics reported are observations by the authors. Other reported studies are cited in the text. Y Only primary roots of T. ingens, T. maximowiczii, T. wilsoniana, and T. dubia have been reported to have root hairs (Botschantzeva 1981).

60


are summarized. It is in this latter area that most of the studies are conducted. We conclude this review by identifying several areas of needed research. Additional research on roots is critical beeause a viable root system is essential for optimal plant growth and development (Whittington 1969). Roots absorb not only water and nutrients but also pesticides and plant growth regulators.

II. ROOT ORIGINS A. Natural Root Development from Seeds Although many floral and vegetable crops are primarily propagated by seeds, only a few ornamental geophytes are propagated in this manner. Examples are: some Allium spp., some Anemone blanda selections, Anemone coronaria, some Dahlia cultivars, some Lilium spp., Ranunculus asiaticus, and some Zantedeschia spp. Consequently, there are few detailed investigations on seedling growth of these genera. The primary roots of flower bulb taxa can be divided into two classes: annual and perennial. With most bulbs, such as Hyacinthus, Narcissus, and Tulipa, the seedling roots exist for one growing season. All subsequent roots are adventitious in nature. In Hippeastrum and Bulbinella, both with branched root systems, the primary roots can be preserved under optimal environmental conditions for a second season of growth. This is beneficial for rapid early growth in the spring (F. Barnhoorn and A. Cohen, personal communications). 1. Dahlia. Root systems of seed dahlias are composed of a primary root and several laterals (Havis 1936; Aoba et al. 1960, 1961). The xylem of the

primary root was tetrarch. The pith, composed of five to ten cells, extended only 2-4 mm into the primary root. No pith was observed in the laterals, which originated from the pericycle that was composed of 1-2 layers of parenchymatous cells. The first adventitious root appeared in the basal part of the cotyledon which, along with the lower nodes of the stem, formed the crown of the plant. The adventitious roots enlarged to form the storage roots. The thickening occurred by increased cell divisions and elongation and was enhanced by 8 photoperiods (Aoba et al. 1960, 1961; Moser and Hess 1968). 2. Gladiolus. The primary root of Gladiolus seedlings formed many

secondary roots after germination and those secondary roots subsequently contracted (Griesbach 1972). This phenomenon in Gladiolus is discussed in detail in Section III. B.

2.


61

3. Narcissus. The cycle from the seed to flowering normally takes 4-6 years (Chan 1952). Germination occurred after the seed was stored for at least 5-6 months. The primary root gradually died and was replaced by two adventitious roots, which originated within the procambial zone at the base of the cotyledon. After the first season's growth, the two adventitious roots also died and were sloughed off. New adventitious roots were formed subsequently on the basal plate of the 1-year-old bulb. This cycle of the formation of new adventitious roots followed by senescence is repeated yearly with Narcissus. 4. ThJipa. The seedling stage of tulip is covered in detail by Botschantzeva (1982). After germination, the radicle was the only root produced and it senesced after the first year of growth. Tulip roots do not branch or contract and most seedlings have no root hairs (Table 2.1). Root hairs were observed only on T. ingens, T. maximowiczii, T. wilsoniana and T. dubia and only during the year of seedling growth (Botschantzeva 1982). Like Narcissus, Tulipa bulbs annually produce adventitious roots from the basal plate.

B. Natural Root Development from Storage Organs Almost all flowering bulbs developed natural asexual reproduction systems (Hartmann et al. 1990). Two of the most common are: (1) annual replacement (e.g. Crocus, Gladiolus, Tulipa) , and (2) offsets (e.g. Muscari, Hyacinthus, and Narcissus). Under outdoor conditions, ornamental bulbs are normally described as either spring flowering or summer/fall flowering (Bryan 1989). These broad groups are based primarily on the hardiness level of the bulbs, which can be quite variable (Sakai and Yoshie 1984) and the time at which the bulbs are planted. There can, however, be intermediate flowering types depending on the prevailing climatic conditions and/or the cultivars or species used. Detailed studies on root development during the annual growth and developmental cycle are limited. For most bulb species, even if they are perennial types, the roots only survive for one growing season. However, for perennial bulbs, such as Convallaria, Dahlia, Hippeastrum, Lilium, and Ranunculus, the roots persist for more than one season provided they are not allowed to dessicate or become diseased. Du Plessis and Duncan (1989) state that the adventitious roots of most Amaryllidaceae species of South Africa have persistent roots, while Hyacinthaceae species roots are seasonal. Theydo not, however, provide data to support this statement. 1. Hyacinthus. Versluys (1927) published an extensive study on H.

62

L. KAWA AND A. DEHERTOGH

orientalis 'Queen of the Blues'. The bulbs were planted in the autumn in water-tight cisterns with sand in which the water level was controlled. She observed that all preformed roots emerged from the basal plate within 6 weeks after planting. Root growth ceased during the winter months, when temperatures were 3.4-4. 7°C. Some roots resumed growth in the spring. All roots of the growing season subsequently senesced and new root primordia were initiated in the persistent basal plate. They started in mid-May and continued through mid-July. The number of roots that emerged after planting was influenced by postharvest storage temperatures. Versluys (1927) found that four weeks of storage at 17°C after 8 weeks at 25.5°C promoted earlier root growth than 12 continuous weeks at 25.5°C.

2. Narcissus. In the second year of growth, the bulb formed four to seven adventitious roots in the basal plate, which varied in root diameter from 0.5-1.0 mm (Chan 1952). The vascular system was either triarch, tetrarch, or pentarch. Endotrophic mycorrhiza were associated with the roots during the second season. Over the subsequent years, the basal plate enlarged until the bulbs reached their maximum circumference size. Each year new roots were formed and some were contractile (Table 2.1). Those roots were shorter and thicker than the noncontractile roots. Narcissus roots do not branch, but they do have root hairs (Chilvers and Daft 1980, 1981). 3. Thlipa. The root system of the tulip bulb has been investigated by Shaub and De Hertogh (1975), who used 'Paul Richter', and by Wilson and Peterson (1982) and Botschantzeva (1982)who used the botanical species, T. kaufmanniana. Root primordia were initiated at the end of June (Fig. 2.2a) and were formed adjacent to the vascular bundles. Growth in the basal plate of the tulip continued through the summer (Fig. 2.2b). By late September or early October the root initials were within 1-2 cells of the epidermis of the basal plate (Fig. 2.2c). Blaauw and Versluys (1925) showed that the development of the root primordia in the bulb was affected by postharvest temperatures, with continuous 13°C until planting being optimal. Tulipa roots emerged and elongated after autumn planting and continued to grow even when the winter soil temperatures reached 3°C (Wilson and Peterson 1982). Roots could extend to lengths of 1 m provided the planting medium was not compacted and the temperatures were above O°C (L. H. Aung and A. A. De Hertogh, unpublished). The root system senesces in late spring as the daughter bulbs mature. 4. Lilium. The adventitious roots grow from the basal plate and they are

2.


63

Figure 2.2. Median longitudinal sections of root tips in the basal plate of a mature mother Tulipa bulb cv. Paul Richter. (A) Root tip (RT) forming beneath vascularbundle (VB)ofbasal plate on August 1. X 156. (B) Developing root tip on August 25. Central cylinder (CC) and root cap (RC) are visible. X 156. (C) Developing root tip on September 12. Note the two remaining cell layers of basal plate (pI) between root cap (RC) and exterior of bulb. X 87 (from Shaub and De Hertogh 1975).

usually initiated 12-18 months before emergence (Feldmaier 1970). New roots were formed during late spring and some initiation took place until autumn. The basal roots of Lilium are contractile (Table 2.1). Most Lilium spp. also produce stem-roots, which grow from the internodes that exist between the bulb and soil surface. These die generally during autumn and are initiated during the following spring. C. Artificially Induced Development With some species, the natural reproductive rate is too low for commercial purposes. Thus, in order to have enhanced multiplication rates and/or to produce disease-free plants, artificial reproductive systems have been developed for some genera (Tables 2.2 and 2.3). In each


64

Table 2.2. Examples of whole tissue organs used for commercial asexual reproduction of flower bulbs. Type of

Taxa

Tissue Used

Conditions Promoting Root Development

Anemone blanda Named cvs. Dahlia cvs.

Tuber cuttings

No information provided.

Stem cuttings

Hippeastrum cvs.

Twin scales

Hyacinth us orientalis

Scooping or scoring

Ulium spp.

Scales

Narcissus cvs.

Twin scales

Nerine spp.

Twin scales

Use of an auxin and only vegetative, herbaceous cuttings should be used. Storage of twin-scales in moist sand or vermiculite at 25-28°C for 3 months. After destroying the apical bud, bulbs are held at 21-30°C until planted. Scales are held in moist vermiculite at 23°C. Twin scales are held in moist vermiculite at 20°C for 12 weeks. Twin scales are held in moist vermiculite at 22-23°C for 8-12 weeks.

instance, the induction and subsequent growth of adventitious roots must be incorporated into the asexual reproductive system, since the bulbs have to be planted either in the greenhouse or field. 1. Whole Tissue Organs. Genera that are commercially propagated by whole tissue organs are summarized in Table 2.2. To maintain their horticultural characteristics, selections of Anemone blanda, e.g. 'Radar' and 'White Splendour', are reproduced by cutting the tubers (Langeslag 1988). Other A. blanda selections are produced from seed, and these have a range of color variability. Dahlia cultivars are propagated by taking stem cuttings from selected mother plants (Langeslag 1988). Normally, 7.5- to IS-em cuttings are taken from the crowns of greenhouse-grown plants in late winter or early spring and dipped in a rooting hormone. Biran and Halevy (1973a) demonstrated that reduced light conditions will promote rooting of cultivars that are easy to root, but not ones that are difficult to root. Also, cuttings rooted best when they were vegetative (Biran and Halevy 1973b). Hippeastrum cultivars are propagated either by offsets, which is the natural system, or artificially by twin-scaling (Vijverberg 1981). Twinscaling is one of the multiplication systems used for many bulbs. They are used for species that do not produce many offsets. Large (>28 cm) bulbs

2.


65

Table 2.3. Examples of tissue culture conditions promoting root formation of ornamental flowering bulbs. Taxa Alstroemeria 'Zebra' Canna indica

Conditions promoting root development

References

MS Zwith 1.0 mgll NAA or 8-16 mgll IBA

Gabryszewska &: Hempel (1985)

Liquid lIz strength MS with

Kromer &: Kukulczanka (1985)

0.1 mgll BA and 0.5 mgll

Crocus chrysanthus

Freesia Gladiolus

Hemerocallis Hippeastrum hybridum Hyacinthus orientalis Iris germanica Iris hollandica Lilium speciosum Muscari armeniacum

Narcissus

Zephyranthes robusta

IBA MS hormone-free with 3% sucrose and 3000 lux for 12 hours MS with low concn. NAA MS with 0.57-5.7 pM IAA MS with 10 mgll NAA lIz strength MS with 0.5 mgll NAA lIz strength MS MS with 5-10 mgll MAA MS with 0.2 mgll BA and 1 mgll NAA MS and light MS and light MS with 0.5-5 J!M NAA MS with high auxin:cytokinin ratio, in dark lIz strength MS with sucrose and no PGR MS with 1.1 pM BA and 0.8 pMIBA modified Knudson's macronutrient's and Heller's micronutrient's with 1.0 gil activated charcoal and 1.0 mgll NAA MS with 4.9 J!M IBA

Fakhrai &: Evans (1989)

Bertaccini et a1. (1989) Bajaj (1990) Ziv et al. (1970) Bertaccini &: Marani (1986) Krikorian et a1. (1990) Mii et al. (1974) Saniewski et al. (1974) Meyer et al. (1975). Hussey (1976) Van Aartrijk et al. (1990) Cumming & Peck (1984)

Seabrook et a1. (1976) Hussey (1982) Seabrook (1990)

Furmanowa &: Oledzka (1990)

ZMS = Murashige-Skoog (1962) medium.

are cut into four segments and then each segment is recut, producing 6080 twin scales. The segments are called twin scales and consist of two scale fragments connected with a piece of basal plate. To develop adventitious roots the twin scales are initially dipped in fungicides and then placed in either moist sand or vermiculite at 25-28°C. The media

66

L. KAWA AND A. DE HERTO GH

must not be allowe d to dry out. The new bulblet will be formed on the basal plate of the twin scale after 3 months . It will develo p roots in approx imatel y 6 weeks after plantin g. Hyacin thus cultiva rs are normal ly propag ated by either scooping out the basal plate or by crosscu tting (Bulb and Corm Produc tion 1984). After scooping, the mothe r bulbs are held at 21-30°C and 85% relativ e humidity. The roots form on the developing bulblets, prior to plantin g of the mother bulbs in Novem ber. Bulblets formed by crosscu tting of the bulbs do not form roots until the following season . Many Lilium species an cultiva rs are propag ated by scales in the fall (Bulb and Corm Produc tion 1984). Health y mother bulbs are lifted, scaled, dipped in fungicides, and placed at 23°C in polyeth ylene bags with moist vermic ulite for 6 weeks. They are subseq uently placed at 17°C for 4 weeks. The roots develo p after the bulblets are planted in either the field or greenh ouse. Narcissus can be propag ated by twin scales; this techniq ue was review ed by Hanks and Rees (1979). The basic proced ures are as follows : (1) Large-sized mother bulbs are selecte d and cleane d. They are sectioned in late summe r (August), and the twin scales must be connec ted by a piece of the basal plate; (2) After cleanin g and cutting, the twin scales are kept in damp tissue paper to preven t them from drying out; (3) The twin scales are dipped in fungicides; (4) The twin scales are placed in O2 and CO2 permea ble polyeth ylene bags with moist vermiculite; (5) The bags are placed for 12 weeks at 20°C. A new bulblet forms on the basal plate of the twin scales. Root develo pment takes place after plantin g of the twin scales at lower temper atures. If the bags with the twin scales are held at temper atures

E

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Figure 3.4. Initiation of primary stolons (open circles), tuber incipients (filled circles), tubers with a diameter >1 cm at final harvest (x), and large tubers with a fresh weight >60 g at final harvest (+). Plants were from the short-day control treatment of a photoperiod experiment (Struik et a1. 1966b) and were grown using the apparatus diagrammed in Figure 3.3 (from Fig. 2a of Struik et al. 1988b).

3.

TUBER FORMATION IN POTATO

99

Figure 3.5. Apparatus used to measure volume of attached tubers. A tuber is enclosed in a plastic ball gauge that consists of two hinged hemispheres. A block of plastic (distorted in the photograph because of refraction of light through the plastic) is attached to the left-hand hemisphere for support. The two hemispheres are clamped together onto an O-ring of silicone rubber to prevent leakage. The stolon of the attached tuber passes through an opening that is padded with waterproof neoprene foam. Tuber volume is calculated from the difference between the weight of water required to fill the empty gauge and the weight required in the presence of the tuber. Measurements are made with automated equipment under standardized conditions and require about two minutes each (Struik et a1. 1988a).

Subjective scales somewhat analogous to those developed for scoring floral stages in Xanthium (Salisbury 1955) have been devised (Fig. 3.8) to permit rating the responses of the buried bud in terms of how strongly the cuttings were induced to tuberize (Ewing 1985; Wheeler et al. 1988; Lorenzen and Ewing 1990). Although subjective, the ratings are well correlated with objective measurements, including percentage of cuttings that tuberize (McGradyet al. 1986; Furumoto et al. 1991), patatin (see Part VII.B) accumulation (Wheeler et al. 1988), and earliness of tuberization (Furumoto et al. 1991). Thus cuttings can be used to test how environmental conditions, genetic differences, the application of growth substances, and other variables affect the degree to which.leaves have been induced to cause stolon or tuber formation. For more information on cuttings see the review by Ewing (1985) and subsequent references (e.g., McGrady et al.

E. E. EWING AND P. C. STRUIK

100

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3 4 5 6 Days after transfer to 2% sue"

7

Figure 3.18. Patatin-promoter activated p-glucuronidase (GUS) in axillary buds and leaves of potato shoot sections cultured in vitro. Potato shoot sections were maintained on a medium containing 8% sucrose under an 8-h photoperiod (SO) for one or three days to induce tuberization. They were then transferred to a medium containing 2% sucrose and exposed to either SO or 16-h photoperiods (LO). The GUS activity was determined in buds (A) and leaves (B) (from Perl et a1. 1991).

activated in both leaves and buds, the response time was more rapid in leaves; yet 2the requirements for activation were more demanding in leaves-both 8% sucrose and subsequent exposure to 16-h photoperiods were required in leaves. 4. Role of patatin. One role of patatin is presumed to be its function as a

158


storage protein and supplier. of N during sprouting and early plant development, but it may play other roles (Prat et al. 1990). The fact that it possesses esterase activity suggests that it may be similar to lipid acyl hydrolase, an enzyme that is able to hydrolyze membrane lipids (Racusen 1984). This function is important during sprouting, because it can stimulate the availability of metabolites by hydrolysis of membranes in old cells. The fatty acids that become available could be used as an energy source; and inasmuch as some fatty acids are known to be potent elicitors of phytoalexin synthesis, it may be that patatin also has a function in resisting pest attacks (Prat et al. 1990). The pH inside the vacuole is considered similar to the isoelectric point of patatin, which would minimize its solubility and activity as an esterase (Willmitzer et al. 1990); but wounding or invading by pests might release patatin so that it can become active in the wound response (Hofgen and Willmitzer 1990). 5. Other proteins. Several other groups of storage proteins are associated with tuberization. Next to patatin, the one most studied is proteinase inhibitor II (see review by Sanchez-Serrano et al. 1990). Proteinase inhibitor II, the monomer of which has a molecular weight of 12 kD, is expressed developmentally in flowers and tubers of healthy, intact plants and can be induced to accumulate in the foliage by wounding. The response to wounding is systemic and moves both acropetally and basipetally. There is evidence that abscisic acid mediates the systemic induction of the gene after wounding and abscisic acid applications can substitute for wounding (Pefia-Cortes et al. 1989). A single promoter drives the constitutive expression of the gene in tubers and the wound-inducible expression in the leaves and stems (Keil et al. 1989). Other proteinase inhibitors that serve as storage proteins have been identified. They consist of at least two multi-gene families and have molecular weights of approximately 22 kD (Mares et al. 1989; Suh et al. 1990). Expression of both types of inhibitor is developmentally regulated in tubers and is wound inducible in leaves (Suh et al. 1991). Accumulation of 22-kD proteins was detected in tubers of leaf-bud cuttings several days after patatin appears (Suh et al. 1991). Like patatin, the 22-kD proteins did not accumulate in the S. brevidens or S. etuberosum petiole bases (HannapeI1990). However, unlike patatin-forwhich gene expression in the bases of petioles was unrelated to dose of tuberizing genomes-expression of one of the 22-kD proteins increased from triploid to pentaploid to S. tuberosum parent, associated with an increased dosage of tuberizing genomes (Hannapel 1990). 6. Significance of storage proteins. The close associations between the

morphological changes that result in tuberization and the expression of

3.


159

genes for storage proteins are striking, but they do not necessarily imply a causal relationship. Deposition of starch and storage proteins is required if tubers are to be useful as storage organs. It might be expected that during the evolution of tuberization the potato plant will develop strategies for a common regulation of the required morphological changes, starch deposition, and storage protein synthesis. This has led to a search for proteins that appear even earlier during tuberization than patatin, which are present in lower concentrations than the storage proteins and might be more directly involved with the changes in cell division and cell enlargement requisite for tuber initiation (Taylor et al. 1991; Hannapel1991b). 7. Enzymatic changes. The predominant form of translocated carbon in

most plants, including potato, is sucrose (see review by ap Rees and Morrell 1990). It has been proposed (Sung et al. 1989) that the ability of an organ to metabolize sucrose is one determinant of sink strength. Compared to mature tubers, growing tubers had a high rate of sucrose hydrolysis from the activity of sucrose synthase (Sung et al. 1989; also see ap Rees and Morrell 1990). There was little relation between size of growing tubers and activity of the enzyme (Sung et al. 1989), but it should be remembered that it is impossible to predict which tubers will stop growing when they reach a certain size and which will continue to grow. Exposure of plants to high temperatures, which may be presumed to have lowered their induction to tuberize (Part III.B), lowered the sucrose hydrolyzing activity of sucrose synthase in tubers (S. A. Wolf and A. Marani, unpublished). Moreover, 'Up-to-Date,' known to be very sensitive to high temperature in terms of effects on tuberization, showed a larger differential response to temperature in sucrose synthase activity than did 'Norchip,' known to be much less sensitive (S. A. Wolf and A. Marani, unpublished). Other evidence of sucrose synthase importance comes from S. demissum (Helder et al. 1991a). The enzyme activity of stolons that contained 7% dry matter was compared to enzyme activity in tubers from the same plant that had a fresh weight of 0.1 g and a 10% dry matter content. There was more than a tenfold increase in sucrose synthase activity in these very small tubers. Exposure of plants to short days caused a decrease in glucose content of stolon tips, but a much greater decrease in fructose content. The rapid decrease in fructose levels might be accounted for by a shift from the vacuole to the cytosol as the main site of sucrose hydrolysis. If acid invertase was acting mainly in the vacuole and sucrose synthase was acting mainly in the cytosol, then phosphorylation of fructose was expected to be more rapid in the cytosol than in the vacuole (Helder et al. 1991a). An increase in the ratio of glucose/fructose

160


concentrations was also associated with tuberization in S. tuherosum: the ratio was less than 2 for stolon tips with a fresh weight of 14 mg. was doubled when fresh weights had doubled. and was >30 when tubers weighing 550 mg were present (Davies 1984b). Aside from its usefulness in increasing sink strength by hydrolyzing sucrose, one product of sucrose hydrolysis by sucrose synthase is UDPglucose (ap Rees and Morrell 1990). UDPglucose is converted to glucose-i-phosphate by the action of UDPglucose pyrophosphorylase (ap Rees and Morrell 1990); and the glucose-i-phosphate plus ATP yields ADPglucose through the action of ADPglucose pyrophosphorylase (ap Rees and Morrell 1990). ADPglucose appears to be the dominant substrate for starch synthase in starch synthesis (ap Rees and Morrell 1990; Anderson et a1. 1990). Another enzyme capable of synthesizing starch is starch phosphorylase. According to the prevailing view this enzyme is associated with starch breakdown in the potato tuber rather than with starch synthesis (Mares et a1. 1985; Davies 1990). although in several studies the activity of starch phosphorylase increased at earlier stages of tuberization than did the activity of ADPglucose pyrophosphorylase (MingoCastel et a1. 1976b; Hawker et a1. 1979; Obata-Sasamoto and Suzuki 1979). Coincident with tuberization of stolons cultured in vitro and the change from small to large starch granules (Part VII .A) was a decrease in soluble phosphorylase activity (Iriuda et a1. 1983). This decrease in the activity of the soluble enzyme did not occur when tuberization was prevented with gibberellic acid. In developing tubers starch phosphorylase was located only in the stroma of amyloplasts. whereas in mature tubers it was present in the cytoplasm in the immediate vicinity of the plastids (Brisson et a1. 1989). The location in the amyloplast stroma would facilitate a synthetic role using glucose i-phosphate as substrate for starch synthesis, or a catabolic role on the growing starch grain to provide glucan primers for starch synthase (Brisson et a1. 1989). Starch synthase (Obata-Sasamoto and Suzuki 1979; Hawker et a1. 1979) and UDPglucose pyrophosphorylase (Sowokinos 1976; Hawker et a1. 1979) also showed increased activities early in the growth of the tuber. Although the increased activity of ADPglucose pyrophosphorylase lagged behind the increases in swelling and starch deposition at the earliest stage of tuberization (Hawker et a1. 1979; Obata-Sasamoto and Suzuki 1979), there is a growing consensus (Mares et a1. 1985; Anderson et a1. 1990; Prat et a1. 1990) that its activity is strategic for the regulation of starch synthesis in the developing tuber. This enzyme is located exclusively in the amyloplasts of developing potato tuber cells (Kim et a1. 1989). A marked lowering in activity of ADPglucose pyrophosphorylase accompanied the cessation of growth of tuber initials (Mares et a1. 1981).

3.


161

Gibberellic acid had an inhibitory effect on its activity, which might help to explain why high temperatures (which in turn lead to high levels of gibberellin activity in buds-see Part III.B) caused decreases in starch production (Marschner et a1. 1984). According to Willmitzer (1991, as reported by Dilworth 1991) transgenic potato plants transformed with the antisense gene for ADPglucose pyrophosphorylase showed reduced rates of starch synthesis. There is evidence for other effects of high temperature on tuber starch synthesis that are apparently distinct from the effects of heat-produced gibberellins on ADPglucose pyrophosphorlyase activity just mentioned (Mohabir and John 1988). Thin slices of tissue from growing potato tubers were incubated in 14C-sucrose solution, and incorporation into starch was measured over time. Uptake, which was linear after one hour, displayed a strong response to temperature of the incubation medium. Arrhenius plots showed an optimum temperature of 21.5°C for starch synthesis in these potato discs, whereas discs from developing cocoyam (Colocasia esculenta L. Schott) corms gave increased incorporation as the temperature was raised to 35°C. Temperature optima for ADPglucose pyrophosphorylase and starch synthase from potato tubers were greater than 35°C (Frydman and Cardini 1966; Kennedy and Isherwood 1975), indicating that direct effects on these enzymes were not responsible for the observed temperature response. Furthermore, amyloplasts isolated from protoplasts prepared from developing tubers did not show the temperature optimum of 21.5°C (Mohabir and John 1988). Why potato tuber slices should show this optimum temperature for starch synthesis is not known. More complete information on enzymes involved in carbohydrate metabolism of the developing potato tuber and potential control sites for starch synthesis can be found in reviews by Mares et a1. (1985), ap Rees and Morrell (1990), and Anderson et a1. (1990). Ap Rees and Morrell (1990) present an excellent discussion of the way in which these processes are regulated, and the possible contributions of genetic engineering toward giving us a better understanding of the control points. C. Relation Between Changes in Shoot and Changes in Stolon Tip How are the physiological changes in the shoots associated with induction to tuberize (Part VI.) translated into the anatomical and biochemical changes associated with tuberization of the stolon tip or buried bud? There are far too many gaps in our knowledge to put all the pieces together, but enough information is emerging so that we can begin to speculate as to the general outline.

162


The induced condition is brought about by and leads to a series of changes regulated at the molecular level: some genes are switched on, presumably others are switched off; and very likely there are changes at the level of translation as well as transcription, with consequent changes in de novo synthesis and activity of enzymes. Whatever the chain of events, there are decreases in activity of gibberellins and increases in the activities of one or more other hormones. (Suggested candidates include tuberonic acid or related compounds, cytokinins, and abscisic acid). The changed hormonal balance has profound effects, directly or indirectly, on the morphology and physiology of the entire plant. In particular, the leaves become thinner and more efficient in photosynthesis per unit leaf dry weight. On a dry weight basis this permits the leaf to accumulate more starch during the day and to export more sucrose. One or more unknown factors, perhaps associated with the changed hormonal balance, switch the export of sucrose from other parts of the plant to underground buds or stolon tips. The higher concentration of sucrose at these underground growing points, in concert with the hormonal changes, promotes the required anatomical changes in the location, plane, and frequency of cell division and in the location and extent of cell enlargement. The high sucrose concentration and hormonal changes elicit the required biochemical changes that lead to starch deposition (increased activity of ADPglucose pYrophosphorylase, favored by the lowered gibberellin activity) and the production of the storage of proteins. A difficulty with this hypothetical scheme is that it assumes an increase in sucrose concentration of the stolon tips just prior to tuber initiation. No such increase is reported (Davies 1984b). One possible reason for difficulty in detecting the presumed increase in sucrose concentration is that the increase might be restricted to a limited number of specific cells, as Vreugdenhil and Helder (1992) point out. The problem could also lie in the timing of assays. The increase in sucrose concentration may persist only for a brief period-buried buds of induced cuttings showed increased starch deposition and cell division one day after cutting (Part VILA). Once the initial increase in sucrose has set off the chain of events that leads to swelling of the stolon, it is reasonable to expect that sucrose levels would drop again so as to maintain a favorable concentration gradient between the source leaf and the developing tuber sink. Random sugar analyses of stolon tips, only some of which will form tubers, might well miss the transitory change. It would be interesting to perform sugar analyses on the buds of induced cuttings at intervals within the first 24 h after cutting, restricting the tissue sampled as narrowly as possible to the expected target zone.

3.


163

D. Competitive Advantage Provided by the Changes during Evolution The changes discussed previously are logical in the context of providing a competitive advantage during evolution of the wild potato. The tuber is a storage organ that permits survival during the period of freezing temperatures in the high Andes where the wild potato evolved. Stolon growth provides a dispersal mechanism-the longer the stolons, the greater the dispersal of new plants, whether formed during the current season from orthotropic shoots (e.g., following damage to the apex of the mother plant), or the following season from overwintering tubers. During the early part of the growing season the best survival strategy is vigorous shoot growth to shade out competing species, so tall plants with relatively long stems and abundant branches have the advantage. Thick leaves may provide more resistance to damage from biotic or abiotic factors. Individual leaf senescence should be delayed to permit more light interception over the course of the growing season. Longer photoperiods and warmer temperatures promote the morphological features just described. Once shoots are well established, some shift to stolon production is logical. This might be triggered in wild potatoes either by a slight shortening of the photoperiod or by the attainment of large leaf areas (Kahn et al. 1983). As the danger of frost increases, survival strategy calls for a shift to tuberization. The shortening photoperiod, cooler temperatures, and perhaps even lower supplies of soil N provide the signal. In response the leaf/stem ratio and specific leaf area increase, maximizing the leaf area produced from the biomass partitioned to shoots. In spite of the thinner leaves the rate of photosynthesis per unit leaf area does not decline, which means a higher rate of photosynthesis per unit of biomass partitioned into shoots. The higher rate of photosynthesis is accompanied by an increase in daytime accumulation of leaf starch and an increase in assimilate export. This is compared to what would be expected from plants that had not developed these evolutionary adaptations if grown under the same environmental conditions. The increased export of assimilate supports the developing tubers. The presence of the strong sink formed by the developing tubers is conducive to continued high rates of photosynthesis. The strong tuber sink also contributes to early shoot senescence, but in nature this does not matter; in the high Andes short days are soon followed by killing frosts.

164


VIII. PATI'ERNS OF STOLON AND TUBER FORMATION A. Significance

A potato plant dug up from a commercial field in midseason (Fig. 3.19) is likely to show a number of stolons that bear tubers, and others that do not. The tubers will vary in size and position on the stolon. Stolons vary not only with respect to tuber production, but also with respect to the main stem node at which the stolon originated, the stolon length, and the degree to which the stolon is branched. The patterns of stolon and tuber frequency, size, and distribution will have important implications for yield and quality of the crop. Yield and size distribution are obviously important; different markets require different tuber sizes, depending on intended use for seed, fresh market, or the various forms of processing. Tubers that form on long stolons high in the ridge are more likely to be exposed to the light and turn green, and tubers set very deep in the ridge require more energy for harvest because of the extra soil that must be handled. Thus the patterns of both tuber and stolon formation have economic consequences.

Figure 3.19. Underground parts of a 'Desiree' plant growing in a commercial field in the Negev region of Israel. Note different positions of tubers on stolons, differences in length of stolons, and wide range of tuber sizes.

3.


165

There are interesting parallels between stolon and tuber formation. Both stolons and tubers form more readily in darkness than in light, although under certain conditions both can be made to develop in the light (Kumar and Wareing 1972). Both stolons and tubers can be produced on tubers in the absence of aerial parts; and the reverse is also true-both can be produced on seedlings or cuttings that lack mother tubers (Fig. 3.12). Finally, there is the evidence for a continuum of responses involving stolonization and tuberization as described in Part V.B.

B. Pattern of Stolon Formation in the Intact Plant All axillary buds, even those above the soil level, possess the potential to form stolons and tubers when they receive the appropriate stimuli. It is clear that either the mother tuber in the absence of aerial parts or leaves in the absence of a mother tuber can produce the stimuli for stolon and tuber formation. Stolons often form even before shoot emergence. Stolons can also form in the absence of a normal mother tuber, for example when TPS or microtubers are planted. Presumably when both the mother tuber and leaves are present both playa role. We turn now to a description of stolon formation in the intact plant grown for mother tubers. On potato plants grown in the usual way from seed tubers, stolons are induced at the underground nodes of the sprout (Plaisted 1957; Booth 1963). According to Plaisted (1957) and Cutter (1992), stolon formation starts at the most basal node and progresses acropetally. In three cultivars examined by WUIT (1977), about half of the stolons formed at the most basal node, with roughly 10% of the remaining stolons at each of the next four higher nodes (Fig. 3.20A). The patterns of stolon formation among the three cultivars investigated were much more uniform than the patterns of tuberization, as is evident in Part VIII.E.2. The effects of environment on stolon number are discussed in Part III.

C. Maintenance of Diagravitropic Growth A stolon tip can be converted into a negatively gravitropic shoot by decapitating the main shoot (Sachs 1893; Lovell and Booth 1969; Kumar and Wareing 1972) or by raising the temperature of the root zone of plants grown with roots and stolons separated (Struik et al. 1989b). The decapitation technique was used to compare apical domes of orthotropic shoots and stolons (Clowes and MacDonald 1987). The former produced cells at twice the latter's elemental rate of mitosis (measured as dividing cells cell-I day-I), although the structure and number of cells were similar. The change in rate could be detected within 12 h of decapitation of a main sprout tip, with youngest leaf primordia most affected.

166


A. Stolons

6 Q)

-g enc:c:

--a-- 'Pentland Crown' -

-0- -

'Desiree'

····il--·· 'Maris Piper'

o

B CJ) '0 ci

2

Z

0...---0---

o+----.---r----,-----r--.,.--::::::IF-=~ B. Tubers

6

~ o

~

\ 4

\

\

15

.2

'0

\

\

\ \ '. \

2

ci

z

..... \ "B;-:'::='--4--=.-:::~:::::::-_~.:-::.:-::.

o !---"':;'==~;t:::=::Ijl=====i"";;:;;';';';';';~---::;:;".:;-:.=a:=-=-1) 1

234

5

6

7

8

Node Number

Figure 3.20. Numbers of stolons and tubers formed at underground nodes of three cultivars. Plants were grown in wood-framed boxes with removable sides. Node 1 is the node nearest the mother tuber. The numbers of stolons (A) and tubers (B) are means of seven harvests (graphs constructed from data of Table 1 in WUIT 1977).

The role of the apex in maintaining diagravitropic stolon growth seems to involve an interplay among levels of auxin, gibberellin, and cytokinin (Booth 1963; Kumar and Wareing 1972; Woolley and Wareing 1972a,b,c). Auxin and gibberellic acid applied to the stumps of decapitated andigena plants favored the diagravitropic outgrowth of aboveground lateral buds as stolons, but only if the plants had been exposed to short photoperiods (Woolley and Wareing 1972c). Application of cytokinin to the stolon tip caused a switch to orthotropic shoot growth (Kumar and Wareing 1972; Woolley and Wareing 1972b). The above-mentioned loss of diagravitropic growth when roots were heated might be explained by increased root activity with associated increase in cytokinin production (Struik et al. 1989b). Rooted cuttings were more likely to form orthotropic shoots rather than stolons (Kumar and Wareing 1972; Woolley and Wareing 1972a), and applications of cytokinin would substitute for the presence of roots in this respect (Kumar and Wareing 1972; Woolley and Wareing 1972a,b). The transport of benzy!adenine toward the stolon tip was

3.


167

decreased threefold by applications of auxin and gibberellic acid to the stumps of stem cuttings (Woolley and Wareing 1972a). D. Stolon Elongation and Branching

The effects of photoperiod and gibberellin activity on stolon growth were considered in Parts IILA and VI.D .1, respectively. Strong induction of the plant to tuberize will cause a cessation of stolon elongation and a conversion to tuber production; treatment of strongly tuberizing plants with moderate levels of gibberellic acid will convert them back to stolon growth (Hammes and Nel 1975). Although the normal reason for the cessation of stolon growth is tuberization, certain temperature combinations restricted stolons without inducing tuberization (Struik et al. 1989b). The resulting stolons were short and died quickly. The degree of branching of stolons can be quite variable. Branching is stimulated by long days (Struik et al. 1988b), high temperatures (Struik et al. 1989b), and high gibberellin levels (Struik et al. 1989d) (the same factors that favor the conversion of the plant away from tuberization toward stolon growth). Under normal growing conditions the later a stolon is initiated, the shorter the interval before tuber initiation becomes dominant (Vreugdenhil and Struik 1989; also see Part VI.C for a discussion of how plant size affects induction to tuberize). Therefore the first stolons formed-typically at the base of the plant-have longer to grow, are more likely to branch, and tend to provide more potential sites for tubers (Lovell and Booth 1969; Struik and Van Voorst 1986). The degree of mechanical resistance encountered by the extending stolon may affect stolon and tuber development. Developing stolons that failed to encounter sufficient mechanical resistance had extremely vigorous stolon growth and delayed tuberization (Lugt et al. 1964; Cary 1986) or secondary stolons and numerous small tubers (Gray 1973; Cary 1986). Vreugdenhil and Struik (1989) reported similar observations. Wheeler et al. (1990) obtained good tuber yields (2.8 kg per plant when each plant occupied 0.4 m2 ) in the absence of mechanical resistance by employing a nutrient film technique; but 29 days after planting they employed 28 cycles of a 12-h photoperiod and cool temperatures to obtain strong induction. This may have been strong enough induction to overcome any problem from the lack of resistance around the stolons, especially since plants were started from in vitro plantlet nodes rather than from mother tubers (Part V.D). It is an open question whether still higher yields would have been obtained by utilizing a medium to provide mechanical resistance. Under field conditions the density of the medium may be increased beyond the optimum through soil compaction. This may reduce tuber


168

number and yield through effects on soil temperature and moisture (Kouwenhoven 1978), as well as through effects on mechanical resistance encountered by the stolons (Vreugdenhil and Struik 1989). The possible role of ethylene in this respect is described in Part VI.D.5 of this review.

E. Effect of Node Position on Tuberization Patterns 1. Cuttings. When more than one node of a multi-node cutting was buried in the soil (Table 3.7), the most basipetal buried bud was by far the most likely to tuberize and to develop the largest tuber (Gregory 1956; Chapman 1958; Kahn and Ewing 1983). This tendency was not explained by orientation with respect to gravity or by assuming that the most basipetal bud was most likely to tuberize (Kahn and Ewing 1983). Thus turning the cutting upside down or laying it horizontally still produced the most tuberization on the oldest bud; and burying the two youngest buds rather than the two oldest ones produced stronger tuberization on the younger of the two buried buds. This is not to say that gravity and bud age Table 3.7. Effects of number and location of buried buds on tuberization. Eight-node apical cuttings were taken from 'Chippewa' plants that had been induced to tuberize. Leaves were excised from the bottom three nodes; and buds of these nodes were also excised, depending upon treatment. Stems were buried up to the middle of the internode above node 6. Data were transformed before statistical analysis (after Table 2 of Kahn and Ewing 1983). Total tuberization (%) Treatment

Buried bud

1

6 7 8 6 7 6

2 3 4 5

6 7

8 7 8

6 7 8

Means 100 100 100 79 100 33 100 92 100 25 100 100

Tuber fresh weight (g)

Significance i Means a a a

NS b a a

1'07 1'49 1'94 0-11 1-39 0-05 1-52 0-30 1-26 0'02 0'31 1-19

Significance b a a

c b a

=

Node 1

2 3 4 5 6 7 8

=

iNS, .. and" indicate nonsignificance, significance at P 0.05, and significance at P 0.01 respectively. For groups of three, means followed by the same letter do not differ significantly at P = 0.05.

3.


169

have no effect on the pattern; but other factors yet to be identified appear more important. 2. Intact plant. Although the basal node of a normally oriented cutting

consistently develops the best tuberization, the pattern in the intact plant is much less predictable. The proportion of stolons that develop tubers is highly variable (Moorby 1967; Wurr 1977; Cother and Cullis 1985), and often the first stolons formed are not the first to tuberize. Cultivars differ not only as to the percentage of stolons that bear tubers, but also with respect to the pattern of tuberization at different nodes. Figure 3.20B shows the distribution of tuber numbers at the nodes of three cultivars. A comparison with Figure 3.20A shows that the percentage of stolons that bore tubers was highest for 'Desiree: and that the difference was primarily at the basal node. Pentland Crown was inferior to the other two cultivars in percentage of stolons developing tubers at nodes 2-5. The pattern of tuber size distribution on the stolon system is also inconsistent and does not always match the pattern described previously for cuttings, although Clark (1921) noticed a tendency for smaller tubers on the upper stolons, and Gray (1973) observed that the tubers on the lower nodes were larger. He also found a shortening of stolon-bearing tubers proceeding upward from the base of the stem. Krijthe (1955) concluded that the third, fourth, and fifth nodes above the base of the stem had the largest tubers. Others (Plaisted 1957; Cother and Cullis 1985) have reported that tubers reaching marketable size were more frequent on the lower than on the upper stolons; but the distribution varied with stolon number. With larger numbers of stolons, fewer marketable tubers were at the lowest stolon positions (Cother and Cullis 1985). Figure 3.21 indicates schematically the complex relationships among the many factors that determine relative tuber size. Factors that influence the competition among tubers on a single stem are presented. Effects of photoperiod (Part lILA), temperature (Part III.B), drought and other environmental factors (Part III.E), and condition of the mother tuber (Part V) on tuber numbers are already described. For more discussion of why tubers are initiated on particular stolon tips, see Vreugdenhil and Struik (1989).

F. Growth Rates of Individual Tubers

The number of tubers initiated commonly exceeds the number that develop to a marketable size. Some are resorbed entirely (Part IX.A), some remain small until haulm maturity, and others grow to variable sizes. Examples of differences in growth rates of individual tubers can be seen in Figure 3.6, where the increases in volumes of three tubers on each

170

l


I

pattern of

1

SJOO formation

pattern of stolon growth

stolon characteristics

~ 1l

l

.

pattern of tuber set

?

position of tuber

I?

differen,e:

initial • tuber actiVity

)

t'Or20 mm that showed second growth. Size classes for the smallest tubers are presented by weight (g). The largest size class is presented in terms of diameter (mm) [after Lommen and Struik (1990)J. Soil cover at 51 DAP

Second growth

Size class

(Ufo)

(Ufo)

Table 6.3.

Continue d. Explant Source

Species

Seedling

Prosopis alba Griseb.

Shoot-ti p or nodal stem segment

P. alba

P. chilensis (MoL) Stuntz.

P. cineraria (L.) Druce

Shoot-ti p or nodal stem segment Shoot-ti p or nodal stem segment

Shoot-tip or nodal stem segment

Root Formati on

Medium

PGR (JLM)

Medium

PGR (JLM)

Acclima tized

Referenc e

MS

5.4-26.9 NAA

MS

4.9 IBA

Yes

Jordan 1987

MS

2.9 lAA + 6.7 BA

-

Not reported

Tabone et aI. 1986

MS

5.4-26.9 NAA

MS

4.9 IBA

Yes

Jordon 1987.

Lateral bud Modifie d MS

17.1IAA + 0.2 KIN

WM

14.8IBA + 0.2 KIN

Yes

Nodal stem segment s

!fzMS

0.5 KIN

-

-

Not reported

Arya and Shekhaw at 1986; Goyal and Arya 1984 Wainwri ght and England 1987

MS

5.4-26.9 NAA

MS

4.9 IBA

Yes

Jordon 1987

Nodal stem segment s (1-2 yrs old)

P. juliflora (Swartz) DC P. tumarug o F. Phil.

Mature

Shoot Formati on

Table 6.3.

Continued. Explant Source

Species

Seedling

Shoot Formation

Root Formation PGR (,uM)

Acclimatized

Reference

1 IBA

Yes, microshoots derived from dormant buds Yes

Davis and Kealthey 1987a; b

1.6 NAA or 1.5 IBA

Yes

Chalupa 1983.

MS

17.1IAA+ 0.2 KIN

Yes

Harris and Puddephat 1989b

17.1IAA+ 0.15 GA

MS

Failed to root

None

MS

None

Mature

Medium

PGR (,uM)

Robinia pseudoacacia L.

Dormant bud or nodal stem segment

MS

0.32-3.3 BA 1/10 MS (no sucrose)

R. pseudoacacia

Nodal stem segment Nodal stem segment (1-2 yr old)

Modified MS

1.1-4.4 BA

l/2MS

9.8 IBA

Modified MS

0.3 IBA+ 1.8-2.7 BA

Modified GO

MS

17.1IAA+ 0.2 KIN

MS

MS

R. pseudoacacia

Sesbania arborea (Rock) Deg. & Deg. S. formosa Mueller

S. grandiflora (L.) Pair.

Nodal stem segment Nodal stem segment Shoot-tip

Medium

Barghchi 1987

Harris and Puddephat 1989b No

Harris and Puddephat 1989b continued

N

co

Ci1

6.

TISSUE AND CELL CULTURES OF WOODY LEGUMES

297

Two basic strategies have been employed to circumvent the deleterious effects of the phenolic-like compounds that accumulate in the medium from explants of several woody legumes. The simplest yet most labor intensive method is to transfer explants frequently to avoid buildup of the phenolic-like compounds. In mature explants of Robinia pseudoacacia, transfers were made every 4-7 days to evade the accumulation of inhibitory compounds in the medium (Davis and Keathley 1987a). Alternatively, explants can be pretreated with antioxidants or antioxidants can be included in the initiation medium. Most commonly, polyvinylpyrrolidone (PVP) or ascorbic acid have been utilized for this purpose. Beneficial effects of antioxidants have been reported for Prosopis (Arya and Shekhawat, 1986) and Dalbergia (Datta et a1. 1983) cultures. However, antioxidants in the medium do not provide an advantageous response in Prosopis tamarugo cultures (Jordan 1987). Cultures of Cercis typically accumulate phenolic-like substances (Geneve et a1. 1990b). The possibility that these compounds were responsible for shoot-tip necrosis in cultures was investigated by using antioxidants, activated charcoal, and Gelrite as a gelling agent to replace DifcoBacto agar. Antioxidants did not reduce phenolic-like exudation or improve subsequent growth. Although cultures grown on Gelrite did not have the typical brown exudation, its elimination had no remedial effect on shoot-tip necrosis. In vitro shoot-tip necrosis has been attributed to calcium deficiency in other susceptible crops (Sha et a1. 1985). Although additional calcium did not alleviate shoot-tip necrosis in Cercis, a nutritional imbalance remains the most likely source of this physiological problem. The success of shoot or root formation in vitro is often related to the maturity of the donor plant (Hackett 1985). Explants obtained from seedlings or plants in the juvenile phase of growth have greater capacity for organ formation. However, selection of superior individuals from woody plants often requires that selection be made from mature trees. The response of explants from mature sources can be limited for both shoot and root formation in vitro (Durzan 1983). Shoot multiplication or shoot elongation have been limited for woody legume explants taken from Ceratonia (Thomas and Mehta 1983), Gleditsia (Rogozinska 1968), Gymnoc1adus (Geneve et a1. 1990a), Prosopis (Tabone et a1. 1986), Robinia (Davis and Keathley 1987a) and Sophora (Froberg 1985). The genetic background of trees can influence the response of explants in culture. Explants taken from several mature trees of Robinia with unknown genetic backgrounds and cultured on the same medium formed shoots and roots at ·different rates (Davis and Keathley 1987a). Rejuvenation of mature trees prior to removing explants is a possible alternative to avoid the recalcitrant organogenic potential

298

R. N. TRIGIANO. R. L. GENEVE. S. A. MERKLE. AND

J. E. PREECE

inherent in explants from a mature source (Bonga 1982). Barghchi (1987) used root cuttings of Robinia to produce adventitious shoots in a presumably juvenile condition for use as explants. These explants showed a greater response for shoot multiplication, shoot elongation, and subsequent root formation than was reported for mature explants of Robinia in other studies (Davis and Keathley 1987a). The effect of ontogenetic age on morphogenetic potential has been directly evaluated between seedling and mature explants of Gymnocladus (Geneve et a1. 1990a). Explants from juvenile sources have the potential for shoot proliferation in vitro, and developing microshoots can be rooted ex vitro. Although explants from mature trees produced multiple buds, these short shoots would not elongate even in the presence of exogenous GA. Explants from mature trees were cultured from shoots derived from basal suckers, which were presumably juvenile, and their response for shoot formation was similar to explants derived from seedlings (Smith and Obeidy 1991; Geneve, unpublished results). There have been additional approaches used to improve in vitro shoot growth, which may be unrelated to ontogenetic age. Thomas and Mehta (1983) improved overall shoot growth and root formation of Ceratonia by the addition of phloroglucinol into the multiplication medium. Phloroglucinol was hypothesized to act SYnergistically with auxin. The promotive effect of phloroglucinol has been observed for other woody species (James et a1. 1980; Jones 1976). However, Dhawan and Bhojwani (1985) working with Leucaena found no benefit on shoot growth with the inclusion of phloroglucinol in the multiplication medium. Growth regulator concentration can also influence successful shoot formation. Explants of a clone of Prosopsis alba treated with 0.44JLM BA produced only leaves. However, by increasing BA concentrations above 44 JLM, explants produced shoots exclusively (Tabone et a1. 1986). There was an interaction between IAA and BA on both shoot number and shoot length, but the high level of BA (44-80 JLM) was important to improved shoot formation. This was a 3- to 4-times greater concentration of cytokinin than was reported for any other micropropagation study with woody legume species (see Table 6.3). The addition of glutamine as a nitrogen source was beneficial to shoot growth for both cultures of Prosopis (Tabone et a1. 1986) and Leucaena (Dhawan and Bhojwani 1985). Glutamine alleviated the problem of leaf drop in Leucaena and also promoted shoot multiplication. Glutamine may benefit the cultures since it is the transported form of nitrogen in many legumes (Tabone et al. 1986). Root formation is often used as an indicator for the juvenile condition. Successful root formation can be a limiting factor in the micropropagalion of several woody legumes including Cercis (Bennett 1987). Prosopis

6.


299

[fabone et al. 1986), Robinia (Davis and Keathley 1987a), and Cladrastis (Weaver and Trigiano 1989). Serial subculturing of shoots proved to be effective in modifying the rooting potential of several woody species (Mullins 1985). This approach may explain the success by Yusnita et a1. (1990) for rooting microcuttings of Cercis canadensis var. alba from microshoots derived from explants that were subcultured for one year. Serial subculturing appears to have a rejuvenating effect and is an alternative strategy for the successful recovery of plants from difficult-topropagate species where ontogenetic age is potentially the limiting factor (Hackett 1985). However, root formation does not appear to be a significant problem for most woody legumes of tropical origin (Table 6.3).

B. Organogenesis In contrast to micropropagation of trees by axillary bud proliferation, organogenesis proceeds de novo via organization of meristems. Adventitious shoots and roots may be initiated either directly from cells within the explant or indirectly from an intervening callus derived from any portion of the plant including meristems (Flick et al. 1983). In vitro propagation by organogenesis generally follows the same four stages outlined previously for axillary bud proliferation, except that establishment of the culture may entail production of callus. For most of the species listed in Table 6.4, the basal medium employed for callus induction and/or shoot formation was either MS or B5 supplemented with auxins and cytokinins. However, in contrast to axillary bud proliferation of woody legumes, initiation of shoots is usually limited to explants obtained from juvenile material; there are very few reports of shoot organogenesis from explants of mature trees. Adventitious root initiation from microshoots of most species was successful and required either transfer to basal medium without growth regulators or an auxin treatment. Many of the difficulties in establishing cultures and initiating shoots, such as phenolic-like exudations, discussed in the section concerning axillary bud proliferation were also encountered with organogenic systems for woody legumes. Similar remedies have been applied including the addition of PVP or other antioxidants to the medium or explants that retard or prevent browning. Phenolic-like inhibition of callus initiation and growth from explants of Dalbergia latifolia was alleviated by utilizing PVP (Ravishankar Rai and Chandra 1988; Rao 1986), and browning of the medium and callusing of adventitious roots was suppressed by PVP and cysteine in cultures of Cajanus cajan (Mehta and Mohan Ram 1980). Shoot formation from hypocotyl callus cultures of Sesbania sesban was only possible after the addition of PVP to the medium (Khattar and Mohan Ram 1982).

Table 6.4.

Continued. Growth regulators (pM) Basal Medium

Callus Formation

Anthers (pollen)

B5

Petiole and stem from mature trees Cotyledon, hypocotyl, leaf and root Hypocotyl Leaf Stem

B5

2.2 2.4-D + 9.2 KIN 2.9 IAA + 4.5 BA

B5

MS MS MS

Cotyldeon

MS

Hypocotyl

MS

Root

MS

Hypocotyl

B5

Species

Explant Z

A. lebbeck (L.) Benth. A. lebbeck

A. lebbeck

A. lebbeck

A. lucida Benth.

Shoot Formation Y 2.9 IAA

Root Formation Y

+ 4.5 BA None

References Gharyal et a1. 1983

Same as callus

None or 0.6 IAA

Gharyal and Maheshwari 1990

11.4 IAA or 10.8 NAA

Same as callus

None or 11.4 IAA

Gharyal and Maheshwari 1983

5.4 NAA + 4.6 KIN 2.2 2,4-D + 4.6 KIN 4.5 2.4-D + 4.6 KIN 13.5 2.4-D + 0.9 BA 0.6 IAA + 0.9 BA 0.6 IAA + 0.9 BA

0.5 NAA + 2.2-4.5 BNM BA IBA 0.5 NAA + 4.5 BA 0.5 NAA + 2.3 KIN

-

+ 9.8

Lakshmana Rao and De 1987

5.7 IAA + 27 NAA 22.5 BA 5.7 IAA + 23 KIN or 5.7 IAA + 22.5 BA 5.7 IAA + 23 KIN

Upadhyaya and Chandra 1983

10 BA

Tomar and Gupta 1988b

1IAA

w

0

~

continued

Table 6.4.

Continued. Growth regulators (p.M)

Species

Basal Medium

Callus Formation x

C. cajan

Cotyledon

B5

-

Cassia fistula L. and C. siamea Lam.

Stem from mature trees

B5

Ceratonia siliqua L.

Cotyledon

MS

Dalbergia lanceolaria L.

Cotyledon and hYPQcotyl Internode from mature trees

MS

10.8 NAA + 2.2 BA or 2.9 IAA + 4.5 BA 5.7 IAA+ 4.9 2iP or 5.7 IAA+ 4.6 KIN 4.5-18 BA

D. latifolia

Internode

MS

D. latifolia

Hypocotyl

MS

D. latifolia Roxb.

w 0 w

Explant Z

MS

2.7 NAA + 4.5 2,4-D + 4.5 BA+ 10% CW 16.1 NAA + 4.4 BA

-

x

Shoot Formation Y

Root Formation Y

References

10 IAA + 10 BA

5 NAA

2.9 IAA+4.5 BA

None or 0.6 IAA

Same as callus

Same as callus Martins-Loucas and Rodriguez-Barrueco 1981

4.5-18 BA

9.8-19.6 IBA

Anand and Bir 1984

0.5-5.4 NAA + 13.5 BA

WM + 5.711.4 IAA or 1/2 MS + 9.824.5 IBA WM + 5.7 IAA + 5.4 NAA + 4.9 IBA-4 days then liz MS 5.7 IAA + 5.4 NAA + 4.9 IAA for 72h then none

Lakshmi Sita et a1. 1986

2.7-5.4 NAA + 22.2 BA

4.5 BA or 2.2 BA + 2.5 2iP

Mehta and Mohan Ram 1980 Gharyal and Maheshwari 1990

Rao 1986

Ravishankar Rai and Jagadish Chandra 1989

continued

(,j)

0

Table 6.4.

Continued.

~

---------_

..

_-----~

Growth regulators (JAM) Species

Explant Z

Basal Medium

D. latifolia

Internode

MS

D. latifolia

Hypocotyl

MS

D. sissoo Roxb. ex DC

Cambium tissue from mature trees

MS

D. sissoo

Nodes

MS

D. sissoo

Root

B5

D. sissoo

Hypocotyl

MS

D. sissoo

Cotyledonary node

MS

Callus Formation

Shoot Formation Y

Root Formation Y

5.4 NAA + 4.5 5.4 NAA + 4.5 BA WM or l/z MS BA or + same as above then liz 27 NAA+ 22.5 BA MS liquid 4.5-9 BA x 4.5-9 BA 5.4-10.8 NAA l/z MS + 5.7 Solidified 2.2-9.0 BA then liquid IAA + 4.9 then IBA + 5.4 NAA solidified. All medium 9.0 2,4-0 + 0.4 BA 2.9 IAA + 2.7 Same as callus Same as callus NAA or 2.9 IAA - x 0.1 NAA or 0.1 1 IBA NAA+O.l BAor 5IAA+0.IBA 13.5-22.5 BA 13.5 BA + 5.7 MS liquid + IAA 17.1 IAA + 16.2 NAA 570 IAA for 5 0.5 NAA + Same as callus min then ex 1.2-4.5 BA vitro to sand bed

References Ravishankar Rai and Jagadish Chandra 1988

Sudhadevi and Nataraja 1987a;b Kumar et al. 1991

Datta and Datta 1983

Mukhopadhyay and Mohan Ram 1981 Sharma and Chandra 1988

Suwal et a1. 1988

Table 6.4.

Continued. Growth regulators (JAM) Basal Medium

Species

Explant Z

Indigofera enneaphylla L.

Cotyledon or hypocotyl?

B5

1. enneaphylla

Cotyledon

B5

Leucafma diversifolia L.

Cotyledon and hypocotyl

MS

2.2 2,4-D + 4.5 BA (liquid then solidified medium) 2.9 IAA + 4.5 BA or 2.2 2,4-D + 4.5 BA 2.9 IAA + 4.5 BA 92,4-D

L. leucocephala (Lorn.) de Wit L. leucocephala

Epicotyl

MS

3 BA

Cotyledon

MS

2.7 NAA + 2.2-22.5 BA

Cotyledon and epicotyl

MS

-

Hypocotyl

L. leucocephala

""0en

Callus Formation

x

Shoot Formation Y

10.8 NAA + 0.9 BA (4 wk) then none

4.5 BA

2.9 IAA

Root Formation Y

+ 5.4 BA 10.8 NAA + 0.9

References Bharal and Rashid 1984

Bharal and Rashid 1979

BA then none same as callus 0.5 NAA + 4.5 BA (direct) or 0.5 NAA + 2.2-4.5 BA or 9-13 BA 3 BA

None-16.2 NAA

Pan and Chang 1987

5IAA

9BA

2.5-4.9IBA

2.2-4.5 2,4-D + 2.3 KIN + 10% CW or 11.4 IAA + 9 2,4-D + 2.3 KIN

Same as shoot

Dhawan and Bhojwani 1987a; 1985 Nagamani and Venketeswaran 1987 Nataraja and Sudhadevi 1984

continued

c.:l

0

Table 6.4.

Continued.

0)

Growth regulators CuM) Species

Explant Z Cotyledon and epicotyl

Basal Medium MS

Cotyledon

MS

Mimosa pudica L.

Cotyledon, hypocotyl, leaf and shoot apex Hypocotyl

B5

Robinia pseudoacacia 1. R. pseudoacacia

Sesbania bispinosa Uacq.) W. F. Wight S. grandiflora (L.) Poir. S. grandiflora

Hypcotyl and internode Leaf disks Cotyledon and hypocotyl Cotyledon and hypocotyl Cotyledon and hypocotyl

2.2-4.5 2,4-D

Shoot Formation Y

Root Formation Y

45 BA

10.8 NAA

4.92iP

No rooting

Same as callus

11.4 IAA

Same as callus

WM + 0.2 KIN + 14.7 BA 1/10 MS + 1 IBA None 10 IBA in the dark 5 IBA

References

+ 2.3 KIN +

L. retusa Benth.

Prosopis cineraria L.

Callus Formation

MS

MS MS B5 BS MS

10% CW or 11.4 IAA + 9 2,4-D + 2.3 KIN 2.7 NAA + 9 BA 2.2 2,4-D or 2.7 NAA + 4.5 BA 1.4 IAA or NAA + 21 KIN 5 NAA + S or 10 BA 1 PIC + 1 BA -

x

0.1 NAA + 1 BA 9 2.4-D + 2.2 BA

10 BA or + 1 NAA 1 PIC + 1 BA 10 BA 0.5-1 BA 22 BA+ 15%CW

liz MS + 9.8 IBA

Nagami and Venketeswaran 1987 Gharyal and Maheshwari 1982 Goyal and Arya 1981

Han et a1. 1990; Han and Keathley 1989 Davis and Keathley 1985 Kapoor and Gupta 1986 Khattar and Mohan Ram 1983 Sinha and Mallick 1989

Table 6.4.

Continued. Growth regulators (JLM)

CJo)

'I

Callus Formation

Internodes and nodes

MS

Same as callus

None

Harris and Moore 1969

Hypocotyl

B5

1.6 NAA + 0.5 BA+ 0.03 GA or 2.2 BA None

1-5 BA

Not reported

Khattar and Mohan Ram 1962

Cotyledon and hypocotyl

B5

1-10 NAA + 5-10BA+1-5 GA

5 BA + 1 IAA + 500 mg/l PVP

Not reported

Hypocotyl

MS

-

0.9 KIN + 2.2 BA

WM + 5.7 IAA + 4.9 IBA + 5.4 NAA + 5.2 IPA for 72h then WM liquid

Explant Z

S. sesban (L.) Merr.

S. sesban

Tamarindus indica L.

o

Basal Medium

Species

x

Shoot Formation Y

Root Formation Y

References

Mascarenhas et aI. 1967

308

R. N. TRIGIANO, R. L. GENEVE, S. A. MERKLE, AND }. E. PREECE

Micropropagation by organogenesis has been suggested as a method used to circumvent plant production problems associated with low seed viability (Lakshmi Sita et al. 1986), long life cycles, and poor vegetative propagation characteristics (e.g. difficult to root) and also to produce rapidly large numbers of plants after genetic selection. The number of important woody legume species reported as being capable of regeneration via organogenesis has increased dramatically in the last decade (Flick et al. 1983). However, in most of these reports (Table 6.4) regeneration protocols have been limited to use of tissues derived from seedlings (e.g. hypocotyls or cotyledons) or explants from juvenile plants. Use of materials from these sources represents a distinct disadvantage to clonal propagation because seeds represent untested genotypes. Furthermore, evaluation of mature characteristics from seedlings is not possible. Other potential disadvantages of organogenic systems involving callus are that deleterious or undesirable genetic changes may be induced during the callus phase of propagation, especially in long-term cultures (Earle and Gracen 1984; Larkin and Scowcroft 1981; Thomas 1981) and that the regeneration capacity of the callus may diminish over long culture periods as reported for D. latifolia (Ravishankar Rai and Chandra 1988).

Although aberrant shoots are not useful for clonal propagation of selected genotypes, regenerated plants exhibiting clonal variation may be exploited for genetic improvement programs. Somaclonal variation (Larkin and Scowcroft 1981) has been reported from cultures of agronomic and horticultural species, but none has been documented from woody legume plants produced by organogenesis. Most determinations of clonal fidelity have been based on the foliage characteristics of newly established plantlets; large-scale evaluations of mature, regenerated populations of trees over time have not been completed. Clonal propagation from explants of mature trees with known characteristics via organogenesis would be highly desirable, but there has been little success. The various techniques used to restore juvenility to plant materials from mature trees have been tested in only a few organogenesis studies of woody legumes. Shoots with presumably juvenile characteristics often grow from stumps and may be used as explants in studies of organogenesis. Adventitious shoots were formed on callus originating from explanted leaves and internode tissue arising from coppice stumps of Acacia melanxylon (Meyer and van Staden 1987). In the same study, adventitious shoots were initiated from callus that formed at the base of explanted axillary buds. Other investigators have taken advantage of the physiologically invigorated nature of new growth produced after a quiescent or dormant stage of the adult tree. Petioles from newly emerged leaves of Albizia

6.


309

lebbeck, Cassia fistula, and C. siamea (Gharyl and Maheshwari 1990) and internodes from new branch growth on mature trees of Dalbergia latifolia (Ravishankar Rai and Chandra 1988; Lakshmi Sita et a1. 1986) and D. sissoo (Kumar et a1. 1991) have been used to initiate morphogenetic cultures. In the case of D. sissoo, callus from cambial explants was used to

initiate suspension cultures. Aggregates of greater than 30 cells were sieved (60 jJ.m) from the suspension culture and transferred to semisolid medium. Vigorous callus growth was reestablished and adventitious shoots formed after 45 days.

c. Somatic Embryogenesis The conditions for initiation of embryogenic cultures of woody legumes and for the production of somatic embryos and plantlets have displayed considerable diversity, similar to herbaceous leguminous species of agronomic importance. However, certain common patterns have emerged that may be useful in defining the primary factors involved in inducing these species to produce embryogenic cultures. All somatic embryogenic cultures of woody legumes have been initiated from seed or seedling tissues (Table 6.5). Embryogenic cultures of Albizia spp. (Gharyal and Maheshwari 1981; Tomar and Gupta 1986; 1988a; 1990) and Acacia koa (Skolmen 1986) were all initiated from seedling hypocotyls; whereas embryogenic cultures of Cercis canadensis (Geneve and Kester 1990; Trigiano and Beaty 1989; Trigiano et a1. 1988), Cladrastis lutea (Weaver and Trigiano 1991) and Robinia pseudoacacia (Merkle and Wiecko 1989) were initiated from immature embryos or immature seeds. Studies employing immature embryo or seed explants established that the developmental stage of the embryo explant was critical in determining the embryogenic response. The potential to initiate embryogenic cultures apparently peaked during the early development of the explanted embryo or seed, but during later ontological stages explants were more likely to produce callus and/or roots than somatic embryos (Trigiano et a1. 1988; Merkle and Wiecko 1989; Geneve and Kester 1990; Weaver and Trigiano 1991). Immature embryo or seed explants produced somatic embryos directly, with no intervening callus production. In cultures of C. canadensis, somatic embryos appeared to originate from epidermal or subepidermal cells of cotyledons (Geneve 1991; Geneve and Kester 1990; Trigiano and Beaty 1989), and also were formed directly on adventitious roots that originated from the explant (Trigiano and Beaty 1989). Robinia pseudoacacia somatic embryos continued to produce new somatic embryos from their radicles on medium lacking growth regulators (Merkle and Wiecko 1989).

t.:l

t-l

Table 6.5.

Somatic embryogenesis in tissue cultures of woody legumes.

0

Growth Regulators CuM)

Comments

References

MS

4.52,4-D

Embryos only

Skolmen 1986

B5

1 BA

Embryos only

Tomar and Gupta 1986

Hypocotyl

B5

None

PlantIets

Hypocotyl Hypocotyl

B5 B5

1 BA lor 10 BA

Embryos only Plantlets

Gharyal and Maheshwari 1981 Tomar and Gupta 1986 Tomar and Gupta 1988b

Species

Explant

Acacia koa A. Gray

Hypocotyl seedling tip Hypocotyl

Albizia amara

(Roxb.) Boivin A. lebbeck (L.) Benth. A. lucida Benth. A. richardiana (Wallich ex Voight) King & Prian A. richardiana

Cereis canadensis L. C. canadensis

C. canadensis Cladrastis lutea (Michaux) K. Koch Robinia pseudoacacia L.

Hypocotyl Immature embryo Immature embryo

Immature embryo Immature embryo

Immature seed

Basal Medium

MS B5 modified WPM modified SH

None or lor 10 BA 1 BA 1 or 5 2,4-D 9 or 13, 2,4-D

modified SH 4.5-22.52,4-D modified SH 4.5-22.5 2,4-D

MS

18 2,4-D

+ 1 BA

Embryos PlantIets Direct and indirect embryos from roots; plantIets Embryos with roots PlantIets

Tomar and Gupta 1986 Geneve and Kester 1990 Trigiano and Beaty 1989

PlantIets

Merkle and Wiecko 1989

Trigiano et al. 1988 Weaver and Trigiano 1991

6.


311

In contrast to the reports of initial direct somatic embryogenesis in cultures of C. canadenesis and R. pseudoacacia, the hypocotyl explants of Albizia spp. and A. koa first produced callus from which somatic embryos were differentiated (except in the case of A. lebbeck, where embryoids emerged directly from cracks in the hypocotyls). Embryogenic callus from A. koa cultures was later employed to initiate embryogenic suspension cultures (Skolmen 1986). Although embryogenic callus has been reported for C. canadensis (Trigiano and Beaty 1989), its ability to produce embryos by repetitive embryogenesis has not been demonstrated. In R. pseudoacacia cultures osmotic stress has produced proembryogenic masses [(PEMs) Halperin 1966]. After the osmotic stress was removed, PEMs diffentiated into somatic embryos at a very high frequency. PEMs have been used to initiate embryogenic suspension cultures of R. pseudoacacia (Merkle, et al. 1991). Another common factor in the studies employing immature embryos or seeds as explants was the use of auxin (2,4-D or IAA) to induce embryogenesis. However, somatic embryogenesis in cultures of Albizia spp. has been induced by BA and in the case of A. lebbeck, somatic embryogenesis was obtained without growth regulators. In woody and herbaceous legumes continued exposure of explants to 2,4-D resulted in somatic embryo abnormalities and lower frequencies of conversion of embryos to plantlets. Studies with alfalfa (Medicaqo sativa L.) by Stuart et al. (1985) and soybean (Glycine max L.) Merr. by Lazzeri et al. (1987) showed that induction with relatively high 2,4-D concentrations increased the frequency of abnormal somatic embryos compared to NAA or lower concentrations of 2, 4-D. Similarly, explants of leguminous woody species exposed to 2,4-D for long periods produced somatic embryos that had abnormal morphology (Geneve 1991; Trigiano et al. 1988; Weaver and Trigiano 1991), or failed to progress past an early stage of development (Skolmen 1986). In contrast, explants exposed to 2,4-D for a short time (Merkle and Wiecko 1989) or not at all (Gharyal and Maheshwari 1981; Tomar and Gupta 1988b) formed embryos that resembled their zygotic counterparts and were capable of forming plantlets.

III. CROP IMPROVEMENT

A. Protoplast Culture Although somatic hybridization and gene transfer are seen as potentially valuable tools for the genetic improvement of woody legumes, relatively little progress has been made with this group of species. Protoplast

312

R. N. TRIGIANO, R. L. GENEVE, S. A. MERKLE, AND

J. E.

PREECE

isolation and culture have only been reported in the following two species: Prosopis cineraria (Shekhawat and Kackar 1987) and Robinia pseudoacacia (Han and Keathley 1988). Prosopis cineraria protoplasts were isolated from roots, hypocotyls. and cotyledons of young seedlings. Dissected cotyledons yielded an average of 0.1 to 0.5 X 106 protoplasts per gram of cotyledon tissue. By culturing in drops of a modified MS medium, cotyledon-derived protoplasts can be induced to regenerate cell walls and divided up to three times before cultures browned (Shekhawat and Kackar 1987). Protoplasts of R. pseudoacacia were isolated from hypocotyl-derived callus, which yielded up to 105 protoplasts per gram fresh weight of callus. Protoplasts cultured in a thin layer of a modified liquid WPM were capable of regenerating cell walls and undergoing cell division, which was sustained by periodically adding fresh medium with lowered levels of osmoticum (Han and Keathley 1988). Protoplasts of R. pseudoacacia have also been isolated from cotyledons of 2-week old seedlings and were induced to regenerate cell walls and divided up to three times by culturing in hanging droplets of modified MS medium (Merkle. unpublished). There have been no reports of plantlet regeneration or morphogenesis from protoplast cultures of woody leguminous species.

B. Androgenesis Bajaj (1990) divides androgenesis into the following two types: (1) direct androgenesis, in which microspores or pollen are induced to form embryos without production of intervening callus. and (2) indirect androgenesis, in which embryos, shoots, or other organized structures are differentiated subsequent to callus formation. All reports of androgenesis in woody legumes (Table 6.6) have been in tropical species. and among these species, both direct and indirect androgenesis have been observed. Cultures of Cajanus cajan, Poinciana regia, Cassia fistula, and Cassia siamea formed multicellular bodies or embryoids directly from repeated divisions of pollen in the explanted anthers (Bajaj and Dhanju 1983; Bajaj et a1. 1980; Gharyal et a1. 1983a; Mohan Ram et a1. 1982). In Cajanus cajan, a suspension of pollen from 2- to 5-week-old cultured anthers formed embryoids or a mixoploid callus. In Albizia lebbeck, however, embryoids were reported to arise from anther-derived callus (Gharyal et a1. 1983b). Mature embryos or plantlets have not been .obtained from pollen-derived embryoids. Instead, the multicellular bodies or embryoids proliferated into callus (Gharyal et a1. 1983a, Mohan Ram et a1. 1982; Bajaj and Dhanju 1983). The only plantlets obtained from androgenesis in woody legumes originated as a callus-derived shoot from an anther culture of A. lebbeck (Gharyal et a1. 1983b). Examination of

Table 6.6.

...

t.:l t.:l

Androgenesis in woody legumes.

Species

Explant

Basal Medium

Albizia lebbeck (L.) Benth.

Anters

MS

A. lebbeck

Anthers

Cajanus cajan (L.) Huth.

Growth Regulators (pM)

Comments

References

2.2 2,4-D + 10.8 NAA + 9.2 KIN

Callus

De and Lakshmana Roa 1983

B5

2.2 2,4-D + 9.2 KIN or 4.5 BA + 2.5 IAA

Embryoids. roots, shoots and plantlets

Gharyal et a1. 1983

Anthers

B5

5 BA + GA + 1 IAA or 10.4 BA + 20 2,4-D + PVP (4 gil)

Callus

Mohan Ram et a1. 1982

C. cajan

Anthers, pollen

MS

22.8 IAA + 9.2 KIN

Embryoids

Bajaj et a1. 1980

Cassia fistula L.

Anthers

MS

22.8 IAA

Embryos

Bajaj and Dhanju 1983

Cassia siamea Lam.

Anthers

M5

9 2.4-D

Embryoids

Gharyal et a1. 1983

Poinciana regia Boj.

Anthers

MS

22.8 IAA + 9.2 KIN

Embryoids

Bajaj and Dhanju 1983

Tamarindus indicia L.

Anthers

Nitsch

Not reported

Embryoids

De and Lakshmana Rao 1983

+ 9.2 KIN

+ 2.3 KIN

314


J. E. PREECE

mitotic figures from root tip squash preparations indicated that the regenerated plants were haploid. C. Genetic Transformation Only a single case of gene transfer in tissue cultures of a woody legume has been reported. Davis and Keathley (1989) inoculated hypocotyl segments and cotyledons of R. pseudoacacia with strains of Agrobacterium tumefaciens and A. rhizogenes and cultured them on MS medium lacking plant growth regulators. Six of the tested Aqrobacterium strains incited tumors, and Southern analyses of four of the resulting tumor lines indicated that T·DNA sequences were present. Callus was obtained from cotyledons inoculated with an A. tumefaciens strain carrying a plasmid with the gene encoding neomycin phosphotransferase (NPT II). This callus proved to be kanamycin·resistant, indicating that the inserted DNA was being expressed. Southern analysis revealed that NPT II sequences had been integrated into the R. pseudoacacia genome. Transgenic black locust plantlets have been regenerated following transformation of callus with A. rhizoqenes (D. E. Keathley, personal communication).

IV. SECONDARY METABOLITE PRODUCTION Woody legumes, like many other plants, produce a variety of secondary products that are useful to humans. Unlike primary metabolites, such as amino acids and nucleic acids that perform vital physiological functions, secondary products do not have direct roles in primary biochemical pathways (Conn 1981) and may be a chemical response of a plant to its environment (Whitaker and Hashimoto 1986). For example, plants that are infected or treated with various biotic or abiotic elicitors produce phytoalexins with antimicrobial activity (Dixon et al. 1983). Secondary products may be exploited as pharmaceuticals, insecticides, agricultural chemicals, or antimicrobial compounds. Plant cell and tissue cultures may be utilized in some cases to accumulate secondary products at rates that are equal to or exceed those in intact plants. A. Alkaloids Many alkaloids have physiological effects on humans and some are important pharmaceutically (Robinson 1983).. Alkaloids have been isolated from green-colored cells grown in suspension cultures of Cytisus

6.


315

canariensis, C. purpureus, C. scoparius, Gentista pilosa, Laburnum alpinum, and Sophora japonica (Table 6.7). When the patterns of

quinolizidine alkaloids in cell cultures of these woody legumes were compared to those in leaves collected from plants growing outdoors, there was a reduced variety of these compounds and the total amount was 1-3 orders ofmagnitude less in the cell cultures (Wink et a1. 1983). Patterns of alkaloids formed in callus and cell suspensions were similar, but the emphasis of the study was on cell suspensions. Cell suspension cultures of all six species contained lupanine as the primary alkaloid. The other major alkaloids in leaves of Cytisus, Genista, Laburnum, and Sophora were not accumulated in cells grown in suspension cultures. Thus the formation of many alkaloids will require more sophisticated techniques to elicit production in cell cultures. The liquid medium in which the cell suspensions of the woody legumes listed above were grown contained alkaloids that were exuded by the cells (Wink et a1. 1983). The amount of alkaloids in the spent liquid medium ranged from 1-70% of that present in the cells from those cultures. Cells from suspension cultures of Laburnum alpinum and Cytisus canariensis contained an S-adenosyl-L-methionine (SAM): cytisine Nmethyltransferase that catalyzes the transfer of a methyl group from SAM to the alkaloid compound cytisine (Wink 1984). Cytisine was assumed to be derived from lupanine via several intermediates. Although the cells from suspension cultures had a relatively active SAM: cytisine N-methyltransferase, they did not contain detectable levels of either cytisine or N-methylcytisine (Wink et a1. 1983). However the cells were metabolically active because after 4 days they were able to degrade 7080% of alkaloids that had been added to the medium (Wink 1984).

B. Anthraquinone Compounds Anthraquinone compounds have a variety of medicinal uses including laxative properties (Rai and Shok 1982). Additionally, anthraquinone is used to manufacture vat dyes and to make seeds distasteful to birds (Windholz et a1. 1983). Rai and Shok (1982) and Rai (1988) demonstrated that visual selection of Cassia podocarpa callus can be used to improve yields of anthraquinone compounds. Initially a soft friable gray to light browncolored callus grew from seedling explants. Two callus lines were produced after three successive subcultures and selection for color-one predominately gray and the other primarily light brown. The total amount of anthraquinones was approximately 40% higher in the brown callus

w

~

Table 6.7.

Secondary metabolites accumulated in cell and tissue cultures of woody legumes.

0)

Species

Explant

Cassia a1ata L.

Seedlings

C. didymobotrya Fresen.

Hypocotyl and stem

C. nodosa Buch. Ham ex Roxb C. podocarpa Guill. 8t Perro

Type of Culture

Medium and Regulators

Substrates

Products

Comments

References

A greater % of these anthraquinone compounds was in bound form than free form Anthraquinones in early exponential phase, whereas flavonids in early stationary phase Most of the chrysophanol was in the bound form Selection of a brown colored callus line yielded higher levels of anthraquinones than plant leaves or other callus

Rai and Shok 1982

+ KIN

None

ChrysophanoI. emodin. aloe emodin

MS 2.4-D

+ KIN

None

Anthraquinones, flavonids

Seedlings

Callus MS 2,4-D

+ KIN

None

Seedlings

Callus MS 2,4-D

+ KIN

None

Chrysophanol, bound form of rhein Several Anthraquinones including rhein and chrysophanol

Callus MS 2,4-D

Cell

Botta et a1. 1989

Rai and Shok 1982

Rai 1988

Table 6.7.

....C..:l

"

Continued.

Species

Explant

C. podocarpa

Seedlings

Cytisus canariensis (L.) O. Kuntze

Shoot tips

C. canariensis (L.)Kuntze

Leaves, stems or flowers

C. purpureus

Leaves, stems or flowers

C. scoparius (L.) Link

Leaves, stems, or flowers

Type of Culture


Products

Comments

References

A brown callus line that was selected yield 40% higher anthraquinones than the original callus This enzyme is involved in quinolizidinealkaloid metabolism Cell suspensions contained fewer alkaloids than leaves Cell suspensions contained fewer alkaloids than leaves Cell suspensions contained fewer alkaloids than leaves

Rai and Shok 1982

+ KIN

None

Chrysophanol, emodin, rhein

1st 2,4-D + KIN, then 2,4-D

None

SAM: cytisin Nmethyltransferase, lupanine

None

Lupanine, tinctorine, 11allylcytisine

None

Tetrahydrorhombifoline, lupanine

None

Sparteine, lupanine

Callus MS 2,4-D

Cell

Substrates

Callus Modified MS Cell 1st 2,4-D + KIN, then IAA, NAA and KIN Callus ModM'ied MS Cell 1st 2,4-D + KIN, then IAA, NAA and KIN Callus Modified MA Cell 1st 2,4-D + KIN, then IAA, NAA and KIN

Wink 1984

Wink et a1. 1983

Wink et a1. 1983

Wink et a1. 1983

continued

t.l:l ....

Table 6.7.

Continued.

CJ:)

_Medium and Regulators

-----_.

Type of Culture

Species

Explant

C. scoparius

Seeds

Callus MS 2,4-0 Cell

Dolichos biflorus L.

Seedling tissues

Gentista pilosa L.

Leaves. stems. or flowers

Laburnum alpin urn (Mill.) Bercht. & J. Pres1.

Shoot tips

__ ..

Products

Comments

References

None

None specified

Khanna and Staba 1968

Callus MS 1st 2,4-0 + KIN. then no regulators

None

A lectin

Callus Modified MS Cell 1st 2,4-0 + KIN, then IAA. NAA, and KIN

None

Tetrahydrorhombifoline, lupanine

Extracts from agar medium. liquid medium. and tissue were inhibitory to Escherichia coli and Staphyloccus aureus Leaf and stem but not seed lectin was detected with no regulators These were new alkaloids for this species. Leaves had more alkaloids than cells from culture These cultures do not accumulate cytisine or Nmethycytisine

Cell

1st 2,4-D + KIN. then 2,4-0

Substrates

Cytisine

SAM: cytisine Nmethyltransferase, lupanine, N-methylcytisine

James et al. 1985

Wink et a1. 1983

Wink 1984

Table 6.7. Continued.

c..;l

~

co

Type of Culture


Species

Explant

L. alpinum

Leaves, stems, or flowers

Sophora Bngustifolia

Seedling

S. angustifolia

Seedling

Callus WM 2,4-D KIN

Tephrosia vogelii Hook. f.

Not given

Callus MS 2,4-D Cell

Modified MS 1st 2,4-D + KIN, then IAA, NAA. and KIN Callus MS 2,4-D + KIN

+

Substrates

Products

Comments

References

None

Lupanine

Cell suspensions contained fewer alkaloids than leaves First report of esterification of exogenous steroids by plant tissues Callus tissue subcultured for three years Rotenoid content was much higher in suspension than callus cultures

Wink et a1. 1983

Progesterone 5-pregnanolone, Pregnenolone pregnenolone palmitate. and 5pregnanolone palmitate None 1-maachiain, 1pterocarpin None

Deguelin, elliptone, rotenone, tephrosium

Furuya et a1. 1971

Furuya and Ikuta 1968 Sharma and Khanna 1975

320


J. E. PREECE

line than in the original mixed callus; the gray callus had a lower anthraquinone content than the mixed callus. Although the brown callus contained elevated levels of anthraquinones, none were released from the tissue into the agar-solidified medium (Rai 1988). The callus contained nine anthraquinone compounds (chrysophanol, emodin, rhein, chrysophanol anthrone, chrysophanol dianthrone, emodin anthrone, rhein anthrone, chrysophanol monoglucoside, and rhein monoglucoside). The free and glycosidic forms of rhein and chrysophanol were 65% of the total anthraquinones in the callus (Rai 1988). The diacetate form of rhein has a therapeutic use as an antirheumatic (Windholz et al. 1983). Therefore, the selected brown callus may have potential economic uses. The phase of cell growth in suspension cultures also influences the yield of anthraquinones. During the early exponential phase (15 days) of CassVJ didymobotrya cells in suspension culture, anthraquinones were the predominant secondary metabolites (Botta et al. 1989). Later, during the early stationary phase (28 days), there was a second maximum accumulation of secondary metabolite in the cells. However, at this later phase flavonoids were the principal secondary metabolites detected. Callus cells similarly produced anthraquinones during early phases of growth (up to 15 days), howeverflavonoids were not detected in the callus but were first observed in suspension cultures that were 10 days old (the late lag phase). Therefore, the type of culture and its age and relative growth phase have profound effects on the amount and quality of secondary metabolites.

c. Rotenoids Rotenone is a naturally occurring selective insecticide and piscicide that does not persist in the environment and causes a minimum amount of injury to pollinating insects (Delfel 1973). Other rotenoids, e.g. delguelin and tephrosin, also are toxic to insects (Windholz et al. 1983). The rotenoid content in 4-week-old suspension cultures of Tephrosia vogelii was as high as 2.8% (Sharma and Khanna 1975). Rotenoids were primarily sequestered in cells and little was present in either spent liquid or agar-solidified medium on which the callus was grown. Similarly, cells of Derris elliptic a in suspension culture contained rotenoids, whereas the spent medium did not (Kodama et al. 1980). Callus of D. elliptica that was 14 months old contained only a minute amount of rotenoids, which decreased with further subcultures to undetectable levels. When adventitious organs (described as "root-like") were induced from Derris stem and leaf explants on medium supplemented with IAA, the new organs contained rotenoids, whereas the accompanying callus had

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almost none (Kodama et al. 1980). These root-like organs synthesized secondary metabolites at a level equivalent to intact plants. Additionally the root-like organs grew in culture better than roots. Thus, there may be some advantages to using organ culture, instead of callus or cell cultures, for the production of secondary metabolites.

D. Steroids Cell suspension cultures of a woody legume, Sophora angu stifolia, have been used to biotransform steroids (Furuya et a1. 1971). The cells esterified both progesterone and pregnenolone to 5a-pregnanolone palmitate as well as other compounds. This was the first report of esterification of exogenous steroids by plant tissues. Thus, plant tissues can be utilized for their ability to transform exogenous compounds, such as steroids, as well as their endogenous secondary metabolites.

E. Antimicrobial Activity Agar discs, extracts from agar and liquid medium, and callus tissue of Cytisus scoparius from old cultures were inhibitory to Escherichia coli and/or Staphylococcus aureus (Khanna and Staba 1968). Tissue or cell cultures of woody legumes, such as C. scoparius, can potentially beused to produce secondary metabolites with antimicrobial activity. Such compounds might have therapeutic applications.

F. Other Secondary Products Other secondary products have been identified in cell and callus cultures or woody legumes. For example, cell cultures of Acacia senegal have the potential to produce gum arabic, an extracellular polysaccharide (Hustache et a1. 1986). Secondary products generated in cell cultures may be useful in studies of taxonomiic relationships, medicinal compounds, plant metabolism, and interactions of cells with the environment. Callus tissues of Sophora angustifolia that were transferred at 6-week intervals for 3 years were extracted with chloroform (Furuya and Ikuta 1968). Following a series of recrystallizations, l-maackiain and 1pterocarpin were identified as secondary products. Furuya and Ikuta (1968) suggested that the presence of 1-maackiain and l-pterocarpin in the plant tissue might be useful for chemotaxonomic studies. Lectin, another secondary product, has been used in cancer research (Windholz et a1. 1983). Leaf and stem lectins were detected in callus that had been initiated from epicotyl, hypocotyl, root, and leaf explants from

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young seedlings of Dolichos biflorus. However, seed lectin was not detected in the callus (James et al. 1985). When exogenous plant growth regulators were in the medium, the level of lectin was minimal and increased when tissues were transferred to medium without exogenous plant growth regulators.

V. IN VITRO STUDIES OF NITROGEN FIXATION Many legumes are characterized by their nitrogen fixing ability, which is mediated through a mutuaIistic association with either Rhizobium or Bradyrhizobium species). The efficiency of infectivity (as measured by the number of nodules formed) and nitrogen fixing ability are often dependent on the interaction of a specific strain of bacteria with the host (e.g. GaIiana et al. 1990; Horvath et al. 1987; Trinick 1968J. Many woody legumes also are capable of fixing nitrogen and over 90% of these species have a tropical origin. They are potentially important components of tropical agroforestry systems and can provide fuelwood, charcoal, pulpwood, and high protein fodder to supplement grass forages; and green manure is provided for improvement of soil nutritional status (Brewbaker 1987). Tissue culture methods have provided opportunities to study the legume-Rhizobium association. These methods furnish controlled environments that permit investigation of the interactions leading to symbiosis as well as physiological, molecular, biochemical, and ultrastructural aspects of the relationship. The in vitro association of Rhizobium with many forage and seed legume cultures was primarily characterized in the 1970s (Gresshoff and Mohapatra 1981), but there are relatively few reports concerning in vitro investigations of woody legume-bacteria interactions. These studies have been restricted to analyses of infection of callus by Rhizobium and in vitro nodulation of aseptically germinated seeds and micropropagated plants. Rango Rao and Subba Rao (1976) successfully established an in vitro symbiotic relationship between Cajanus cajan and Rhizobium by coincubating the bacterium isolated from roots of the host plant with root callus. The infection threads that are commonly associated with in situ establishment of Rhizobium on C. cajan were limited to the peripheral areas of the callus masses and were not well organized or continuous. Meristematic areas within the callus were often associated with infection threads and these cells, which contained numerous bacteriods, were larger than those not associated with bacteria. Acetylene reduction, a measure of nitrogenase activity, can be demonstrated in isolated infected callus masses.

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The interaction of Rhizobium with callus cultures isolated from nodules of Sesbania rostrata, which forms nodules on both roots and stems (Dreyfus and Dommergues 1980), has also been investigated. Duhoux and Alazard (1983) reported that bacteria multiplied in intercellular spaces and "pseudo infection threads" similar to those reported for Cajanus-Rhizobium co-culture were also formed. Infection was accompanied by development of fibrillar material that is also present in the intact plant relationship. Secretion of filamentous pecticaceous materials by Glycine max (soybean) callus cells in response to Rhizobium infection is also been reported and is thought to bind the bacteria to the host cell wall (Reporter et a1. 1975). However, with the SesbaniaRhizobium interaction, infection of individual cells and bacteriod development within cells were rare. Acetylene reduction from infected callus was diminished compared to nodules from intact plants (Duhoux and Alaz~rd 1983). Nitrogen-fixing woody legumes are capable of growth without nodule development provided that adequate nitrogen is present in the soil (Chaturvedi 1983). However, if low-input agroforestry and/or plantation systems are to be developed and maintained, it would be advantageous if these legumes formed nodules and fixed nitrogen. Ideally, the soil would contain sufficient Rhizobium inoculum to establish nodulation, but sites either deficient in Rhizobium or lacking compatible strains of the bacterium have been described for Leucaena leucocephala (Dhawan and Bhojwani 1987a;b). Bacteria may be incorporated into the soiL added to seed, or in the case of micropropagated plants, introduced during the acclimatization or hardening phase of propagation. Addition of a highly efficient, compatible strain of Rhizobium at this time could ensure nodulation and potentially enhance the survival rates of plants located on marginal sites or in soil lacking Rhizobium (Dhawan 1988; Dhawan and Bhojwani 1987a;b). In a study with L. leucocephala, rooted microshoots were transferred aseptically to sterile quartz sand and inoculated with Rhizobium strain NGR 8 (Dhawan and Bhojwani 1987b). Nodule formation was observed in about 80% of the plantlets after 5 weeks of incubation. However, aseptically grown seedlings inoculated with bacteria nodulated after only 2 weeks under similar conditions. Reduced photosynthetic ability and infection sites (lateral roots) were suggested as reasons for the apparent lag in nodule formation on micropropagated plants. Nodulated plants were established in soil, but the authors did not provide any information about the performance of the trees under field conditions.

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VI. CONCLUDING REMARKS

Woody legume species represe nt a highly divergent taxonomic grouping of plants that occupy various niches in ecosystems throug hout the world. These plants are especially import ant in tropical and semiar id environments producing food, proven der for wildlife and domesticated animals, and wood for construction, pulp, and fuel. These multipurpose plants positively influence soil nitrogen status, a factor that often limits agricultural endeav ors in tropical areas. Woody legumes also produc ea wide variety of useful second ary metabolites. In vitro techniques are being developed to facilitate production and extract ion of these important compounds. Although there has been little investment in the development of superio r or elite lines for many of the import ant woody legume species , progress has been made during the last decade developing in vitro propagation techniques. Axillary bud multiplication and somati c embryogenesis methodologies probably offer the best opport unity for clonal propag ation, although information regarding the latter techniq ue is scanty. The recalci trant nature of mature specimens to in vitro culture techniques is a primar y obstacle to clonal propag ation of selected trees. Application of existing methods to restore juvenility to superio r specimen trees or development of alternative means that permit initiation of proliferating cultures from explan ts from mature individuals are needed before any significant practic al application of micropropaga ted trees for improvement of agriculture and forestry can be made. Lack of suitable and reliable regeneration techniques for many woody legume s also impedes incorporation of the use of protop lasts and gene transfe r technologies for tree improvement. Heightened awaren ess of the potential import ance of woody legumes (especially in tropical ecosystems), concern for the rapid deforestation of large tracts of rainforest, and decimation of trees for fuelwood in semiarid climates, should continu e to provide suffici ent incentives for researc h and development of in vitro propag ation techniques for woody legume species.

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Khattar, S., and H. Y. Mohan Ram. 1983. Organogenesis and plantlet formation in vitro in Sesbania grandiflora (L.) Pers. Indian J. Expt. BioI. 21:251-253. Khattar, S., and H. Y. Mohan Ram. 1982. Organogenesis in the cultured tissues of Sesbania sesban, a leguminous shrub. Indian J. Expt. BioI. 20:216-219. Kodama, T., T. Yamakawa, and Y. Minoda. 1980. Rotenoid biosynthesis by tissue culture of Derris e11iptica. Agr. BioI. Chern. 44:2387-2390. Kumar, AS., T. P. Reddy, and G. M. Reddy. 1985. Genetic analysis of certain in vitro and in vivo parameters in pigeonpea (Cajanus cajan L.). Theor. Appl. Genet. 70:151-156. _ _ . 1983. Plantlet regeneration from different callus cultures of pigeonpea (Cajanus cajan L.). Plant Sci. Lett. 32:271-278. Kumar, A, P. Tandon, and A. Sharma. 1991. Morphogenetic responses of cultured cells of cambial origin of a mature tree-Dalbergia sissoo Roxb. Plant Cell Rpt. 9:703-706. Lakshmana Rao, P. V., and D. N. De. 1987. Tissue culture propagation of tree legume Albizia lebbeck (L.) Benth. Plant Sci. 51:263-267. Lakshmi Sita, G., S. Chattopadhyay, and D. H. Tejavathi. 1986. Plant regeneration from shoot callus of rosewood (Dalbergia latifolia Roxb.). Plant Cell Rpt. 5:266-268. Larkin, P. J. and W. R. Scowcroft. 1981. Somaclonal variation-a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60:197-214. Lazzeri, P. A., D. F. Hildebrand, and G. B. Collins. 1987. Soybean somatic embryogenesis: Effects of hormones and culture manipulations. Plant Cell Tissue Organ Cult. 10:197208. Lloyd, G., and B. McCown. 1980. Commercially feasible micro-propagation of mountain laurel, Kalmia latifolia, by use of shoot tip culture. Comb. Proc. IntI. Plant Prop. Soc. 30:421-427. Martins-Loucao, M. A. and C. Rodriguez-Barrueco. 1981. Establishment of proliferating callus from roots, cotyledons and hypocotyls of carob (Ceratonia siliqua L.) seedlings. Z. Pflanzenphysiol. 103:297-303. Mascarenhas, A F., S. Nair, V. M. Kulkarni, D. C. Agrawal, S. S. Khuspe, and U. J. Metha. 1987. Tamarind. p. 316-325. In: J. M. Bonga, and D. J. Durzan (eds.), Cell and tissue cuIture in forestry. Vol. 3:. Case histories: gymnosperms, angiosperms, and palms. Marinus Nijhoff, Dordrecht. Mathur, I., and N. Chandra. 1983. Induced regeneration in stem explants of Acacia nilotica. Current Sci. 52:882-883. McCown, D. D., and P. A. Barker. 1989. Micropropagation of a Kentucky coffeetree (Gymnoc1adus dioicus) clone for urban use. J. Arbor. 15:255. Mehta, U., and H. Y. Mohan Ram. 1980. Regeneration of plantlets from the cotyledons of Cajanus cajan. Indian I. Expt. BioI. 18:800-802. Merkle, S. A., H. D. Wilde, A. T. Groover, D. T. Carraway, and B. A. Watson-Pauley. 1991. Embryogenic suspension cultures for genetic transformation and mass propagation of hardwood forest trees. IntI. Symp. on Appl. of Biotechnol. to Tree Cult., Protection and Utilization. USDA Forest Service, Northeastern Forest Ext. Stat. Gen. Tech. Rpt. NE-152. p. 116. Merkle, S. A., and A. T. Wiecko. 1989. Regeneration of Robinia pseudoacacia via somatic embryogenesis. Can. J. For. Res. 19:285-288. Meyer, H. I., and J. van Staden. 1987. Regeneration of Acacia melanoxylon plantlets in vitro. S. Afr. Tydskr. Plantk. 53:206-209. Mittal, A., R. Agarwal, and S. C. Gupta. 1989. In vitro development of plantlets from axillary buds of Acacia auriculiformis-a leguminous tree. Plant Cell Tissue Organ Cult. 19:65-70. Mohan Ram, H. Y., U. Mehta, I. V. R. Rao, and M. Narasimham.1982. Haploid induction in legumes. p. 541-543. In: A. Fujiwara (ed.), Plant Tissue Culture 1982. Ipn. Assoc.

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Plant Tissue Cult., Tokyo. Mukhopadhyay, A., and H. Y. Mohan Ram. 1981. Regeneration of plantlets from excised roots of Dalbergia sissoo. Indian I. Expt. BioI. 19:1113-1115. Mullins, M. G. 1985. Regulation of adventitious root formation in microcuttings. Acta Hort. 166:53~1. Murashige. T. 1974. Plant propagation through tissue culture. Annu. Rev. Plant Physiol. 25:135-166. Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497. Nagmani, R., and S. Venketeswaran. 1987. Plantlet regeneration in callus cultures of Leucaena. p. 285-291. In: I. M. Bonga and D. I. Durzan (eds.). Cell and Tissue Culture In Forestry. Vol. 3. Case histories: gymnosperms, angiosperms and palms. Martinus Nijhoff, Dordrecht. NAS. 1979. Tropical legumes: resources for the future. National Academy of Sciences, Washington. D.C. Nataraja, K., and A. M. Sudhadevi. 1984. In vitro induction of plants from seedling explants of subabul (Leucaena leucocephala Lamk.). Indian I. Plant Physiol. 27:255258. Pan. F. I., and S. H. Chang. 1987. In-vitro culture of Leucaena diversifolia. Bul. Taiwan For. Res. Inst. New. Ser. 2:217-236. Polhill, R. M., P. H. Raven and C. H. Stirton. 1981. Evolution and systematics of the leguminosae. p. 1-26. In: Advances in Legume Systematics. Royal Botanic Garden, Kew, England. Phukan, M. K.• and G. C. Mitra. 1983. In vitro regeneration of Albzzia odoratisima Benth., a shade tree for the tea plantation of North-east India. Two and a Bud 30:54-58. Rai. P. P. 1988. Anthraquinone formation callus cultures of Cassia podocarpa. I. Nat. Prod. 51:492-495. Rai, P. P., and M. Shok. 1982. Anthracene derivatives in tissue cultures of Cassia species indigenous to Nigeria, p. 277~278. In: Fujiwara.A. (ed.), Plant Tissue Culture 1982. Proc. 5th IntI. Congo Plant Tissue & Cell Culture. Ipn. Assn. Plant Tissue Cult. Ranga Rao. G. V. and M. N. V. Prasad. 1991. Plantlet regeneration from the hypocotyl callus of Acacia auriculiformis-multipurpose tree legume. I. Plant Physiol. 137:625627. Ranga Rao, V., and N. S. Subba Rao. 1976. Studies on the interaction of legume root callus with Rhizobium. Z. Pflanzenphysiol. 80:14-20. Rao. K. S. 1986. Plantlets from somatic callus tissue of the east indian rosewood (Dalbergia latifolia Roxb.). Plant Cell Rpt. 5:199-201. Ravishankar Rai, V., and K. S. Iagadish Chandra. 1989. Micro-propagation of indian rosewood by tissue culture. Annu. Bot. 64:43-46. _ _ _ . 1988. In vitro regeneration of plantlets from shoot callus of mature trees of Dalbergia latifolia. Plant Cell Tissue Organ Cult. 13:77-83. Reporter, M .• D. Rareed. and G. Norris. 1975. Binding of Rhizobium japonicum to cultured soybean root cells: morphological evidence. Plant Sci. Lett. 5:73-76. Robinson. T. 1983. The organic constituents of higher plants. Cordus Press, North Amherst. MA. Rogozinska. J. H. 1968. The influence of growth substances on the organogenesis of honey locust shoots. Acta Soc. Bot. Pol. 37:485-491. Roy, S. K. and S. K. Datta. 1985. Clonal propagation of a legume tree Albizia procera through tissue culture. Bangladesh J. Bot. 14:127-131. Schenk, R. U. and A. C. Hildebrandt. 1972. Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot.

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Trinick, M. J. 1968. Nodulation of tropical legumes. I. specificity in the Rhizobium symbiosis of Leucaena leucocephala. Expt. Agr. 4:243-253. Umboh, I., 1. Setiawan, H. KamiI., S. Yani, and J. Situmorang. 1989. L'appIication de techniques de culture in vitro a la multiplication d'especes forestieres tropicales en Indonesie. Bul. Soc. Bot. Fr. 136:179-184. Upadhyaya, S., and N. Chandra. 1983. Shoot and plantlet formation in organ and callus cultures of Albizzia lebbek Benth. Annu. Bot. 52:421-424. Wainwright, H., andN. England. 1987. Themicropropagation ofProsopisjulif1ora (Swartz) DC: establishment in vitro. Acta Hort. 212:49-53. Weaver, L. A., and R. N. Trigiano. 1991. Plant regeneration of Cladrastis lutea (Fabaceae) via somatic embryogenesis. Plant Cell Rpt. 10:183-186. Weaver, 1. A., and R. N. Trigiano. 1989. Tissue culture of yellowwood. Proc. Southern Nurserymen's Res. ConL 34:15-18. Whitaker, R. J., and T. Hashimoto. 1986. Production of secondary metabolites. p. 264-286. In: Evans, D. A., W. R. Sharp, and P. V. Ammirato (eds.), Handbook of Plant Cell Culture, Vol. 4. MacMillan, New York. White, P. R. 1963. A handbookofplanttissue culture. Jacques CottellPress, Lancaster, PA. Williams, R. R., A. M. Taji, and J. A. Bolton. 1985. Specificity and interaction among auxins, light, and pH in rooting of Australian woody species in vitro. HortScience 20:1052-1053. Windholz, M., S. Budavari, R. S. Blumetti, and E. S. Otterbein (eds.), 1983. The Merck Index 10th ed. Merck, Rathway, NJ. Wink, M. 1984. N-methylation of quinolizidine alkaloids: an S-adenosyl-L-methionine: cytisine N-methyltransferase from Laburnum anagyroides plants and cell cultures of L. alpin urn and Cytisus canariensis. Planta 161:339-344. Wink, M., L. Witte, T. Hartmann, C. Theuring, and V. Volz. 1983. Accumulation of quinolizidine alkaloids in plants and cell suspension cultures: Genera Lupinus, Cytisus, Baptisia, Genista, Laburnum, and Sophora. Planta Medica. 48:253-257. Winton,1. 1. 1978. Morpohogenesis in cIonalpropagationofwoodyplants. p. 419-426. In: T. A. Thorpe (ed.), Frontiers of plant tissue culture. Univ. Calgary, Calgary, Canada. Woods, A. 1985. The potential for the in vitro propagation of a number of economically important plants for arid areas. p. 333-342. In: G. E. Wickens, J. R. Gookin, and D. V. Field (eds.), Plants for arid lands: Proc. Kew IntI ConL on Econ. Plants for Arid Lands. Allen and Unwin, London. Yanxiu, Z., Y. Dunyi, and P. J. C. Harris. 1990. In vitro regeneration of plantlets from explants and callus of Acacia salicina. Nitrogen Fixing Tree Res. Rpt. 8:113-115. Yusnita, S., R. 1. Geneve, and S. T. Kester. 1990. Micropropagation of white flowering Eastern redbud (Cercis canadensis var. alba L.). J. Environ. Hort. 8:177-179. Zimmerman, R. H. 1986. Regeneration in woody ornamentals and fruit trees. p.243-258. In: I. K. Vasil (ed.), Cell Culture and Somatic Cell Genetics: Vol. 3. plant regeneration and genetic variability. Academic Press, New York.

7 Polyamines In Horticulturally Important Plants Miklos Faust and Shiow Y. Wang Fruit Laboratory, Beltsville Agricultural Research Center Agricultural Research Service Beltsville, MD 20705

I. II.

III.

IV.

V. VI.

Introduction Overview A. Types of polyamines B. Biosynthesis of polyamines Polyamines and Plant Development A. Embryogenesis B. Rapid Growth C. Flower Initia tion D. Pollen Formation E. Fruit Development F. Root Formation G. Internode Elongation Stress-Induced Changes in Polyamine Content A. Nutrient Stress B. Chilling Stress C. Other Stresses Polyamines and Senescence Conclusions Literature Cited

I. INTRODUCTION

Aliphatic polyamines are ubiquitous amines common in all cells including those in plants. Although polyamines are among the oldest organic compounds known to science, their role in metabolic activities of plant cells has been investigated widely only in the last decade. Polyamines promote growth of some tissues, stabilize membranes, minimize stress of various organs, and delay senescence of detached leaves. They are involved in cell division, embryogenesis, root formation, floral initiation and development, and fruit development and pollen formation (Evans and Malmberg 1989). Investigators used a wide range 333

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of agronomically important plants, convenient test plants, and occasionally plants of horticultural importance. This review intends to unite the knowledge on the occurrence of polyamines and their possible roles in horticulturally important plants. The biosynthesis and the role of polyamines in plants have been the subjects of several reviews and books (Galston and Kaur-Sawhney 1980, 1987a,b; 1990; Bagni et a1. 1982; Galston 1983; Slocum et a1. 1984; Galston and Smith 1985; Smith 1985, 1990; Bagni 1986; Bachrach and Heimer 1989; Evans and Malmberg 1989; Flores et a1. 1989; Flores et a1. 1990). These reviews serve as sources of information on polyamines not covered here.

II. OVERVIEW

A. Types of Polyamines Among the free polyamines there are three which have significance for this review. They are:

Diamine, putrescine H3 N+ - (CH21 - NHt Triamine, Spermidine H3 N+ - (CH21 - NHt - (CH2 1 - NHt H+3 N+ - (CH2 )3

Tetraamine, Spermine - NHt (CH21 - NHt - (CH21 - NH 3

There are other polyamines found in plants and algae (Smith, 1990; Kuehn et aI. 1990) but they are not discussed in this review. Polyamines may conjugate with other compounds. Putrescine was found to be conjugated with hydroxycinnamoy!, alkylcinnamoy!, caffeoy!' and feruloyl; spermidine with caffeoyl; and agmatine, a biosynthetic intermediate in putrescine biosynthesis, with cumaroyI. In some plants the concentration of conjugates may exceed the concentration of free amines (Evans and Malmberg 1989). At cellular pH, polyamines are in most cases [but not always) ionized and associate with anionic macromolecules, such as DNA, RNA, phospholipids, or certain proteins. Polyamines are also bound to ribosomes (Galston and Kaur-Sawhney 1990). They may stabilize the double helix structure of DNA or interact with anionic P residues on membranes. Polyamines may be bound to proteins. Examples are Helianthus tuberosus (Serafini-Fracassini et aI. 1988, 1989) and petunia (Mizrahi

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335

et a1. 1989) among horticulturally important plants. In apple fruit, conjugated spermine not soluble in TCA is greater than free spermine for at least six weeks after bloom, whereas conjugated putrescine and spermidine decrease rapidly (Biasi et a1. 1988). Considerable quantities of bound polyamines were found in Solanum tuberosum but not in Petroselinum hortense, Helianthus annuus or Cyperus rotundus (Felix and Harr 1987). Changes in free polyamines also occur in post pollination placentae and ovules of potato (Olson and Nowak 1988). Variation of polyamine content in different plant species and in various parts within a plant can be considerable. Felix and Harr (1987) list polyamine content of various parts of the seed and seedlings of 30 species with concentrations ranging from trace to 2.5 p.mol (g FWr1 • In general, rapidly dividing tissues contain high concentrations of polyamines. This was the case in growing buds and leaves of Phaseolus vulqaris (Bagni 1970; Palavan and Galston 1982;), in tomato ovary after pollination (Cohen et a1. 1982), when potato buds begin to sprout (Kaur-Sawhney et a1. 1982), when apple buds resume growth after dormancy (Wang et a1. 1985), and in rapidly proliferating callus tissues (Montague et a1. 1978, 1979; Bagni and Serafini-Fracassini 1979; Heimer et a1. 1979). In some cases, when it was examined, the extracellularly administered polyamines were compartmentalized. Putrescine was absorbed into cytoplasmic fraction of carrot cells, whereas spermine was present in the cell wall (Pistocchi et a1. 1987). In Santpaulia petals, polyamines are largely taken up into the vacuole (Pistocchi et a1. 1987). In carrot cell cultures 68.9% of putrescine was in the cytoplasm, whereas 73% of spermidine and 77.5% of spermine were bound to the cell wall (Bagni and Pistocchi 1990).

B. Biosynthesis of Polyamines A schematic illustration of polyamine biosynthesis is presented in Figure 7.1. Putrescine may be formed directly from ornithine by ornithine decarboxylase (ODC), or indirectly, through a series of intermediates, including agmatine (Agm), from arginine by arginine decarboxylase (ADC). The respective functions of the two pathways of putrescine biosynthesis, via ODC and ADC are not clear (Smith 1990). Which route the plant uses to produce putrescine may depend on the species or the conditions. In Helianthus tuberosus, putrescine is synthetized through ODC (D'Orazi and Bagni 1987) although in cotyledons of Cucumis sativus it is synthesized through ADC (Suresh et a1. 1978). The difference may lie in the biosynthesis of ornithine and arginine and the compartmentalization involved in handling these compounds (Shargool et a1. 1988). In general, when cell division is affected, changes are usually noted in ODC

336

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<E0.1% ovule set). He felt there was sufficient variability in the backcross generations for progress to be made. From investigating backcrosses of V. vinifera X V. rotundifolia hybrids to V. vinifera Jelenkovic and Olmo (1969a) found seedlings that ranged from completely sterile to as fertile as standard V. vinifera cultivars. They also found segregation for fruit quality, flavor, bark type, tendrils, diaphragm, flower cluster size, and leaf shape. Wood type of all the seedlings was characteristic of V. rotundifolia. They observed that F1 hybrids could be used as males or females when crossing to V. vinifera, but only as females when crossing to V. rotundifolia. Nesbitt (1962) and Jelenkovic and Olmo (1969b) investigated polyploid

388

R.G.GOLDY

hybrids between Euvitis and V. rotundifolia. Nesbitt investigated allotriploids and autoallohexaploids while Jelenkovic and Olmo studied colchicine-produced allotetraploids. Nesbitt theorized allotetraploidy may be the most promising means of combining the gene pools, but when studied by Jelenkovic and Olmo the doubled F1 hybrids had low, erratic fertility. Jelenkovic and Olmo found that what held true at the diploid level was generally true at the tetraploid: V. rotundifolia characters were primarily dominant; the two species would only hybridize when V. vinifera was used as the female; and tetraploid hybrids are crossable among themselves, only as females to V. rotundifolia, and as females and males to V. vinifera. Olmo (1971) investigated use of V. vinifera X V. rotundifolia hybrids backcrossed two generations to V. vinifera wine grapes and found some which produced acceptable wine. Many vines from this second backcross generation still had low fertility but enough variation existed that fertile types could be selected. In this generation fruit could be handled and processed like V. vinifera fruit and some had the berry shedding characteristic of V. rotundifolia, a trait which could aid mechanical harvest. Many studies have been aimed at trying to move desirable traits from Muscadinia into Euvitis; however, there are traits in Euvitis,which could improve Muscadinia. These include the possibility of improving pigment quality of Muscadinia-types by introgressing the high-quality pigments from V. vinifera (Goldy et a1. 1986), or attempting to move seedlessness from V. vinifera into Muscadinia (Goldy et a1. 1988). Both areas are discussed in Sections IV.C.3 and 4. Of all the work that has gone into producing and evaluating V. vinifera x V. rot,undifolia hybrids, little has been of value to the commercial industry. To date, only two clones have been released as rootstocks: 'VR 039-16', a cross between 'Almeria' (V. vinifera) and V. rotundifolia male No.2 (Lider et a1. 1988a): and VR 043-43, a cross between 'Hunisa' (V. vinifera) and V. rotundifolia male No.2 (Lider et a1. 1988b). Both clones showed resistance to dagger nematode (Walker et a1. 1989). Since grape fanleaf virus is vectored by these nematodes, it was thought that scions on these rootstocks would not suffer degeneration due to fanleaf. Since these plants were released they have acquired fanleaf (Walker et a1. 1989). Bouquet (1981) showed muscadines were not immune to fanleaf, so these plants either escaped field inoculation, or their resistance to dagger nematode has been overcome. B. Cytogenetics Cytogenetic studies of Euvitis x V. rotundifolia hybrids were initiated to determine reasons for the difficulty in obtaining hybrids and hybrid

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389

sterility. Sax (1929) reported members of Euvitis had 38 somatic chromosomes and V. rotundifolia had 40. The n number of chromosomes for Euvitis and Muscadinia is therefore 19 and 20, respectively. This is high for a diploid species; many cytotaxonomists believe any plant having n numbers greater than 11 are of polyploid ancestry (Goldblatt 1980). Sax (1929) felt chromosome difference alone could not account for the high degree of sterility observed, and states: "A cytological study of these F1 hybrids should be of considerable interest." The first cytogenetic study of these hybrids was by Patel and Olmo (1955) who found F1 hybrids had a chromosome number of 2n = 39 and that functional gametes were n =20 ± 1, with 20 the most frequent. When studying meiotic chromosome pairing of 3 hybrids they found 13 bivalents (range 6-18); 10 univalents (3-15); and 2 quadrivalents (0-4), leading them to propose a genomic constitution of 13RrRr + 7AA for V. rotundifolia, 13R R + 6BB for V. vinifera, and 13RrRv + 6A + 7B for the F1 hybrid. The R chromosomes are homologous between V. vinifera and V. rotundifolia and the A and B represent two genomes from different, unknown ancestral species. They also state sterility is not only due to chromosome differences but mainly to abnormal pairing and irregular distribution. Meiotic irregularities, such as lagging chromosomes, bridge-fragment formation, and chromosome elimination were common in hybrids, indicating presence of structural dissimilarities like inversions and translocations in the common genome. Nesbitt (1966) also observed a lack of synchrony of meiotic stages in anthers. Jelenkovic and Olmo (1968) reported that the fertility of the population of F1 seedlings they studied was directly correlated to chromosomal pairing in meiosis 1. Patel and Olmo (1955) concluded that 13 chromosomes from V. vinifera were homologous enough to pair to 13 from V. rotundifolia, and the 7A and 6B chromosomes were left on their own to form univalents. This indicated Vitis species are secondary polyploids with an ancient basic chromosome number of probably 6 and 7, and Euvitis is (6 + 7) + 6 =19, and Muscadinia is (6 + 7) + 7 = 20. This makes both subgenera ancient hexaploids that have undergone diploidization to produce normal bivalent pairing. When F1 hybrids were backcrossed to V. rotundifolia Patel and Olmo (1955) found that in 20 seedlings, 18 had chromosome numbers of 2n = 40, one was 2n = 39, and one was 2n = 41. They also found Fz and F3 hybrids were weak due to a high degree of genetic imbalance. In a larger study on backcross generations to V. vinifera, Jelenkovic and Olmo (1969a) found variation in fertility directly related to chromosomal pairing: higher fertility was observed in progeny having a low frequency of univalents and high sterility in progeny with a low frequency of bivalents. In backcrosses they observed frequent occurrences of multivalents, and bridges V

V

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and laggards were observed in seedlings having a high degree of sterility. Sterility problems associated with F1 hybrids and backcross generations greatly restricts interchange of genetic material between the two subgenera. Fertility could be enhanced using the proper parents, and it could gradually be restored through backcrossing, but this was a slow process made even slower with the juvenility expressed in grapes. It was thought sterility problems in hybrids, such as Euvitis x Muscadinia had been solved when it was discovered that fertility could be restored by doubling the chromosome number of the hybrids or parents prior to hybridization with colchicine (Dermen 1954). Dermen (1954) was the first to show V vinifera and V. rotundifolia responded to chromosome doubling using colchicine, and indicated future work would include attempts at doubling F1 hybrids. The colchiploid V. rotundifolia plants generally had larger fruit, earlier maturity, thicker foliage and canes, slower growth, thick roots, and some were quite susceptible to leaf spotting. They also proved to be more susceptible to winter injury than diploid vines (Loomis and Fry 1965). Patel and Olmo (1956) were the first to report on actually doubling the F1 hybrid. They accomplished this by treating seedlings at the cotyledon stage with a 0.25% aqueous solution of colchicine for 1-4 days. Of the 208 seedlings treated they identified 22 polyploids. Identification was based on stomate size and since this measures only the L1 layer, it is unclear whether a1122 plants obtained were solid or only chimeral polyploids. A later report by Jelenkovic and Olmo (1969b) used five of these plants to study meiosis in allotetraploids, so at least five also had a polyploid LIl layer. Dermen (1958; Dermen and Scott 1962) treated buds on an interspecific F1 plant (NC 6-16) with colchicine and produced one branch with a doubled chromosome number (2n = 4x = 78) in all tissues except the epidermis. The year after treatment, one flower cluster appeared on the tetraploid branch and developed into a cluster containing 33 berries, whereas, only two berries developed on a diploid branch. More flower clusters developed in subsequent years on the tetraploid branch, with several producing fruit; none developed on diploid branches. From 33 berries, 48 seeds were obtained, resulting in 42 seedlings; all proved to be tetraploid and were indistinguishable from each other. Dermen's statement that the original tetraploid plants were "fully fertile ," was questioned by Jelenkovic and Olmo (1969b) since only a few seedlings were obtained when the colchiploids were used in crosses (Dermen 1964). Tetraploid seedlings investigated by Dermen (1964) had enough variation to indicate that segregation for V. vinifera and V. rotundifolia traits was taking place. Dermen (1958) attempted hundreds of 4x-4x crosses using 4x female

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muscadines and twice using 4x Euvitis as the female. No cross produced fruit, indicating that sterility barriers are increased with tetraploids. In a later study (Dermen 1964) 4x crosses failed when similar 2x crosses set fruit. Despite backcross success at the diploid level by Dunsten (1962b), Dermen (1964) felt the future in Euvitis X Muscadinia was breeding at the tetraploid level, a statement supported by Nesbitt (1962). However, the repeated failures Dermen experienced in trying to cross 4x V. vinifera with 4 x V. rotundifolia indicates doubling chromosome number should be done in the F1 and not the parental generation. Doubled F1s should not be evaluated for cultivar potential, but treated as a base population to begin breeding. No auto- or allotetraploid muscadine has proven successful in commercial cultivation. Dermen et a1. (1970) obtained 12 tetraploid plants directly from a diploid cross of 'Red Malaga' (V. vinifera) with V. rotundifolia. Five were fertile and seven sterile. One fertile plant had 2n =70, the others had 2n = 78. Pollen from the 2n = 78 plants were uniform in size and stained over 90% with acetocarmine. The 2n = 70 plant lacked pollen in some flowers but in others it was uniform and 50% stained. Seedlings from the 2n = 78 plants had 2n numbers of 39,66, and 78. The 39 chromosome seedlings were thought to be parthenogenic and the 66-chromosome seedlings resulted from fusion of a 27-chromosome egg with a 39-chromosome sperm. The 70-chromosome plant was thought to result from fusion of a 30-chromosome egg with a 40-chromosome sperm. Jelenkovic and Olmo (1969b) report great variation in allotetraploid hybrids between Euvitis and Muscadinia, but low and erratic fertility. When used as females, berry set ranged from 0.0-16.0% and ovule set 0.0-4.0%. When used as males, berry set ranged from 0.0-26.5 and ovule set from 0.0-7.8%. There were year-to-year differences but no seedlings resulted from 6800 self-pollinations. Although pollen stained well in the hybrids (up to 85.9%), germination was poor (1.1% was the best). Intercrossing hybrids produced from 0.0-12.6% berry set, and 0.0-3.4% ovule set. Generally poor results were reported when they crossed hybrids with different ploidy levels and with different genotypes. They also got poor results when crossing autotetraploids between V. vinifera and V. rotundifolia, but were able to obtain berry set when V. vinifera was used as the female; something Dermen (1964) was not able to obtain.

Jelenkovic and Olmo (1969b) summarized crossability reactions between V. vinifera and V. rotundifolia and established three "essential facts": 1. Whenever the maternal diploid plant contains two chromosomal complements of V. rotundifolia, the generative nucleus of V. vinifera or VR hybrids (diploid Fl hybrid) fail to

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fertilize the V. rotundifolia egg. If the maternal diploid contains only one or a partial chromosomal complement of V. rotundifolia, fertilization succeeds with V. vinifera and VR pollen. 2. In allotetraploids at the ratio of 2:2 of V. vinifera-rotundifolia complements in the maternal plant, pollenofautotetraploid V. vinifera and VVRR hybrid s fertilize the egg. With 4 V" rotundifolia complements in the female no setting was obtained with tetraploid V. vinifera or VVRR hybrids. 3. In a maternal plant with 2:2 VR complements, pollen of diploid V. vinifera fertilizes the egg. Pollen ofVVR fertilizes diploid but not tetraploid V. rotundifolia. The cross RR X VVRR (diploid V. rotundifolia X tetraploid F1 hybrid) produced berry set but all seeds were found to be floaters and nonviable.

They also propose a hypothesis for success based on chromosomal ratio: "The hypothesis implies that success by which: (a) V. vinifera (V) pollen fertilizes eggs depends on the ratio of the chromosomes of the two species in the maternal parent, (b) V. rotundifolia (R) eggs can be fertilized with pollen of a plant containing chromosomes ofboth species, but depends on the ratio ofV:Rin the pollen parent."Thus, if the ratio ofV:Rin the maternal plant is lor more, then pollination with V. vinifera will be successful; if it is less than 1, V. vinifera pollination will probably fail. In the pollen parent, if the ratio is 1 or less, pollen will be functional on V. rotundifolia, but if it is greater than 1, pollination should fail. No test of this hypothesis could be found. Allotetraploids studied by Jelenkovic and Olmo (1969b) had the same crossability patterns as diploids. No relationship was found between chromosome pairing and fertility at the tetraploid level, in contrast to results in diploid backcross generations (Jelenkovic and Olmo 1969a). Lagging chromosomes were common in Anaphase I and hybrid sterility was chromosomal and genic. They concluded that breeding at the diploid level appears more efficient than at the tetraploid level unless the diploid F1 is completely sterile, a statement supported by Bouquet (1980b).

C. Specific Characteristics of Euvit1s Hybrids

X

Muscadinia

Although reasons for hybridizing Euvitis and Muscadinia were well intentioned, breeding progress for scion characteristics is yet to be realized, even after more than 100 years of attempts. Early researchers could not foresee how difficult the initial cross would be, nor could they foresee how or why hybrids would be so sterile. 1. Pest resistance. Original intent behind Euvitis/Muscadinia crosses was pest resistance. Discussion in this section will be limited to the above-ground portion of the plant and its problems; rootstocks are discussed in the next section.

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Although Muscadinia germplasm has been evaluated for response to several disease and insect pests (Table 8.3) relatively little evaluation of F1 hybrids has been carried out. Olmo (1986) discusses potential but presents little data on actual performance. He observed that hybrids did not show sYmptoms of Eutypa dieback or PD. An active program to transfer powdery mildew resistance was initiated (Rombough 1977) and V. vinifera-type vines homozygous for resistance have been isolated. Walker et a1. (1985) also report on an F1 hybrid that was resistant to grapevine fanleaf virus. If the measure of success is release of resistant cultivars containing V. rotundifolia genes, then not much success has been realized. Introgression of pest resistance into commercial cultivars has been slowed by sterility, something intolerable in scion cultivars. It also appears when sufficient v: vinifera traits are recovered in backcrosses to V. vinifera; many hybrids lose the desired resistance of V. rotundifolia suggesting selective chromosome elimination with little recombination. 2. Rootstocks. Using V. rotundifolia as a rootstock has been of interest for almost a century (Bouquet 1980a). Its use, however, has been limited due to its poor rooting characteristics and its graft incompatibility with many Euvitis. Graft compatibility can be improved using green grafts but results depend on the muscadine genotype (Bouquet and Hevin 1978; Bouquet 1980a). There is interest in using Euvitis-Muscadinia hybrids as rootstocks since muscadines are tolerant of many of the pests, which plague Euvitis (especially V. vinifera) and rootstocks do not depend directly on fruit quantity and quality. A successful rootstock does not have to be fertile, just easily propagated, graft compatible, and tolerant to root pests. Two hybrid rootstock cultivars have been released (Lider et a1. 1988a, 1988b). They can be propagated from dormant cuttings, are graft compatible to V. vinifera, and contribute to comparable fruit quantity and quality. Hybrids have been specifically evaluated for tolerance to phylloxera (Davidis and almo 1964; Firoozabady and almo 1982), root knot nematode (Bloodworth et a1. 1980; Firoozabady and almo 1982b), and dagger nematode (Lider et a1. 1988a, 1988b; Walker et a1. 1989). Tolerance and/or immunity can be found for all three pests in hybrid populations. Along with a high degree of tolerance or immunity, Davidis and Olmo (1964), found hybrids to root easier than V. rotundifolia, and formation of phylloxera lesions was related to how closely the anatomical structure of the root resembled V. rotundifolia. They also found that the contribution of V. rotundifolia to resistance was dominant, and in tetraploids one genetic complement was enough to produce considerable tolerance. Firoozabady and Olmo (1982a), however, did not find all seedlings

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equally tolerant, and they attribute this to the highly heterozygous background of the hybrids they studied. Olmo (1986) states that some phylloxera immune seedlings have fruit of V. vinifera quality, but further selection was needed to obtain commercially acceptable cultivars. Olmo (1986) reports several F1 hybrids were screened for resistance to dagger nematode, and although all plants did not produce good root systems some highly resistant types were identified. The two rootstock clones released by Lider et a1. (1988a, 1988b) are reported to be immune to dagger nematode. Bloodworth et a1. (1980) did not find a Muscadinia level of resistance to root knot nematodes in several Euvitis X Muscadinia progenies backcrossed to Euvitis. Declining nematode populations were observed in some complex F1 hybrids, which showed comparable resistance to Muscadinia when inoculated with three root knot species. Firoozabady and Olmo (1982b) found similar results and calculated a heritability estimate of 0.391 ± 0.06. Both reports indicate that efforts appear promising and that progress can be expected. 3. Processed quality. In wine grapes quality of the processed product is

of vital importance. The intent of producing V. vinifera X Muscadinia hybrids, and subsequent backcrosses to V. vinifera is to produce a grape having Muscadinia pest tolerance without affecting V. vinifera wine quality. Aside from sterility problems this is difficult due to the strong aroma and flavor found in Muscadinia, which is also present in progeny with V. vinifera, although diluted to varying degrees. Organoleptic qualities of Muscadinia, however, could benefit ~ vinifera-type grapes by broadening flavor and aroma characteristics. By the second backcross generation with several V. vinifera wine types, Olmo (1971) obtained clones that had adequate crop load and that produced acceptable wine. Fruit from these vines had acceptable sugar (significantly above Muscadinia), acid and pH levels, and received wine scores of between 0 and 6 on a 0-10 scale with 10 being best and 5 indicating good commercial quality. Fruit could be handled like V. vinifera since they did not have the thick skin and mucilaginous pulp of Muscadinia. Some hybrids retained the abscission properties of Muscadinia, which would aid mechanical harvest. A promising area is transfer of high quality pigments from Euvitis (especially V. vinifera) to V. rotundifolia. Pigmentation of V. rotundifolia is poor while V. vinifera is excellent. Goldy et a1. (1986) found 12 complex hybrids to contain monoglucoside pigments from Euvitis and diglucoside pigments from V. rotundifolia. Monoglucoside pigment ranged from 19.5-55.0% (% of total). They also varied in individual pigments: Mv, 2.344.3%; Pt, 0.7-30.3%; Pn, 0.8-69.2%; Cy, 0.0-17.2%; and Dp, 0.5-52.3%

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(% of total) and contained acylated pigments ranging from 0.0-51.1% (%

of total). They ranked seven hybrids and three V. vinifera for desirability of use in breeding for improved pigmentation and found three hybrids better than 'Petite Verdol', one better than 'Cabernet Sauvignon', but none better than 'Petite Sirah'. One of the hybrids they evalauted came from a cross that theoretically should have produced nonpigmented fruit. This suggests complimentation indicating the mutation for nonpigmentation in Euvitis may be different than the mutation in Muscadinia. This is possible since the two subgenera appear to have diverged early in their evolution. However, the possibility of a mistake in the pedigree and/or pollen contamination cannot be ruled out. Lamikanra's report (1989) is incorrect in its conclusions on pigmentation since the clones used were pure Euvitis. He was unaware of this at the time (0. Lamikanra, personal communication). 4. Fresh fruit quality. The only genetic study of table grape charac-

teristics of V. vinifera xV. rotundifolia hybrids is that of Firoozabady and Olmo (1987). From more than 1000 offspring from 46 families they determined narrow-sense heritabilities (h2) for cluster weight (h2 = 0.12), cluster compactness (h Z =0.55), berry weight (hZ =0.49), skin texture (hz = 0.75), pulp texture (h2 = 1.04), total soluble solids (hz =0.34), juice acidity (hZ = 0.15), general vigor (h Z = 0.10), and crop weight (h Z = -0.08). Heritability was estimated by regressing the average performance of each seedling on the average performance of its midparent. They also found a high correlation between crop weight and cluster weight (0.61 ± 0.04), skin tenderness and pulp firmness (0.42), and berry weight with cluster weight, skin tenderness, and pulp firmness (0.26,0.39 ± 0.06, and 0.49 ± 0.06, respectively). Total soluble solids were negatively correlated to berry weight (r = -0.37 ± 0.06), skin tenderness (-0.32 ± 0.06), pulp firmness (-0.21), and acidity (-0.21). Other correlations were found but at lower levels. They suggest that some of these traits are closely linked, which could enhance or retard progress depending on the direction of selection, and that a selection index approach may be useful for improvement. Firoozabady and Olmo did not evaluate seedlessness, presently the most important character for a successful table grape. Since no useable source exists in V. rotundifolia, transferring it from Euvitis seems to be the only route available. The necessity of using Euvitis as the seed parent hinders transfer of seedlessness to V. rotundifolia. Using standard breeding procedures, a seeded Euvitis female with seedless ancestry would be pollinated with V. rotundifolia and a series of progeny intercrosses and/or backcrosses undertaken to recover seedless V.

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rotundifolia types. Since all progeny would not have the genets) for seedlessness, progeny testing would be necessary. This is extremely inefficient due to long generation time and low fertility of F1 hybrids (Goldy et a1. 1988). Recently developed embryo rescue techniques for seedless grapes (Cain et a1. 1983, Emershad and Ramming 1984, Spiegel-Royet a1. 1985; Gray et a1. 1987; Emershad et a1. 1989, Ledbetter and Ramming 1989) provides a possible means of overcoming the need for progeny testing. If seedless V. vinifera grapes were pollinated with V. rotundifolia pollen, and the embryos rescued, any resulting plant should possess the genets) for seedlessness, although it might not be seedless itself since it had a seeded V. rotundifolia parent. In a study by Goldy et a1. (1988) of embryo rescue from seedless V. vinifera grapes pollinated with V. rotundifolia pollen, 19 hybrids were obtained from an estimated 16,000 pollinations, indicating this is a possible means of transferring seedlessness to V. rotundifolia. Another means proposed for developing seedless V. rotundifolia-types utilizes protoplast fusion (D. J. Gray, personal communication; Lee and Wetzstein 1988). This process would develop allotetraploids between a seedless Euvitis and a seeded V. rotundifolia. Further breeding at the tetraploid level would be necessary to recover seedless types. Given the difficulty others have found breeding muscadines at the tetraploid level, the success of this method is questionable but deserves further investigation. Some already existing hybrids between Euvitis and Muscadinia might contain seedless alleles. Therefore, their pedigree should be closely examined to identify them and use them in intercrosses, selecting seedless progeny.

5. Other traits. Williams (1923) found stem characteristics of F1 hybrids generally intermediate between the parents, but in some cases there was a greater resemblance to V. rotundifolia. Reports on inflorescence size of hybrids usually indicate a greater resemblance to the V. rotundifolia parent. In an inheritance study of inflorescence in Euvitis X Muscadinia hybrids, Dunstan (1967) describes an inflorescence type, which closely resembles Euvitis.

v. FUTURE PROSPECTS The muscadine industry is stable at present but its future is uncertain. There will always be a demand for muscadines, but whether it will

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remain steady, increase, or decrease is unknown. Historically muscadines have been used primarily in wine production, with some wineries producing award-winning, 100% muscadine wines. Recent interest is in pasteurized juice, and if it is marketed properly and on a large enough scale, it may increase demand. Wine and juice are primarily marketed in the southeastern United States where demand for the product is greatest. This is unfortunate since they should appeal to segments of the entire population, and should be of national, not just regional, interest. The future of muscadine breeding and research is even more uncertain. Active breeding programs are dwindling; the program in North Carolina was discontinued in 1990, causing North Carolina growers to rely on Florida and Georgia for new cultivars, which may not have the necessary winter hardiness. The Florida and Georgia breeding programs will no doubt be evaluated for their economic impact when those positions are vacated. Uncertainty of the muscadine industry and breeding/research could not come at a worse time. Muscadine breeders are on the verge of making tremendous improvement for the species. Much germplasm has been evaluated for its breeding potential for disease tolerance, pigment quantity and quality, flower type, and yield potential. It is only a matter of time to develop clones superior in these traits. Breeders are making progress in improving muscadines as table grapes by identifying clones with thin skin and firm pulp. If these can be combined with seedlessness, an almost completely new fruit will be developed. Currently those cultivars used in the juice industry are types that have been developed for wine. Breeding should be done to develop superior juice types to aid this segment of the industry. There is a great need for physiological research on muscadines, especially in the areas of fruit set, ripening, and dormancy. Investigating these traits in muscadines may help to better understand their mode of operation among plant species. This may be particularly true for chill units vs. heat units and how they relate to dormancy. Results from studies in these areas will assist breeders in knowing what can be accomplished in breeding, and the proper steps needed to accomplish it. Euvitis X Muscadinia crosses will continue to be a source of frustration, but positive results will be achieved if enough time and effort are devoted. More work needs to be done on the cytogenetics of these hybrids now that new clones and techniques have been developed. Electrophoretic and restriction fragment length polymorphism (RFLP) analysis may shed light on the evolution of the Vitis genus. Continued improvement in muscadines will require the combined efforts of growers, processors, marketing specialists, and public researchers.

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LITERATURE CITED Anonymous. 1990. The world viticultural situation in 1989. Bul. O.LV. 63:717-718. Antcliff, A. J. 1980. Inheritance of sex in Vitis. Annu. Amelior. Plantes 30:113-122. Armstrong, W. D., T. A. Pickett, and M. M. Murphy, Jr. 1934. Muscadine grapes: Varieties and some properties of juices. Ga. Agric. Expt. Stat Bul. 185. Bailey, L. H. 1934. The species of grapes peculiar to North America. Gentes Herb. 3:154244. Balerdi, C. F., and J. A Mortensen. 1969. Performance of muscadine grapes (Vitis rotundifolia Mich.) in central Florida. HortScience 4:252-253. ___ . 1974. Suitability for mechanical harvest in cultivars of muscadine grape (Vitis rotundifolia Michx.). Proc. Fla. State Hort. Soc. 86:342-344. Ballinger, W. E., E. P. Maness, W. B. Nesbitt, and D. E. Carroll, Jr. 1973. Anthocyanins of black grapes of 10 clones of Vitis rotundifolia, Michx. J. Food Sci. 38:909-910. Ballinger, W. E., E. P. Maness, W. B. Nesbitt, D. J. Makus, and.D. E. Carroll, Jr. 1974. A comparison of anthocyanins and wine color quality in black grapes of 39 clones of Vitis rotundifolia Michx. J. Am. Soc. Hort. Sci. 99:338-342. Ballinger, W. E., W. F. McClure, W. B. Nesbitt, and E. P. Maness. 1978. Light-sorting muscadine grapes (Vitis rotundifolia Michx.) for ripeness. J. Am. Soc. Hort. Sci. 103:629-634. Barritt, B. H., and J. Einset. 1969. The inheritance of three major fruit colors in grapes. J. Am. Soc. Hort. Sci. 94:87-89. Bloodworth, P. J., W. B. Nesbitt, and K. R. Barker. 1980. Resistance to root-knot nematodes in Euvitis X Muscadinia hybrids. Proc. Third Int. Symp. Grape Breeding. Univ. Calif., Davis. p. 275-292. Bouquet, A. 1980a. Differences observed in the graft compatibility between some cultivars of muscadine grape (Vitis rotundifolia Michx.) and European grape (Vitis vinifera L. Cabernet Sauvignon. Vitis 19:99-104. Bouquet, A 1980b. Vilis X Muscadinia hybridization: A new way in grape breeding for disease resistance in France. Proc. Third IntI. Symp. on Grape breeding. p. 42-61. _ _ . 1981. Resistance to grape fanleaf virus in muscadine grape inoculated with Xiphinema index. Plant Dis. 65:791-793. Bouquet, A, and M. Hevin. 1978. Green-grafting between muscadine grape (Vitis rotundifolia Michx.) and bunch grapes (Euvitis spp.) as a tool for physiological and pathological investigations. Vitis 17:134-138. Boyle, J. A, and L. Hsu. 1990. Identification and quantification of ellagic acid in muscadine grape juice. Am. J. Enol. Vitic. 41:43-47. Brooks, H. J., and D. W. Barton. 1983. Germplasm maintenance and preservation, p. 1120. In: J. N. Moore and J. Janick (eds.), Methods in Fruit Breeding. Purdue Univ. Press, West Lafayette, IN. Brooks, R. M., and H. P. Olmo. 1972. Register of new fruit and nut varieties: 2nd ed. Univ. of California Press, Berkeley. Brown, W. L. 1940. The anthocyanin pigment of the Hunt muscadine grape. J. Am. Chern. Soc. 62:2808-2810. Cain, D. W., R. L. Emershad, and R. E. Tarailo. 1983. In-ovulo embryo culture and seedling development of seeded and seedless grapes (Vilis vinifera L.). Vitis 22:9-14. Carroll, D. E. 1985. Muscadine grapes: Factors influencing product quality, p. 177-197. In: H. E. Pattee (ed.), Evaluation of Quality of Fruits and Vegetables. AVI, Westport, CT. Carroll, D. E., W. E. Ballinger, W. F. McClure, and W. B. Nesbitt. 1978. Wine quality versus ripeness of light-sorted Carlos muscadine grapes. Am. J. Enol. Vitic. 29:169-171. Carroll, D. E.. W. B. Nesbitt, and M. W. Hoover. 1975. Characteristics of red wines of six

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cultivars of Vitis rotundifolia Michx. J. Food Sci. 40:919-921. Chaparro, J. X., R. G. Goldy, B. D. Mowrey, and D. J. Werner. 1989. Identification of Vitis vinifera L. x Muscadinia rotundifolia Small hybrids by starch gel electrophoresis. HortScience 24:128-130. Clayton, C. N. 1975. Diseases of muscadine and bunch grapes in North Carolina and their control. N.C. Agr. Expt. Sta. Bul. 451. Clayton, C. N., and W. H. Ridings. 1970. Grape rust, Physopella ampelopsidis, on Vitis rotundifolia in North Carolina. Phytopathology. 60:1022-1023. Comeaux, B. L. 1984. Taxonomic studies on certain native grapes of eastern North Carolina. PhD dissertation, North Carolina State University, Raleigh. Comeaux, B. L., W. B. Nesbitt, and P. R. Fantz. 1987. Taxonomy of the native grapes of North Carolina. Castanea 52:197-215. Cowart, F. F., and E. F. Savage. 1944. The effect of various treatments and methods of handling upon rooting of muscadine grape cuttings. Proc. Am. Soc. Hort. Sci. 44:312314.

Daniel, E. M., A. S. Krupnick, Y.-H. Heur, J. A. Blinzler, R. W. Nims, and G. D. Stoner. 1989. Extraction, stability, and quantification of ellagic acid in various fruits and nuts. J. Food Compo Anal. 2:338-349. Davidis, U. X., and H. P. Olmo. 1964. The Vi tis vinifera X V. rotundifolia hybrids as Phylloxera resistant rootstocks. Vitis 4:129-143. Daykin, M. E., and R. D. Milholland. 1984. Ripe rot of muscadine grape caused by Colletotrichurn gloeosporioides and its control. Phytopathology 74:710-714. Dearing, C. 1917a. Muscadine grape breeding. J. Hered. 8:409-424. _ _ . 1917b. The production of self-fertile muscadine grapes. Proc. Am. Soc. Hort. Sci. 14:30-34. _ _ . 1938. Muscadine grapes. U.S. Dept. of Agr. Farmers, Bul. 1785. Dermen, H. 1954. Colchiploidy in grapes. J. Hered. 45:159-172. _ _ . 1958. Sterile hybrid grape made fertile with colchicine. Fruit Var. Hort. Dig. 12:3436. _ _ . 1964. Cytogenetics in hybridization of bunch- and muscadine-type grapes. Econ. Bot. 18:137-148. Dermen, H. and D. H. Scott. 1962. Potentials in colchiploid grapes. Econ. Bot. 16:77-85. Dermen, H., F. H. Harmon, and J. H. Weinberger. 1970. Fertile hybrids from a cross of a variety of Vitis vinifera with V. rotundifolia. J. Hered. 61:269-271. Detjen, L. R. 1917a. Breeding southern grapes. J. Hered. 8:252-258. _ _ . 1917b. Inheritance of sex in Vitis rotundifolia. N.C. Agr. Expt. Sta. Tech. Bul. 12. _ _ . 1919a. The limits in hybridization of Vitis rotundifolia with related species and genera. N.C. Agr. Expt. Sta. Tech. Bul. 17. _ _ . 1919b. Some F1 hybrids of Vitis rotundifolia with related species and genera. N.C. Agr. Expt. Sta. Tech. Bul. 18. Dunstan, R. T. 1962a. Nouvelles notes sur une vielle historie: les hybrides Euvitis X V. rotundifolia. Bul. de l'Office International de la Vigne et du vin. Paris. 378:993-1000. _ _ . 1962b. Some fertile hybrids of bunch and muscadine grapes. J. Hered. 53:299-303 (Corrigendum. 1963. 54:25). _ _ . 1964. Hybridization of Euvitis X Vitis rotundifolia: backcrosses to muscadine. Proc. Am. Soc. Hort. Sci. 84:238-242. _ _ . 1967. Inheritance of inflorescence in bunch grape-muscadine hybrids. J. Hered. 58:235-237. Einset, J. and C. Pratt. 1975. Grapes, p. 130-153. In: J. Janick and J. N. Moore (eds.), Advances In Fruit Breeding. Purdue Univ. Press, West Lafayette, IN. Emershad, R. L. and D. W. Ramming. 1984. In-ovulo embryo culture of Vitis vinifera

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L. 'Thompson seedless'. Am. J. Bot. 71:873-877. Emershad, R. 1., D. W. Ramming, and M. D. Serpe. 1989. In Ovulo embryo development and plant formation from stenospermic genotypes of Vitis vinifera. Am. J. Bot. 76:397402.

Espino, R. R. C., and W. B. Nesbitt. 1982. Infection and development of Plasmopara viticola (B. et C.) Berl. et de T. on resistant and susceptible grapevines (Vitis sp). Philipp. J. Crop Sci. 7:114-116. Fennel, J. L. 1940. Two North American species of Vitis. J. Wash. Acad. Sci. 30:15-19. Firoozabady, E., and H. P. Olmo. 1982a. Resistance to grape phylloxera in Vilis vinifera X V. rotundifolia grape hybrids. Vitis 21:1-4. ___ . 1982b. The heritability of resistance to root-knot nematode (Meloidogyne incognita scrits Chit.) in Vitis vinifera X V. rotundifolia derivatives. Vitis 21:136-144. ___ . 1987. Heritability and correlation studies of certain quantitative traits in table grapes, Vitis spp. Vitis 26:132-146. Flora, L. F. 1978. Influence of heat, cultivar and maturity on the anthocyanidin-3,5diglucosides of muscadine grapes. J. Food Sci. 43:1819-1821. Fry, B. O. 1963. Production of tetraploid muscadine (V. rotundifolia) grapes by gamma radiation. Proc. Am. Soc. Hort. Sci. 83:388-395. ___ . 1964. Fertile interspecific hybrids Vilis rotundifolis X Vitis vinifera. Ga. Agr. Expt. Sta. Mimeo. Series N .S. 200. ___ . 1967. Value of certain varieties and selections in the breeding of high quality, largefruited muscadine grapes. Proc. Am. Soc. Hort. Sci. 91:213-216. Fuchigami, L. H., and C-C. Nee. 1987. Degree growth stage model and rest-breaking mechanisms in temperate woody perennials. HortScience 22:836-845. Galletta, G. J. 1983. Pollen and seed management, p. 23-47. In: J. N. Moore and J. Janick (eds.), Methods In Fruit Breeding. Purdue Univ. Press, West Lafayette, IN. Gohdes, C. 1982. Scuppernong, North Carolina's grape and its wines. Duke Univ., Durham NC. Goldblatt, P. 1980. Polyploidy in angiosperms: Monocotyledons, p. 219-239. In: W. H. Lewis (ed.), Polyploidy: Biological Relevance. Plenum Press, New York. Goldy, R. G. 1987. Grape breeding in North Carolina. Proc. of the Vit. Sci. Symp. 1987. Florida A & M Univ., Tallahasee. p. 158-171. ___ . 1988. Variation in some yield determining components in muscadine grapes and their correlation to yield. Euphytica 39:39-42. Goldy, R. G., W. E. Ballinger, and E. P. Maness. 1986. Fruit anthocyanin content of some Euvitis X Vitis rotundifolia hybrids. J. Am. Soc. Hort. Sci. 111:955-960. Goldy, R. G., W. E. Ballinger, E. P. Maness, and W. H. Swallow. 1987a. Anthocyanin content of fruit, stem, tendril, leaf, and leaf petioles in muscadine grape. J. Am. Soc. Hort. Sci. 112:880-882. ___ . 1987b. Pigment correlations between fruit and vegetative tissue in 10 selections of muscadine grape. J. Am. Soc. Hort. Sci. 112:883-885. Goldy, R., R. Emershad, D. Ramming, and J. Chaparro. 1988. Embryo culture as a means of introgressing seedlessness from Vitis vinifera to V. rotundifolia. HortScience 23:886889. Goldy, R. G., E. P. Maness, H. D. Stiles, J. R. Clark, and M. A. Wilson. 1989. Pigment quantity and quality characteristics of some native Vitis rotundifolia Michx. Am. J. Enol. Vitic. 40:253-258. Goldy, R. G., and W. B. Nesbitt. 1985. 'Nesbitt' muscadine grape. HortScience 20:777. Goldy, R. G., D. W. Ramming, R. L. Emershad, and J. X. Chaparro. 1989. Increasing production of Vi tis vinifera X V. rotundifolia hybrids through embryo rescue. HortScience 24:820-822.

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Goode, D. K., Jr., G. W. Krewer, R. P. Lane, J. W. Daniell, and G. A. Couvillon. 1982. Rooting of dormant muscadine grape cuttings. HortScience 17:644-645. Graves, C. H., D. Griffin, and L. Wilson. 1988. Occurrence and potential significance of vascular Agrobacterium spp. of muscadine and approaches for control of associated problems. Proc. third viniculture short course 1988. Miss. Agr. For. Expt. Sta. Miss. State. p. 32-35. Gray, D. J., and L. C. Fisher. 1985. In vitro shoot propagation of grape species, hybrids and cultivars. Proc. Fla. State Hort. Soc. 98:172-174. Gray, D. J., L. C. Fisher, J. A. Mortensen. 1987. Comparison of methodologies for in Ovulo embryo rescue of seedless grapes. HortScience 22:1334-1335. Hamann, D. D., and D. E. Carroll. 1971. Ripeness sorting of muscadine grapes by use of low-frequency vibrational energy. J. Food Sci. 36:1049-1051. Harman, F. N. 1943. Influence of indolebutyric acid on the rooting of grape cuttings. Proc. Am. Soc. Hort. Sci. 42:383-388. Hedrick, U. P. 1908. The grapes of New York. State of New York Dept. Agr. 15th Annu. Rpt. Vol. 3. Part 2. Hopkins, D. L., H. H. Mollenhauer, and J. A. Mortensen. 1974. Tolerance to Pierce's disease and the associated rickettsia-like bacterium in muscadine grape. J. Am. Soc. Hort. Sci. 99:436-439. Hrazdina, G. A, A J. Borzell, and W. B. Robinson. 1970. Studies of the stability of the anthocyanidin-3,5-diglucosides. Am. J. Enol. Vitic. 21:201-204. Huang, P.-Y., R. D. Milhollond, and M. E. Daykin. 1986. Structural and morphological changes associated with the Pierce's disease bacterium in bunch and muscadine grape tissues. Phytopathology. 76:1232-1238. Husman, G. C., and C. Dearing. 1913. The muscadine grapes. U.S. Dept. Agr. Bur. Plant Ind. Bul. 273. Husmann, G., and C. Dearing. 1916. Muscadine grapes. U.S. Dept. of Agr. Farmers' Bul. 709. Jabco, J. P., W. B. Nesbitt, and D. J. Werner. 1985. Resistance of various classes of grapes to the bunch and muscadine grape forms of black rot. J. Am. Soc. Hort. Sci. 110:762-765. Jelenkovic, G., and H. P. Glmo. 1968. Cytogenetics of Vitis III. Partially fertile F1 diploid hybrids between v. vinifera L. XV. rotundifolia Michx. Vitis 7:281-293. ___ . 1969a. Cytogenetics of Vitis IV. Backcross derivatives of V. vinifera L. XV. rotundifolia Michx. Vitis 8:1-11. _ _ . 1969b. Cytogenetics of Vitis V. Allotetraploids of V. vinifera L. X V. rotundifolia Michx. Vitis 8:265-279. Johnson, D. T., J. R. Meyer, and R. L. Mayes. 1986. Evaluation of hereon laminated dispensers baited with Z,Z-3,13-octadecadien-1 01 acetate for suppression of the grape root borer, Vitacea polistiformis (Harris), (Lepidoptera Cesiidae), populations in grapes. J. Entomol. Sci. 21:231-236. Kasimatis, A. N., B. E. Bearden, and K. Bowers. 1981. Wine grape varieties in the north coast counties of California. Div. Agr. Sci., Univ. of California, publication 4069, Richmond, CA. Kasimatis, A. N., L. P. Christensen, D. A. Luvis, and J. J. Kissler. 1980. Wine grape varieties in the San Joaquin Valley. Div. Agr. ScL, Univ. of California, publication 4009, Richmond, CA Lamikanra, O. 1988. Development of anthocyanin pigments in muscadine grapes. HortScience 23:597-599. ___ . 1989. Anthocyanins of Vitis rotundifolia hybrid grapes. Food Chern. 33:225-237. Lane, R. P., and L. F. Flora. 1979. Effect of ethephon on ripening of 'Cowart' muscadine grapes. HortScience 14:727-729.

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Lane, R. P., and M. A. Owen. 1989. 'Loomis' muscadine grape. HortScience 24:398-399. Lane, R. P., and R. P. Bates. 1987. 'Golden Isle' muscadine grape for wine. HortScience 22:165-166. Lanier, M. R., and]. R. Morris. 1979. Evaluation of density separation for defining fruit maturities and maturation rates of once-over harvested muscadine grapes. ]. Am. Soc. Hort. Sci. 104:249-252. Lawrence, G. H. M. 1951. Taxonomy of vascular plants. MacMillan, New York. Ledbetter, C. A, and D. W. Ramming. 1989. Seedlessness in grapes. Hort. Rev. 11:159184. Layne, R. E. C.1983. Hybridization, p. 48-65. In: J. N. Moore and]. Janick (eds.), Methods In Fruit Breeding. Purdue Univ. Press, West Lafayette, IN. Lee, N., and H. Y. Wetzstein. 1988. Protoplast isolation and callus production from leaves of tissue-cultured Vitis-spp. Plant Cell Rpt. 7:531-534. _ _ . 1990. In vitro propagation of muscadine grape by axillary shoot proliferation. J. Am. Soc. Hort. Sci. 115:324-329. Lider, L. A, H. P. Olmo, and A. C. Goheen. 1988a. Hybrid grapevine rootstock. U.S. Pat. Plant 6,166. Lider, L. A., H. P. Olmo, and A. C. Goheen. 1988b. Hybrid grapevine rootstock named 'VR 043-43'. U.S. Pat. Plant 6,319. Loomis, N. H. 1948. A note on the inheritance of flower type in muscadine grapes. Proc. Am. Soc. Hort. Sci. 52:276-278. Loomis, N. H., and B. O. Fry. 1965. Cold injury to muscadine grapes in Georgia and Mississippi. Fruit Var. Hort. Dig. 19:35-36. Loomis, N. H., and C. F. Williams. 1957. A new genetic flower type of the muscadine grape. J. Hered. 48:294,304. Loomis, N. H., C. F. Williams, and M. M. Murphy. 1954. Inheritance of flower types in muscadine grapes. Proc. Am. Soc. Hort. Sci. 64:279-283. Lyrene, P. M. 1987. Breeding rabbiteye blueberries. Plant Breed. Rev. 5:307-359. Mainland, C. M., W. B. Nesbitt, and R. D. Milholland. 1977. The effect of ethephon on detachment and keeping quality of 'Carlos', 'Magnolia' and 'Noble' muscadine grapes (Vitis rotundifolia Michx.). Proc. of the Plant Growth Reg. Working Group; Fourth Annual Meeting, Hot Springs AR. McGiffen, K. C., and H. H. Neunzig. 1985. A guide to the identification and biology of insects feeding on muscadine and bunch grapes in North Carolina. N.C. Agr. Res. Ser. Bul. 470. Raleigh, NC. Millardet, A. 1901. Note sur la fausse hybridization chez les amplidees. Rev. Vitic. 16:677680. Moore, M. O. 1987. A study of selected taxa of Vitis (Vitaceae) in the southeastern United States. Rhodora 89:75-91. Mortensen, E. 1952. Grape rootstocks for southwest Texas. Texas Agric. Expt. Sta. Progr. Rpt. 1475. Mortensen, J. A. 1971. Breeding grapes for central Florida. HortScience 6:149-153. _ _ . 1981. Sources and inheritance of resistance to anthracnose in Vitis. J. Hered. 72:423-426. Mortensen, J. A, and C. F. Balerdi. 1974. Muscadine grapes for Florida: yields and other characteristics of 48 cultivars. Proc. Fla. State Hort. Soc. 86:338-341. Mortensen, J. A., and J. W. Harris. 1988. Muscadine and bunch grape fresh fruit taste panels during 21 years with 101 cultivars. Proc. Fla. State Hort. Soc. 101:229-232. _ _ . 1989. Yields and other characteristics of muscadine grape cultivars at Leesburg. Proc. Fla. State Hort. Soc. 102:223-226. Mortensen, J. A, L. H. Stover, and C. F. Balerdi. 1977. Sources of resistance to Pierce's

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_ _ . 1964. Les Composes phenolique du raisin et du vin. Ann. Physiol. Veg. 6:211. Ridings, W. H., and C. N. Clayton. 1970. Melanconium fuligineum and the bitter rot disease of grape. Phytopathology. 60:1203-1211. Robinson, W. B., L. D. Weirs, J. J. Bertino, and L. R. Mattick. 1966. The relation of anthocyanin composition of color stability of New York state wines. Am. J. Enol. Vitic. 17:178-842. Rogers, D. J., and J. A. Mortensen. 1979. The native grape species of Florida. Proc. Fla. State Hort. Soc. 92:286-289. Rombough, L. J. 1977. Inheritance of resistance to powdery mildew (Uncinula necator Burr.) in the hybrid (Vilis vinifera L. x Muscadinia rotundifolia Small). MS Thesis, Univ. Calif., Davis. Sax, K. 1929. Chromosome counts in Vitis and related genera. Proc. Am. Soc. Hort. Sci. 26:32-33. Sharpe, R. H. 1954. Rooting of muscadine grapes under mist. Proc. Am. Soc. Hort. Sci. 63:88-90. Sherman, W. B., and R. B. Nevins. 1963. A morphological study of fruit abscission of the muscadine grape, Vitis rotundifolia Proc. Assoc. Southern Agr. Workers Inc. 60:209. (Abstr.) Shulman, Y., G.Nir, L. Fanberstein, and S. Lavee. 1983. The effect of cyanamide on the release from dormancy of grapevine buds. Sci. Hort. 19:97-104. Shulman, Y., G. Nir, and S. Lavee. 1986. Oxidative processes in bud dormancy and the use of hydrogen cyanamide in breaking dormancy. Acta Hort. 179:141-148. Simpson, K. L., T. C. Lee, D. B. Rodriguez, and C. O. Chichester. 1976. Metabolism in senescent and stored tissues, pp.779-842. In: T. W. Godwin (ed.), Chemistry and Biochemistry of Plant Pigments. Academic, New York. Sims, C. A., and J. R. Morris. 1984. Effects of pH, sulfur dioxide, storage time, and temperature on the color stability of red muscadine grape wine. Am. J. Enol. Vitic. 35:35-39. _ _ . 1985. A comparison of the color components and color stability of red wine from Noble and Cabernet Sauvignon at various pH levels. Am. J. Enol. Vitic. 36:181-184. Sims, C. A., and J. R. Morris. 1986. Effects of acetaldehyde and tannins on the color and chemical age of red muscadine (VHis rotundifolia] wine. Am. J. Enol. Vitic. 37:163-165. Sistrunk, W. A., and J. R. Morris. 1982. Influence of cultivar extraction and storage temperature, and time on quality of muscadine grape juice. J. Am. Soc. Hort. Sci. 107:1110-1113. _ _ .1985. Quality acceptance of juices of two cuItivars of muscadine grapes mixed with other juices. J. Am. Soc. Hort. Sci. 110:328-332. Small, J. K. 1913. Flora ofthesoutheastern United States. 2nd ed. (published by the author). Smit, C. J. B., H. L. Cancel, and T. O. M. Nakayama. 1971. Refrigerated storage of muscadine grapes. Am. J. Enol. and Vitic. 22:227-230. Snyder, E. 1937. Grape development and improvement. USDA Year. of Agr. 1937:631-664. Spiegel-Roy, P., N. Sahar, J. Baron, and U. Lavi.1985. In vitro culture and plant formation from grape cultivars with abortive ovules and seeds. J. Am. Soc. Hort. Sci. 110:109-112.

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9 Nitrogen Metabolism in Grapevine* Kalliopi A. Roubelakis-Angelakis Department of Biology, University of Crete P.o. Box 1470, 71110 Heraklio, Greece

w. Mark Kliewer Department of Viticulture and Enology, University of California Davis, California 95616

I. Introduction II. Uptake of Nitrogenous Compounds A. Uptake of Ammonium B. Uptake of Nitrate C. Uptake of Amino Acids D. Uptake of Nitrogen by Grapevines III. Biosynthesis of Nitrogenous Molecules A. Reduction of Nitrate B. Assimilation of Ammonia C. Synthesis of Amino Acids D. Synthesis of Proteins E. Other Enzyme Proteins F. Polyamines IV. Nitrogenous Compounds A. Perennial Parts B. Vegetative Parts C. Reproductive Parts V. Storage and Reallocation of Nitrogen A. Nitrogen Reserve Partitioning B. Nitrogen Nutrition Cycle C. Nitrogen Allocation Modeling VI. Translocation of Nitrogenous Compounds A. Xylem Transport of Nitrogen B. Phloem Transport of Nitrogen VII. Diagnosis of Nitrogenous Status VIII. Future Research Directions Literature Cited

·Concentrations of nitrogenous substances when necessary were converted appropriately for comparative purposes. 407

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I. INTRODUCTION Nitrogen plays the most important role of all nutrients in plant growth. It is the plant nutrient most likely to be deficient in grapevines although it

is the nutrient most commonly applied to vineyards to increase productivity. Considerable new information has been obtained over the past twenty years on the uptake, translocation, distribution, partitioning, and storage of nitrogenous compounds in grapevines. Considerable new information has also been developed on synthesis and degradation of amino acids and other nitrogenous compounds in grapevines and the enzymes associated with these reactions. However, in spite of the widespread usage of nitrogen fertilization in vineyards, the physiological and biochemical effects of nitrogen on shoot and fruit growth, fruit bud initiation, flowering, fruitset, and crop yield are still poorly understood. Grapevines differ from herbaceous plants and many other woody plants in that they do not form terminal buds at the end of shoots, but they can continue to grow late into the season. The individual flower parts are formed after budbreak, unlike most deciduous fruit trees. Nitrate and ammonium ions are the most common forms of nitrogen available to plants in soils. The relative importance and magnitude of each of these ions depends on the genetic, developmental, and physiological status of each plant as well as on soil properties, such as texture, structure, water content, and pH. Also, the metabolism and distribution of both nitrate and ammonium ions and the partition of their products in plants are multifactor-dependent processes. Some of these factors, such as light, temperature, and nutrient species and concentration, may affect or even regulate the reactions of certain enzymatic systems. Other deal with the demand for the intermediate metabolites and their cellular localization, and still others with unspecified parameters or conditions. There have been several reviews on nitrogen metabolism of woody plants, including that of Titus and Kang (1982) on apple, Kato (1986) on citrus, and Korcak (1989) on blueberries and other calcifuges. Other recent general reviews on nitrogen metabolism of plants include Oaks (1986), Oaks and Long (1991), and Durzan and Steward (1983). Also, information on the uptake, distribution, partitioning, redistribution and seasonal dynamics of nitrogen and on the amino acids and protein metabolism in grapevines has been recently reviewed by Conradie (1991), Wermelinger (1991), Williams (1991), and Roubelakis-Angelakis (1991).

This review emphasizes the metabolism and biochemistry of nitrogenous compounds in grapevines, particularly the reduction of nitrate, ammonia assimilation, amino acid and protein synthesis, and

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recent work on polyamines. In addition, uptake, translocation, partitioning, storage and remobilization of nitrogenous compounds in grapevines as well as means of diagnosing the nitrogen status of grapevines are reviewed. It becomes apparent that many areas of nitrogen metabolism in grapevines are grossly deficient. This is particularly true for storage proteins. Practically nothing is known on the amounts and forms of storage proteins in grapevines and how they are degraded, transported, and recycled during the season. In leaves and stems of most plants, approximately 60% of the nitrogen is present as proteins with the remainder mainly accounting for as water-soluble amino acids (Parsons and Tinsley, 1975). There is increasing evidence that proteins play an important role in overwintering nitrogen storage in woody deciduous plants, but the nature of these proteins are unknown for grapevines. In some woody perennials, specific proteins in the shoots have been shown to undergo marked increases in concentration prior to overwintering and then are mobilized from these the following spring (Oaks et al. 1991). Other areas in urgent need of more research will be pointed out in this review.

II. UPTAKE OF NITROGENOUS COMPOUNDS Ions diffuse into cell walls of the epidermal cells and active ion uptake may occur at the plasmalemma of these cells. The ions may then be transported across the cortex, endodermis, and pericycle in the symplast. Ions may also move passively into the continuum of cell wall material of the cortex cells and then be absorbed across the plasmalemma of the cortical and endodermal cells, thus entering the symplast. Casparian strips essentially restrict apoplastic movement from the free space of the cortex to the free space of the stele. Solutes entering the vascular tissue of roots must therefore overcome these strips by being absorbed across the plasmalemma of the epidermal, cortical, or endodermal cells and then moving through the symplast (Haynes 1986c). Absorption of ions across the plasmalemma of root cells is generally accepted to be an active process that often overcomes an unfavorable electrochemical gradient through the expenditure of energy, and it may be accomplished by protein carriers (Haynes 1986c). The factors that affect mineral nutrient acquisition by plants are discussed by Clarkson (1985). The maintenance of appropriate cellular concentrations of nutrients appears to be the purpose of the regulation of carrier activity and control of efflux. The forms of organic and inorganic nitrogenous compounds present in soil and their inter-conversion have been reviewed by Cameron and Haynes (1986), Firestone (1982), Goh and Haynes (1986), Havelka et al.

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K. A. ROUBELAKIS-ANGELAKIS AND W. MARK KLIEWER

(1982), Haynes (1986a,b), Ladd and Jackson (1982), MelIilo (1981), Nelson (1982), Stevenson (1982), and Woodmansee et a1. (1981). Nitrate and ammonium ions are the most common forms of nitrogen available to plants. The relative importance of each of these ions depends on the genetic, developmental, and physiological status of each plant as well as on soil properties, such as texture, structure, water content, temperature, source of N organic matter, biological fixation and pH (Barker 1989). Because both nitrogen deficiency and nitrogen excess may contribute to reduced Yield and poorer fruit quality, it is essential to know the factors that affect nitrogen uptake, accumulation, and metabolism in grapevines and other horticultural crops. Most plants are capable of nitrate and ammonia uptake from the root environment (HaYnes and Goh 1978). However, nitrate is considered the major form because it is more available in most soils than ammonia, which is rapidly converted to nitrate (nitrification) by microorganisms (Haynes 1986c). Little nitrification occurs at low pH or low soil temperature. Increased ammonia levels are available for plant uptake under these root environments. Calcifuge (acid-loving) plants are adapted to take advantage of high soil ammonia at low pH. These plants utilize ammonia in preference to nitrate (Haynes and Goh 1978; Korcak 1989). The influence of soil and plant nitrogen and different nitrogen sources and the interaction of various forms of nitrogen with other elements in the soil on NHt and NO; utilization has been reviewed by Korcak (1989). A. Uptake of Ammonium

The time-dependent uptake of NHt by plants can be characterized as biphasic. The initial phase represents a passive exchange-absorption phenomenon in the negatively charged free space of roots (Nye and Tinker 1977). The second phase of uptake represents active absorption of NHt and it appears to have a multiphasic pattern (Dogar and van Hai 1977; Nissen et a1. 1980). The Km values are generally in the range from 10 to 70 J.1.M (Lycklama 1963; Fried et a1. 1965). Vrnax and Krn for NHt uptake differ among plant species. The mechanisms of NHt uptake is not fuHy resolved. There is limited information indicating a similarity between NHt uptake and the uptake of monovalent ions, especially K+ (Berlier et a1. 1969; Epstein 1972; Hassan and van Hai 1976). There is a possibility that NHt and K+ ions share a common uptake system (Epstein 1972). Potassium uptake is either directly linked to an ATPase that acts as an electrogenic H+/K+ pump or is mediated by a specific carrier and occurs with simultaneous cotransport of protons maintained by a membrane-bound ATPase (Clarkson and Hanson 1980; Hodges 1976; Lin 1979; Poole 1978; Spanswick 1981).

9.


411

However, pH affected differently the uptake of K+ and NHt by young rice plants (Zsoldos and Haunold 1982). Also, Lycklama (1963) found that ammonia absorption was highly temperature dependent with a 27°C optimum for rye grass in nutrient solution at a pH 4.0-6.5. However, absorption was independent of temperature at a pH of between 6.5 and 8.5. Lycklama (1963) suggested that this pH/temperature interaction reflected ammonia being taken up as NH4 0H between pH 6.5 and 8.5 rather than as NHt or NH 3 at pH 4.0-6.5. B. Uptake of Nitrate Uptake of nitrate exhibits an initial lag phase and a subsequent linear phase (Huffaker and Rains 1978; Jackson 1978). The latter phase is inducible and depends on a critical internal NO; concentration (Jackson 1978). Uptake of NO; by plants is an active process and it is inhibited by inhibitors of respiratory and oxidative phosphorylation (Rao and Rains 1976) and of RNA and protein synthesis (Jackson et al. 1973; Tomkins et al. 1978). Huffaker and Rains (1978) proposed that transport is linked to a membrane-bound ATPase. Ammonium ions appear to have an inhibitory effect on NO; uptake. This effect is caused by the cytoplasmic concentration of NHt in root cells Uackson et al. 1973) or by the amino acid concentration (Doddema and Otten 1979; Heimer and Filner 1971). Criddle et al. (1988) evaluated the multiple interactions among NO;, NHt, NO; and urea; they suggested that several regulatory features influence the net influx of the N species tested: (1) competitive effects were seen; (2) urea had strong interactive effects on the uptake of the others, although it was not taken up at a measurable rate; and (3) the uptake of total N appeared to be under regulatory control. In contrast, recently, it was suggested that accumulation of nitrate and ammonium are unrelated phenomena, that the rate of nitrate accumulation is strongly stimulated by light operating through phytochrome, while ammonium accumulation was not affected by light in short-term experiments and only weakly in long-term light (Heckt and Mohr 1990). Nevertheless, further research is required to elucidate the exact mechanism(s) involved in NO; uptake.

c. Uptake of Amino Acids Within plant cells different compartments, such as chloroplasts, cytosol, mitochondria, and vacuoles, are involved in amino acid metabolism and storage. At times of increasing demand, stored amino acids are mobilized and transported to the cytosol. In plant cells amino acid transport occurs via a H+ gradient-produced energy driving force,

412


and the carrier binds both the ion and the substrate (see Reinhold and Kaplan 1984 for review). The available experimental data indicate that there are two transport channels; the first a general transport system available to all amino acids, and the second a system with much higher affinity for basic than for other amino acids (Berlin and Mutert 1978). Protoplasts isolated from in vitro-grown virus-free axenic 'Thompson Seedless' leaf blades had an average size of 28 J.1m and a respective volume of 1.2Xl0-14 m3 (Theodoropoulos and Roubelakis-Angelakis 1989). Arginine uptake rate was 100 pmoles/Hf viable protoplasts per minute, which corresponded to an intracellular concentration of labeled compound of 8.3 pM. Uptake was linear for at least 60 minutes. Kinetics analysis revealed a biphasic uptake curve (Fig. 9.1). The high affinity component had a Km of 2.2 mM. The optimum pH value was 5.5. Two carrier systems, one for basic and neutral and one for acidic amino acids were identified. Use of inhibitors revealed that those associated with ATP metabolism inhibited arginine uptake, and proton motive force appeared to be the predominant energy source (Theodoropoulos and RoubelakisAngelakis 1989). In vacuoles, a three- to six-fold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate, an ATP analogue not being hydrolysed by ATPase. ATP-stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. The results suggested the existence of a uniport translocator specific for neutral or basic amino acids that is under the control of metabolic effectors (Dietz et al. 1990).

10

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Figure 9.1. Concentration dependence of [14Cj-L-arginine uptake by grapevine protoplasts and Lineweaver-Burk plot (inset) (from Theodoropoulos and RoubelakisAngelakis 1989).

9.


413

D. Uptake of Nitrogen by Grapevines Recently, Lohnertz (1991) reviewed the characteristics of soil nitrogen and nitrogen uptake by grapevines. The soil nitrate during the winter is remarkably reduced and often does not show a treatment response. Although there is a relationship of nitrate content to the nitrate fertilization during the year, this association is reduced by leaching during winter until budburst. In spring, the content of nitrate in viticultural soils changes relatively rapidly. Analysis of nitrate content in March does not forecast the nitrate supply of the vines for the subsequent growth period. Nitrate content can be affected by yearly variations in climate, soil heterogeneity, and system of soil tillage. The total loss of nitrate in the soil by plant uptake, as can occur with agricultural crops, does not occur in the vineyard (Lohnertz 1991). The N uptake by grapevine cannot be determined from the changes of nitrate in the soil. Over a nine-year period, soil nitrate concentrations higher than 60 kg N03"-N' ha-1 at budburst were found in 50% of the test plots in German soils. Thus, these soils did not require spring fertilization. In grapevine, the uptake of nitrogen from the soil is closely linked to the physiological status of plants. Before budbreak there is no remarkable uptake of N into the woody parts of grapevine. A significant rate of uptake of nitrogen starts soon after budbreak and peaks about four weeks after flowering when the uptake rates are 1.5-1.6 kg N· ha-1 ·d-1 (Lohnertz et al. 1989). Another peak is found shortly after harvest with an uptake rate of about 1.0 g N· ha-1 ·d-1 • Also, nitrates as nitrogen source resulted in higher K+ concentration in roots and stems of grapevines as compared to NHt (Ruhl 1989). Furthermore, studies on the uptake of nitrogen by field grown vines indicated that it is best to be applied between bloom and veraison or the early postharvest period rather than during dormancy or budbreak (Peacock et a1. 1982, 1989, 1991). There are no reports indicating that grapevine can absorb both nitrates and ammonia from the soil. The only work has been performed with calluses grown in vitro from either leaf, root, or green shoot segments of "Thompson Seedless" (K. A. Loulakakis and K. A. RoubelakisAngelakis, unpublished; Roubelakis-Angelakis et a1. 1991). Ammonium, as a sole nitrogen source in the culture medium, was insufficient to support callogenesis either from leaf, shoot, or root segments of grapevine. In the presence of 20 mM KN0 3 maximum callogenic response was found at 5-15 mM ammonium for leaf explants, at 0-10 mM for shoot explants, and at concentrations lower than 5 mM for root explants (Fig. 9.2). This may indicate that grapevine tissues have differential ability to assimilate ammonium or that the species of nitrogen affects the biosynthesis of molecules, which are related to cell proliferation. In in vitro-grown

414


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Figure 9.2. Effect of ammonium concentration on the fresh weights of grapevine calluses from leaf, shoot and root explants grown on culture media containing 20mM KN0 3 plus 020 mM NH 4 CI (from Roubelakis-Angelakis et a1. 1991).

'Thompson Seedless' plants, addition of 6 mM N~Cl to nitrate (16 mM) containing medium resulted in an increase in NADH-GDH activity of 43%, 39%, and 66% in leaf, shoot, and root enzyme, respectively (Loulakakis and Roubelakis-Angelakis 1990b). To reduce injury from soil pest and tolerate unfavorable soil conditions it is common in viticulture to use rootstocks resistant or tolerant to these problems. Rootstocks show differences in the size and spatial distribution of roots in the soil (Perry et al. 1983; Southey and Archer 1988; Swanepoel and Southey 1989). Differences in rooting pattern of the rootstock may affect the uptake of water and nutrient elements by the vine, as has been shown for potassium (Ruhl 1989). Williams and Smith (1991) found no differences in the concentrations of total nitrogen in the organs of Cabemet Sauvignon grafted onto four rootstocks, which suggests similar nitrogen uptake rates by the different rootstocks. III. BIOSYNTHESIS OF NITROGENOUS MOLECULES Most plants including grapevine, can effectively utilize either ammonium or nitrate ions. The productivity of a species, when grown on ammonium salts, is related to its ability to detoxify ammonia and depends directly on the availability of keto acids and indirectly on the supply of photosynthates. In contrast, nitrate is relatively innocuous and nitrate assimilation is regulated by carbohydrate oxidation and associated with organic acid production. Consequently, nitrate can be accumulated to relatively high concentrations without detriment to the plant.

9.


415

Ammonium may be toxic at high concentrations, although Christensen (1984) has reported ammonia concentrations in grapevine leaf petioles as high as 1700 ppm without any apparent injury. Normally, ammonia is detoxified in the roots by conversion to amino acids or amides and only traces are detectable in the xylem exudate (Givan 1979; Hill-Cottingham and Lloyd-Jones 1979), although Dintscheff et aI. (1964) found ammonium ions to be one of the major N constitutents reaching the shoots, leaves, and clusters.

A. Reduction of Nitrate Once nitrates enter the root cells, they are reduced and incorporated into organic molecules, or stored or translocated to aerial plant organs for further use. The reduction of nitrate to the useable (ammoniacal) form requires eight electrons (Haynes 1986c). In nonchlorophyllous tissues these eight electrons are derived from carbohydrate oxidation. In chlorophyllous tissues, two electrons are derived from carbohydrate oxidation while six electrons come from the trapping of light energy (via ferredoxin). Characteristics of the enzymes involved in nitrate and nitrite reduction and discussion of the biochemical and physiological aspects of nitrate metabolism have been presented by several authors (Hewitt 1975; Hewitt and Notton 1980; Hewitt et aI. 1978; Haynes and Goh 1978; Haynes 1986c). The first step in the assimilatoryreductionofN03 inhigherplants is catalysed by nitrate reductase (NR) [EC 1.6.6.1. (NADH specific); EC 1.6.6.2. (either NADH or NADPH specific) more prevalent in green algae; and EC 1.6.6.3. (NADH specific) present in molds]. The enzyme has a high molecular weight ranging from 220-600 kD depending on species; contains flavin adenine dinucleotide (FAD), cytochrome bss7 , and molybdenum (Notton and Hewitt 1978; Guerrero et aI. 1981). The proposed pathway of electrons from NAD(P)H to nitrate is NAD(P)H FAD - CyfoS7 - Mo - NO;;. The regulation of NR is very complex and appears to differ among species and even among different plant organs. Substrate concentrations, light, NHt and certain amino acids appear to exert control on NR activity. Variations in NR activity can be attributable to NR-degrading protein and to NR-specific binding protein. Furthermore, the size of the metabolic pool of N03 and its transport as well as the availability of reductants, temperature and water stress could affect NR activity (Haynes 1986c). The enzyme responsible for the reduction of nitrite to ammonium in photosynthetic cells is ferredoxin-nitrite reductase (NiR) (EC 1.7.7.1.) and in nonphotosynthetic organisms is NAD(P)H-NiR (Ee 1.6.6.4.). The reaction of nitrite reduction to ammonium involves the transfer of 6

416


electrons. Fd-NiR is composed of a single polypeptide chain of about 600 amino acids and has a molecular weight of approximately 62 kD (Hucklesby et al. 1978). Despite the importance of utilization of nitrates in grapevines, information is scarce on the enzymology of their reduction. This is partially due to the fact that enzymatic studies in perennial species, including grapevines, are complicated by the high concentration of secondary metabolites, phenolics, and so forth in plant tissues. In vivo assays or intact-tissue infusion methods for detecting enzyme activity have been applied often for overcoming these problems. Nitrate reductase activity was found in 'Thompson Seedless' leaf tissue by the in vivo assay (Perez and Kliewer 1978). Optimum enzyme activities required high concentrations of nitrates (0.1-0.2 M), whereas concentrations higher than 0.4 M inhibited NR activity. Addition of 1% 1propanol to the reaction mixture slightly increased NR activity; it was suggested that this compound could facilitate substrate diffusion through cut edges of leaf fragments. However, at higher concentrations it suppressed NR activity, possibly by allowing phenols and/or inhibitors to come in contact with the enzyme (Perez and Kliewer 1978). The inducible nature of NR in Vilis spp. was confirmed by growing 'Pinot Noir' grapevines in a soil-sand-peat mixture without N-fertilization for 3 months before 16 mM NOa- treatment (Perez and Kliewer 1982). NR activity was also detected in a partially purified preparation from 'Thompson Seedless' leaves. In vitro activity was lower compared to the in vivo activity (Perez and Kliewer 1978). NR activity in the leaves of 'Riesling' grapevines followed a diurnal rhythm with maximum activity between 11:00 A.M. and 12 noon, and relatively low activity during the night (Schaller 1984). NR activity was higher in leaves sprayed with Mo+ salts and there was a positive relationship between NR activity and the Cu2 + content of leaves. The level-of nitrate in leaves was not inversely correlated to NR activity. In addition, NR activity was determined in leaves and grapes of several cultivars of Vilis vinifera during berry development (Perez and Kliewer 1982). NR activity showed large differences among cultivars; a negative relationship between NR activity and nitrate content in grape juice was generally found. NR during rapid shoot growth was higher than during veraison (Schalleret al. 1985, 1986). Higher nitrate levels occurred in grape stems and penduncles, whereas NR activity predominated in the leaves (Schaller et al. 1985). B. Assimilation of Ammonia Ammonia is the primary inorganic nitrogen form involved in the synthesis and catabolism of organic nitrogen. Although ammonia is

9.


417

critical for plant growth and development, it is potentially very toxic. Ammonia assimilation is the only way plants can reduce elevated ammonia levels. Unlike many other molecules or ions, ammonia is difficult to compartmentalize because it is very membrane mobile. Consequently, plants are unable to use compartmentalization as a protection strategy against elevated ammonia. This strategy is used with other harmful materials where the vacuole serves to isolate them from the cytoplasm with movement restricted by the tonoplast. Ammonia content in plant cells varies as it can be derived from several routes, which depend on the overall metabolic potential of the cell. Before about 1973, the key reaction in ammonia assimilation was considered to be catalyzed by glutamate dehydrogenase (GDH) (EC 1.4.1.2) (Miflin and Lea 1982). This enzyme catalyses the synthesis of glutamate from a-ketoglutarate and ammonia and it is widely distributed in plants. However, since 1973 the glutamine synthetase/glutamate synthase pathway has been considered as the primary route of ammonia assimilation in higher plants (Miflin and Lea 1982; Wallsgrove et al. 1977). Plant glutamine synthetase (GS) (EC 6.3.1.2) is octameric and twodimensional electrophoresis has shown that the cytosolic enzyme in Phase01us vulgaris is composed of up to three isoelectric focusing variants of the 40-kD subunit (a, p, and y), which occur in varying proportions in different organs of the plant (Lara et al. 1984). The a, p, and y subunits are specified by three distinct but homologous genes (gIn-a, gln-p, gln-y) which are differentially expressed during plant development (Cullimore et al. 1984; Gebhardt et al. 1986; Turton et al. 1988). One gene (g1n-p) is expressed preferentially in roots, whereas 810-y is strongly induced during nodule development. Recently, Forde et al. (1989) studied the regulatory properties of g1n-y gene's promoter region in transgenic plants Lotus corniculatus. Molecular analysis of the GS genes in Pisum sativum has uncovered a multigene family whose individual members encode a single chloroplast GS2 polypeptide, and several distinct GS polypeptides, all of which are encoded in the nucleus (Tingey et al. 1987, 1988). The single nuclear gene for chloroplast GS2 is expressed predominantly in leaves in a lightdependent fashion. The physiological role of chloroplast GS2 in the reassimilation of photorespiratory ammonia, correlates with the preferential accumulation of GS2 mRNA in leaves of plants grown under photorespiratory conditions (Edwards and Coruzzi 1989). For cytosolic GS. the two distinct types of cytosolic GS polypeptides (38kD and 37kD) are encoded by homologous but distinct members of the pea GS gene family (GS 299 and GS 341) (Tingey et al. 1988). Recently, the fourth and final member of the pea GS gene family has been characterized (GS 132) and shown to encode a cytosolic GS family, which is nearly identical

418

K. A. RQUBELAKIS-ANGELAKIS AND W. MARK KLIEWER

to the GS 314 gene (E. L. Walker and G. M. Coruzzi, unpublished). Glutamate synthase (GOGAT) exists in higher plants in three forms: (1) NADH-GOGAT (EC 1.4.1.14.), and (2) NADPH-GOGAT (EC 1.4.1.13.), which are found in nonphotosynthetic cells (Fowler et a1. 1974; Suzuki et a1. 1982), and (3) ferredoxin-GOGAT (EC 1.4.7.1), which is present in photosynthetic tissues (Stewart and Rhodes 1978; Suzuki and Gada11982) and also in the roots (Miflin and Lea 1975; Suzuki et a1. 1982). The enzyme consists of a single polypeptide chain with a molecular weight from 140 to 180 kD (Tamura et a1. 1980; Wallsgrove et a1. 1987). Glutamate dehydrogenase (EC 1.4.1.2.) catalyzes the amination of aketoglutarate to form glutamate in the expense of NAD(P)H. The enzyme is a metalloprotein, and is localized in the mitochondria. It shows a 7isoenzymic pattern and a molecular weight from 220 to 270 kD (Miflin and Lea 1982). Differences in ammonia affinity (Km) between assimilation enzymes have been used to identify their relative importance in ammonia assimilation. For example, glutamine synthetase has a much higher affinity of ammonia (0.001-{).002 mM Km) than glutamate dehydrogenase (1D-80 mM Km) (Stewart and Rhodes 1978). Consequently, the GS/GOGAT pathway was considered the primary assimilation path rather than GDH. Other evidence is based on experiments that used inhibitors and FSN] or 14 [ C], or mutants (Wallsgrove et a1. 1987) or combination of them. Methionine sulfoximine (MSX, an irreversible inhibitor of glutamine synthetase), amino-oxyacetate (AOA, a transaminase and glycine decarboxylation inhibitor), and azaserine (glutamate synthase inhibitor) are the common inhibitors used in ammonia assimilation experiments. Inhibitor studies can be difficult to interpret. Experiments that use labeled substrates plus inhibitors have provided more complete information than using inhibitors or labeled substrate alone (Oaks 1985). Ammonia assimilation enzymes in grapes have received some research attention. Glutamine synthetase (Roubelakis-Angelakis and Kliewer 1983b) and glutamate dehydrogenase (Ghisi et a1. 1984; Nutsubidze and Oganesyan 1985; Roubelakis-Angelakis and Kliewer 1983a) activity have been detected in grape leaf and root extracts. These enzymes are also present in berry extracts (Ghisi et a1. 1984). However, RoubelakisAngelakis and Kliewer (1983b) were unable to detect GOGAT activity in grape roots. This could reflect problems with enzyme extraction rather than the absence of enzyme. If, in fact GOGAT activity is low in grape tissue, other ammonia assimilation pathways (e.g., glutamate dehydrogenase) could be more important in grape than other plants. Recently, Loulakakis and Roubelakis-Angelakis (1990a,b,c; 1991) and Roubelakis-Angelakis et a1. (1991) have further studied the structure, function and some regulatory properties of GDH from grapevine tissues

9.


419

and calluses. The amination reaction was fully activated by about 100 JlM Ca 2+ although the deamination reaction was not affected by the addition of Ca 2+. Leaf. shoot, and root GDH showed a 7-isoenzymic pattern (Loulakakis and Roubelakis-Angelakis 1990b). The enzyme consists of two subunits, a and (J, with similar antigenic properties but with different molecular weight and charge. The two subunits have a molecular weight of 43.0 and 42.5 kD, respectively. The holoenzyme is hexameric and is resolved into 7 isoenzymes by native gel electrophoresis. Twodimensional native/SDS-PAGE revealed that the 1 and 7 isoenzymes are homohexamers and the isoenzymes 2-6 are hybrids of the 2 subunits following an ordered ratio. The total quantity of a-and (J-subunit and the isoenzymic pattern was altered by the exogenous nitrogen source. GDH from calluses grown on nitrate or glutamic acid contained a slightly greater amount of the (J-subunit and of the more cathodal isoenzymes, whereas the a-subunit and the more anodal isoenzymes predominated in callus grown in the presence of either ammonia or glutamine (Fig 9.3). The anabolic reaction was correlated with the a-subunit and the catabolic reaction with the (J-subunit; this suggested that each isoenzyme exhibits anabolic and catabolic functions of different magnitude. The isoenzymic patterns did not obey the expected binomial distribution proportions (Loulakakis and Roubelakis-Angelakis 1991; Roubelakis-Angelakis et al. 1991). Furthermore, Loulakakis and Roubelakis-Angelakis (1992) by using 35S-methionine showed that the increase in NADH-GDH activity in the presence of ammonium was accompanied by an increase in the activity staining of the more anodal isoenzymes, which are hexameric containing mainly a-subunits. This increase was due to de novo synthesis of the a-subunit (Fig. 9.4). The results further confirmed the model for the structure and the physiological function of the GDH isoenzymes (Loulakakis and Roubelakis-Angelakis 1991). Although GS/GOGAT is still considered as the primary ammonia assimilation path, Yamaya and Matsumoto (1985) and Yamaya and Oaks

o

2 10 15 5 Ammonium concentration. mM

20

Figure 9.3. Effect of ammonium concentration on the glutamate dehydrogenase isoenzymic pattern in grapevine shoot calluses (from Roubelakis-Angelakis et al. 1991).

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Figure 9.5. Chromatography profile of the intracellular labeled soluble compounds extracted after accumulation of 14C-L-arginine at 20 mM for 120 minutes (from Theodoropoulos and Roubelakis-Angelakis 1989).

in grapevine tissues, in addition to those mediating ammonia assimilation reactions (glutamate dehydrogenase, glutamine synthetase, and glutamate synthase). Arginine, one of the more abundant amino acids, is of special importance in Vitis spp. because it is a major N-storage compound and also it participates in the biosynthesis of other amino acids, guanidines, and polyamines (Fig. 9.6). Ornithine transcarbamoylase (OTC, EC 2.1.3.3.); arginosuccinate synthetase and lyase (ASA synthetase and lyase, EC 6.3.4.5. and 4.3.2.1., respectively); and arginase (EC 3.5.3.1.) mediate the reactions of the Krebs-Henseleit or urea cycle of arginine metabolism. All four enzymes were present in leaves, berries, germinating seeds, and seedlings of grapevine (Roubelakis and Kliewer 1978a,b,c). The Km values forOTC were 3.5 mM and 5.5 mM for carbamoyl phosphate and ornithine, respectively. Optimum pH values for enzyme activity were 8.4-8.8 for OTC, 7.3-7.8 for ASA synthetase, 7.5-8.0 for ASA lyase, and 9.4-9.8 for arginase. In developing grape berries maximum aTC and arginase activity was found at veraison and decreased thereafter (Roubelakis-Angelakis and Kliewer 1981). In germinating seeds and seedlings OTC activity increased during the first 2 weeks and remained almost constant thereafter for the following 3 weeks. Arginase activity increased rapidly during seed germination, reached a maximum after about 3 weeks and decreased thereafter (Roubelakis-Angelakis and Kliewer 1978d). There was a tendency for higher aTe activity in grape berries with increasing exogenously supplied nitrogen concentration, especially at veraison and maturity,

422

Figure 9.6.


Metabolic fate of arginine in plant cells (from Roubelakis-Angelakis 1991).

whereas arginase activity was unaffected (Roubelakis-Angelakis and Kliewer 1981). Aspartic acid, the product of transamination between glutamic acid and oxaloaceatic acid, is also abundant in grapevine tissues. It may represent a storage form of oxaloaceatic acid, which can in turn be transformed into either malic acid or into carbohydrates (Kluba 1977). Glutamate oxaloacetate transaminase activity (L-aspartate: aketoglutarate aminotransferase, GOT, EC 2.6.1.1.), which catalyzes the reversible interconversion between glutamate and aspartate and their two keto-analogues was present in grapevine leaf and root tissues (Roubelakis-Angelakis and Kliewer 1984). The enzYme was purified and characterized from grape, was dimeric, and showed a 4-enzYmic pattern in native PAGE (Sauvage et al. 1991). Aspartase activity (L-aspartate ammonia-lyase EC 4.3.1.1), which catalyzes the reversible deamination of aspartate to fumaric acid and ammonia was found in berries. Malate exhibited an activation effect and several electrolytes exhibited an inhibitory effect on enzyme activity, suggesting that aspartase may playa key role in the regulation of the organic acid pool in grapes (Robin et al. 1987). An enzYme that mediates a reaction involved in the metabolism of the amino acid phenylalanine is phenylalanine ammonia-lyase (PAL, EC 4.3.1.5). It channels phenylalanine away from the synthesis of proteins toward that of many phenolic compounds. PAL activity was present mostly in the epidermal cell layers of grape berries (RoubelakisAngelakis and Kliewer 1985, 1986; Kataoka et al. 1986). During the ripening period, PAL activity was high during the early stages of berry development and declined thereafter in white cultivars, whereas in pigmented cultivars a second increase was present in PAL activity

9,


423

coinciding with rapid color accumulation in berries (Kataoka et al. 1986). Exogenously applied ethephon and sucrose in detached 'Cardinal' berries in the light caused an increase in PAL activity, whereas in berries kept in darkness enzyme activity decreased over the 72-h experimental period (Roubelakis-Angelakis and Kliewer 1986).

D. Synthesis of Proteins The transcription of DNA sequences into mRNA, the translation of mRNA, the initiation, the elongation, and the termination of polypeptide chains are the molecular events that lead to protein synthesis in cells. Proteins are of three types: (1) structural, (2) metabolic, and (3) storage (Miege 1982). The purification and characterization of proteins from grapevine tissues are quite difficult because these tissues are very rich in phenolic compounds, other secondary metabolites, and oxidizing enzymes, which tend to bind and inactivate proteins during cell lysis and fractionation (Roubelakis-Angelakis, unpublished data; Yokotsuta et al. 1988). Total protein content in organs of in vitro grown 'Thompson Seedless' vines was 6.25,3.12, and 1.30 mg/g fresh weightfor leaf, shoot, and root, respectively (Roubelakis-Angelakis 1991). The SDS-PAGE profile of total proteins from the same tissues revealed several differences (Fig. 9.7). Soluble protein content in grape berries increases with maturity. The reported soluble protein concentrations in grape juice for various cultivars grown in different geographical regions range from 1.5 to 260 mg/liter (Hsu and Heatherbell1987; Koch and Sajak 1959; Yokosuta etal. 1988). The SDS-PAGE profile of soluble proteins in grape berries consists of numerous protein bands with molecular masses from 11 to 70 kD (Murphey et al. 1989; Nakanishi et al. 1986). In general, greater amounts of soluble proteins are found during warm rather than cool seasons. This may be of special importance for the quality of the grape products. In grapevine leaves, the soluble protein fraction was maximal two weeks before a nthesis to 2 weeks after anthesis a nd minimal during the 4th to 7th week following anthesis. The overall value ranged from about 0.41.5 mg/g fresh weight (Ghisi et al. 1984). Grapevine leaf protoplasts were able to incorporate labeled methionine throughout their culture period. The rate of protein synthesis was higher immediately following isolation of protoplasts, and it showed a second increase the 4th day of culture. These proteins were considered shock induced and cell-wall-related proteins, respectively (Katsirdakis and Roubelakis-Angelakis 1991).

424


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14.4-

Figure 9.7. SDS-PAGE of total proteins from Vitis vinifera 1. cv 'Thompson Seedless' leaf. shoot. root. and callus tissues (Siminis and Roubelakis-Angelakis. unpublished).

E. Other Enzyme Proteins Peroxidase isoenzymes have been implicated in several plant reactions, such as indole-3-acetic acid oxidation (Hinnman and Lang 1965), polysaccharide cross-linking (Fry 1986), cross-linking of extensin monomers (Everdeen et al. 1981), lignification (Grisebach 1981), phenol oxidation and pathogen defense (Hummerschmidt et al. 1982), and cell elongation (Goldberg et al. 1986). Two major groups of isoperoxidases were revealed in grapevine leaf, shoot, and root tissues by IEF (Siminis and Roubelakis-Angelakis 1990). Peroxidase activity in grapevine protoplasts remained low during culture; two isoperoxidases in the basic and two in the acidic region appeared during culture (C. 1. Siminis, A. K. Kanellis, and K. A. Roubelakis-Angelakis, unpublished). In grapes, peroxidase activity increased sharply beginning six weeks after anthesis (Schaeffer 1983). The increase was greater with ethephon treatment. Peroxidase activity was associated with two isoenzymes at Rf 0.68 and 0.75 (Kochhar et al. 1979). Also, a homogeneous peroxidase (Lee et al. 1984) and four anionic isoperoxidases (Sciancalepone et al. 1985) have been characterized from grapes. Peroxidase activity was also correlated to in vitro rhizogenesis in grapevine explants (Mato et al. 1988; Tabakakis and Roubelakis-Angelakis, unpublished data).

9.


425

Catalase and superoxide dismutase (SOD) enzymes implicating in oxidative status of cells have also been found in grapevine tissues. Catalase activity in cultured 'Thompson Seedless' protoplasts was low during culture and it was separated into two bands by immunoblotting (Siminis, Kanellis and Roubelakis-Angelakis, unpublished material). Also, catalase activity in 'Perlette' buds increased to a maximum in the fall and decreased thereafter during the winter. The rate of bud ~prouting was negatively correlated with the activity of catalase (Nir et a1. 1986). Polyphenoloxidase (PPO), which catalyzes the oxidation of phenolic compounds was found in grape juice (Sanchez-Ferrer et a1. 1989; Yokotsuta et a1. 1988). The enzyme activity was dependent on cultivar, developmental stage, environmental conditions, and conditions of enzyme extraction and assay. Phosphoenolpyruvate (PEP) carboxylase, PEP carboxykinase, and malic enzyme, which catalyze the synthesis of malic acid, are present in grape berries and exhibit higher activities in unripe berries (Hawker 1969; Lakso and Kliewer 1975; Maynhardt 1965; Possner et a1. 1981; Ruffner and Kliewer 1975; Ruffner et a1. 1984; Takanishi and Chrelashvili 1984).

Also, activities of ACP-glucose-starch glucosyltransferase and ATPglucose pyrophosphorylase are low in young berries and increase two- to three-fold during maturity, whereas UDP-glucose pyrophosphorylase and amylase increase six- to seven-fold during berry development (Downton and Hawker 1973). Beta-glycosidase activity increases markedly in grape berries from veraison to maturity and is evenly distributed between skin and pulp (Aryan et a1. 1987; Biron et a1. 1988). Activity of a-L-arabinofuranosidase or a-L-rhamnopyranosidase increased during maturation (Gunata et a1. 1988). Acid invertase activity was inversely correlated to leaf area and sucrose concentration in grapevine leaves (Ishikawa et a1. 1989), whereas in grape berries 4 isoforms of invertase were present with different pH optima. Pectin methyl esterase (Datunashvili et a1. 1976), lipoxygenase (Caryel et a1. 1983; Zamora et a1. 1985), amino-, carboxy-, and dipeptidase (Pallavicini and Peruffo 1977), alcohol dehydrogenase (Molina et a1. 1986; Nicolas et a1. 1987), nicotinamide nucleotide transhydrogenase (Spettoli and Bottacin 1981), starch synthetase (Hawker and Downton 1974), acid phosphatase (Schaffer 1982). and proteolytic enzymes (Peruffo and Pallavicini 1975) activities have all been found in grapevine tissues. F. Polyamines

The polyamines, spermidine and spermine, are nitrogenous compounds that occur ubiquitously in the plant kingdom together with their

426


diamine precursors, putrescine, and agmatine. In addition to these, others that are closely related have been found, both structurally and metabolically (Altman et al. 1983; Smith 1982a,b, 1986). Polyamines stimulate the growth of several higher plants, suggesting that the endogenous concentrations of these amines can be growth limiting (Smith 1982a,b, 1986). The possibility that polyamines act merely as sources of nitrogen when stimulating growth must be excluded at the low concentrations used of less than 100 P.M. The physiological role of polyamines can be explained by their ionic binding with nucleic acids, which may promote transcription and translation, or by the interaction with anionic groups on membranes, preventing leakage and causing stabilization under conditions of stress (Smith 1986). Polyamines that have been reported to occur in either leaves or fruit or callus of grapevines are putrescine, spermidine, spermine, cadaverine, and norspermidine (Adams 1991; Adams et al. 1990; Broquedis et al. 1989; ChristakisHampsas and Roubelakis-Angelakis 1990; Murty et al. 1971). Polyamines have also been implicated in morphogenetic phenomena. In leaf grape segments of 'Thompson Seedless' grown in vitro on basal medium-promoting morphogenetic expression (Katsirdakis and Roubelakis-Angelakis 1991), putrescine was found mainly in the free and soluble conjugated forms, whereas the insoluble-conjugated form was low. An increase of putrescine occurred when roots appeared (ChristakisHampsas and Roubelakis-Angelakis 1990) (Fig. 9.8). The endogenous concentrations of putrescine in 'Thompson Seedless' protoplasts cultured in GCWR culture medium (Katsirdakis and Roubelakis-Angelakis 1992) were 33.8±4.0, 31.1±0.8, and 29.3±3.4 nmol'10-6 leaf protoplasts for soluble, conjugated, and bound putrescine, respectively (M. D. Christakis-Hampsas and K. A. Roubelakis-Angelakis, unpublished material). During culture, these protoplasts showed a maximum uptake rate of labeled putrescine from the second to the fifth day. Uptake was dependent on external pH value. At 5.5 J.tM concentration of labeled putrescine, the optimum pH value ranged from 5.0 to 5.5, whereas at 50 mM it was 8.0. Presence of CaCh at concentrations from 0.01 to 10 mM in the assay medium had no effect on uptake rate of labeled putrescine. The distribution of the labeled polyamine following a 7-h uptake period from an 11-J.tM external concentration during a 7-day sampling period ranged from 13% to 31% and from 69% to 87% in the 27,500-g supernatant and pellet, respectively (M. D. Christakis-Hampsas and K. A. RoubelakisAngelakis, unpublished material). Various reports indicate that polyamine levels and activities of enzymes mediating the biosynthesis of polyamines may increase in response to various plant stresses (Evans and Malmberg 1989). Potassium deficiency resulted in a 20-fold increase in putrescine levels and a six-fold

9.

NITROGEN METABOLISM IN GRAPEVINE (x

427

100) 24 PUT. Total

SPO. Q)

20

SPM y

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4

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Weeks of culture

Figure 9.8. Total polyamines during callogenesis in grapevine leaf segments (from Christakis-Hampsas and Roubelakis-Angelakis 1990).

increase in arginine decarboxylase activity in barley and oats (Richards and Coleman 1952; Young and Galston 1984). Forshey and McKee (1970) detected putrescine in fruit-tree tissues under conditions of potassium deficiency. In grapevines, Hoffman and Samish (1971) found that amine level was proportional to the severity of the potassium deficiency; the amine content of deficient 'Semillon' leaves increased 19-fold over normal leaves. Adams et al. (1990) reported that 'Thompson Seedless' and 'Flame Seedless' leaves showing potassium deficiency symptoms at fruit set contained 21- to 3D-fold higher free putrescine levels with no increase in the amount of the bound form. They provided evidence that "spring fever" is a false potassium deficiency but there was a similarity to true potassium deficiency in that putrescine levels temporarily increased although potassium levels in the tissues were at normal levels. Feeding studies using intermediates in the pathway from arginine to putrescine showed that normal leaves have the enzymes necessary to convert agmative and N-carbamoylputrescine to putrescine (Adams 1991). Whether plants accumulate putrescine as a mechanism against high ammonium

428


concentrations under stress conditions remains to be proven (Adams 1991). IV. NITROGENOUS COMPOUNDS

The concentration of nitrogenous compounds in grapevine organs depends on genetic factors, environmental conditions, and cultural practices (e.g. Alexander 1957; Alleweldt et a1. 1984; Araujo and Williams 1988; Bell et a1. 1979; Castor 1953a,b; Dimotakis 1958; Flanzy and Poux 1965; Gallander et a1. 1969; Juhasz 1983; Juhasz and Kozma 1984, 1986; Kharchidze and Matikashvili 1973; Kliewer 1967, 1971; Kliewer and Nassar 1966; Kliewer and Ough 1970; Kliewer et a1. 1966; Kubota and Shimamura 1989; Lafon-Lafourcade and Guimbertreau 1962; Lohnertz 1988, 1991; Pandey et a1. 1974; Schaller et a1. 1985; Wermelinger 1991; Wermelinger and Koblet 1990; Williams 1987b, 1991).

A. Perennial Parts Roots have the highest and more fluctuating N concentration ranging from 0.4-1.7% (Alexander 1957; Lohnertz 1988; Schaller et a1. 1985). The nitrogen content in roots remains constant early in the growing season and increases thereafter. In 'Cabernet Sauvignon' grapevines nitrogen concentration in the root system increased throughout the growing season (Williams and Biscay 1991). The concentration of soluble N in roots increases during dormancy and reaches maximum just prior to budbreak and decreases thereafter (Kliewer 1967; Schaller et a1. 1985). Labeled nitrogen in the insoluble fraction of roots was higher at 27°C than at 13°C, and higher temperature resulted in an increased soluble and amino nitrogen and a decrease in the insoluble nitrogenous fraction (Kubota and Shimamura 1989). In trunk and canes, total N concentration ranges from 0.3-0.7% and its fluctuations parallel that of roots. The accumulation of N in the parts of the grapevine, trunk, canes, and roots begins before grape maturation and reserves continue to increase until the end of the vegetation period (Lohnertz 1988; Schaller et a1. 1985). Especially in roots, N reserves increase by either N uptake from the soil or by translocation of nitrogenous compounds from the aging parts (Alexander 1957; Conradie 1986; Lohnertz 1988; Schaller et a1. 1985). Williams and Smith (1991) found no effect of rootstock ('ARG#l,' 'Rupestris du Lot' or 'S04') on the concentration of nitrogen in the organs of 'Cabernet Sauvignon'. The concentration, late in the growing season, in mg per g dry weight, was 3.0, 2.7, and 2.4 for roots, trunk, and canes, respectively (Williams and Smith 1991).

9.


429

B. Vegetative Parts Total N in the shoots starts increasing after budbreak and continues to be at the (more or less) same levels up to the end of the vegetation period when it shows an increase due to retranslocation of N from senescing leaves (Alexander 1957; Conradie 1990; Lohnertz 1988; Wermelingerand Koblet 1990; Williams 1987a,b). Williams (1987b) found a linear increase in vine nitrogen concentration from budbreak to 1000 growing degree days later. During this time the accumulation of nitrogen was primarily in the stems and leaves. In leaves the maximum amount of N is reached at full leaf expansion and remains constant until senescence. During much of the season, the nitrogen in the leaves greatly exceeds shoot levels. The nitrogen concentration of vegetative grapevine tissues starts at high levels sharply declining to a relatively constant level thereafter (Alexander1957; Bettneretal.1986). During the growing period, nitrogen concentration of leaves decreases from about 6% in young leaves to 3% in mature leaves and to 1% in aging leaves (Fig. 9.9). The concentration of N late in the growing season, in milligrams per gram dry weight, in the shoots and leaves of 'Cabernet Sauvignon' vines was 20.6 and 3.6, respectively (Williams and Smith 1991). The shoot N concentration and protein content decrease steadily after bud break up to veraison, when it shows a slight increase (Dintscheff et al. 1964). Shoots represent an N sink until the end of the main vegetative growth, when they become instead a source until the beginning of maturation and they terminate the season as a sink (Lohnertz 1988). Shoots have also higher nitrogen concentration, up to 4500 ppm, than the woody tissues; it peaks before anthesis, decreases until veraison and reaches

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UJ

a::

u. 90

~

z

UJ

g 70 a::

I-

Z ..J

«

I-

0

I-

50 7

9

11 LEAF

13

15

17

19

AGE (WEEKS)

Figure 9.9. Changes oftotal nitrogen concentration in "Chen in blanc" leaves [RoubelakisAngelakis and Kliewer. unpublished).

430


the maximum value at the end of the vegetative period (Lohnertz 1988). Williams and Smith (1985) found a correlation between net CO2 assimilation rate and nitrogen content of leaves from 'Thompson Seedless' during leaf senescence. They suggested that leaf nitrogen content could be used as an indication of grapevine leaf photosynthetic capacity subsequent to fruit harvest. Maximum photosynthetic rate was at greater than 3% (dry weight basis) leaf nitrogen concentration.

C. Reproductive Parts Cultivar, degree of maturity, rootstock, temperature, mineral nutrition, crop level, trellising system, and diseases may influence the nitrogenous composition of grapes. In flower clusters nitrate concentration was 120 ppm and reached a peak of 5300 to 8600 ppm at flowering. The berries showed a maximum nitrate concentration before veraison of about 100 ppm. The skin portion of the berries had the highest nitrate concentration (Shaller et a1. 1985). There are two phases of intense N incorporation in grape berries: the first one starts about two weeks before the "pea size" stage of the berries and the second starts at the beginning of maturation and lasts about two weeks (Lohnertz 1988). Towards the end of fruit ripening, the concentrations of soluble and total nitrogen increase again; at harvest half of the N present (Nassar and Kliewer 1966) in the annual structures of grapevines was located in the reproductive parts (Alexander 1957; Kliewer 1968; Lohnertz 1991; Nassar and Kliewer 1966; Wermelinger and Koblet 1990).

In grape berries during maturation, the organic nitrogen in the fruit steadily increases, including total amino acids and protein, while ammonia decreases (Peynaud 1947; Peynaud and Maurie 1953). In immature fruit, ammonium ions account for more than half the total nitrogen. Peynaud (1947) found 19-144 mg of ammonia and 156-879 mg/liter of total nitrogen in grapes grown in Bordeaux. In California, ammonia concentration in wine cultivars ranged from 1.5-18.8 mM, with an average value of 7.7 mM (Ough 1969). Synthesis of amino acids, peptides, and proteins occurs mainly during the last six to eight weeks of berry ripening and during this period ammonia decreases sharply. Kliewer (1968) found that total free amino acids in the juice of 18 grape cultivars increased two- to five-fold during ripening and ranged from 2 to 8 g/liter juice (as leucine equivalents). Kliewer (1968, 1969, 1970) found that the amino acid fraction (amino acids and low molecular weight peptides) accounted for 50 to 90% of the total nitrogen in the juice of 78 grape cultivars and the non-amino acid

9.


431

fraction accounted for 10 to 56%. Eight free amino acids (alanine, yaminobutyric acid, arginine, aspartic acid, glutamic acid, proline, serine, and threonine) accounted for 29 to 85% of the total nitrogen in the juice of grapes and for 50 to 95% of the total free amino acids present. Using microbiological assays, Castor (1953a,b) determined the free amino acids in the juice of seven grape cultivars. Glutamic acid was the most prominent (mean 690 mg/liter; range 270-1070) followed by arginine (mean 400 mg/liter; range 70-1130), histidine, leucine, isoleucine, valine, aspartic acid, phenylalanine, and tryptophan; each averaged 50-100 mg/liter juice. The importance of proline, serine, and threonine was shown later (Alleweldt et a1. 1984; Castor and Archer 1956, Juhasz 1985), when the main amino acids of the 'French Colombard' juice were (given as mg/liter) proline 3490, arginine 1030, serine 480, glutamic acid 270, and threonine 210. A similar range of amino acids in grape juice has been confirmed by many workers (Dimotakis 1958; Gallander et a1. 1969; Lafon-Lafourcade and Peynaud 1959; Lafon-Lafourcade and Guimberteau 1962; Nassar and Kliewer 1966; Juhasz and Kozma 1984). However, there are some notable differences in the amino acid composition of grapes from that cited in the previously-mentioned literature. Terelji (1965) found much more arginine than proline in the juice of 'Servanti'. Kliewer (1969, 1970) showed that arginine was the predominant amino acid in 33 grape cultivars, proline was predominant in 40 cultivars, and fJ-alanine was the major amino acid in 4 cultivars. Annual precipitation and light intensity also affected proline concentration. The cultivars in which alanine was predominant had some American species in their parentage. The amino acids reported in the juice of grapes and their concentrations are given in Table 9.1. Concentrations of arginine and proline differ by as much as ten- to twelve-fold among cultivars and from two- to six-fold between early- and late-harvested fruits of the same cultivar. Ough (1968) reported that the level of proline in grapes ranged from 304-4600 mg/liter and that the 'Cabernet' group of cultivars ('Cabernet Sauvignon' and 'Merlot') are particularly high in proline. Several investigators have also shown that the level of proline in grapes is very closely related to fruit maturity (Lafon-Lafourcade and Guimberteau 1962; Kliewer and Ough 1970). Proline is usually present in larger amounts in grapes during warm rather than cool years (Flanzy and Poux 1965). Generally other amino acids are largely unaffected by temperature. Juhasz (1985) found that in four vinifera cultivars, proline showed an intensive accumulation in grape berries, increasing a 25- to 30-fold during berry ripening. He suggested that this increase was due to proline biosynthesis from other amino acids and that the intensive carbohydrate synthesis during the late season hinders its synthesis. Also, the amount of arginine in 'Thompson

432

Table 9.1.

K. A. RQUBELAKIS-ANGELAKIS AND W. MARK KLIEWER Levels of amino-acids in grape musts. Concentration (mg/liter)

Amino acids a-Alanine p-Alanine a-Aminobutyric acid y-Aminobutyric acid Arginine Asparagine Aspartic acid Citrulline Cysteine Cystine Glutamic acid Glutamine Glycine Histidine Homoserine Hydroxyproline Isoleucine Leucine Lysine Methionine Norvaline Phenylalanine Pipecolic acid Proline Serine Threonine Tyrosine Tryptophan Valine Source:

(California) Five wine grape cultivars Z

(France) Merlot and Cabernet Sauvignon

(California) 18 Grape cuItivars x

Thompson seedless

350

47

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