Horticultural Reviews (Volume 14)

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

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CHICHESTER /

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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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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.

HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

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.

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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.

HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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.

HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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.

HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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.

HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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.

HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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.

HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

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 <100 m altitudes (Santos 1978), while in Ecuador it has been collected up to 800 m (Andersson, 1985b). H. angusta is found at altitudes from 200-1000 m (Santos 1978) and is seen to grow and flower in sheltered conditions in south Florida even with temperatures down to aoe (Ball 1987b, 1988). The increased interest in the genus may stimulate introduction of other species tolerant to cool conditions, if not to frost and freezing. Specific physiological traits are described as follows. 1. Juvenility. Juvenility has not been reported in heliconia, although it

probably exists in the seedling stage. Flowering can begin after the plant has achieved sufficient size to support the energy requirements of reproduction. It has been shown that a or4leaves must be unfurled on the pseudostem before the apex is capable of responding to a photoperiodic stimulus (Criley and Kawabata 1986; Lekawatana 1986; Lekawatana and Criley 1989). Within a species the number of leaves subtending inflorescences seems to be a fairly constant character in field situations, although many more leaves have been observed in some pot trials. 2. Abscission. Flower and bract abscission are two undesirable traits in

the commercial cut-flower trade and suggest to the recipient that the product is old. On the other hand, lack of flower abscission leads to an accumulation of decaying floral tissues (both unsightly and unpleasantly odorous) which necessitate removal by flower growers before shipping. Although only 1 or 2 flowers open in an inflorescence on a given day and abscission occurs within 24 h (Stiles 1975, 1978), the adaptive significance of "one-dayness" is uncertain. In the wild, flowers in a localized patch often abscise synchronously, a trait consistent with synchronous visitation by hummingbird pollinators (Dobkin 1987). Near synchronous pollination and a resulting increase in ethylene production and/or increased ethylene sensitivity in floral tissues are hypothesized as the bases for synchronous floral abscission (Dobkin 1987). Asynchronous abscission is explained on the basis of asynchronous (or lack of) pollination as well as differences in pollen tube growth rate between self and nonself pollen. This may be a mechanism to reduce the extent of selfing in self-compatible species, but the necessary experiments are yet to be conducted. In some species, particularly those with bract liquids (see the following

18

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

paragraphs), the perianth does not abscise but withers within the day and decomposes over time. Remarking on this peculiarity, Skutch (1933) wrote, "The entire arrangement seems a great and foul extravagance of a riotous tropical nature, and is as forbidding as it is bizarre." Bract abscission occurs on only a few species, such as H. episcopalis, under field conditions. For such species their value as cut flowers is limited to local use rather than export shipment. In Hawaii H. chartacea exhibits scattered postharvest bract and cincinnus abscission at different times of the year. This remains a problem, although there is a suggestion of ethylene involvement (R. Paull, University of Hawaii, personal communication). 3. Senescence. In a number of species, bract necrosis begins while the

inflorescence is still opening (e.g., H. imbricata, H. mariae, H. necrobracteata). However, abscission does not always accompany senescence. Flower senescence has been suggested (Dobkin 1987) to be a result of ethylene generation during the growth of pollen tubes down the style, and it culminates in rapid abscission of the epigenous perianth from the ovary whether or not fertilization has occurred. The relatively short vaselife recorded for a number of species is suggested to be the result of insufficient water uptake by the inflorescence (Tjia and Sheehan 1984; Tjia 1985; Donselman and Broschat 1986). Inflorescence senescence is characterized by one or more of the following characteristics: bract tip necrosis, dulling of bract color, withering of bracts or rachis, or flower abscission. Senescence is somewhat delayed by holding flowers at 15-18°e, but a lifespan close to that of an inflorescence on an intact plant is never achieved. 4. Bract liquid accumulation. The erect bracts of heliconias often con-

tain considerable amounts of liquid which may protect submerged fruits and flowers from herbivory (Skutch 1933; Stiles 1979; Wootton and Sun 1990). The origin of the liquid is often assumed to be trapped rainwater as many species flower during the rainy season. Bronstein (1988) determined that this fluid was actively secreted in H. imbricata, with a totally drained bract capable of replacing up to 15 ml in 24 h. One indication that this was of plant origin was its pH of 7.8, while rainwater in the region had a pH of 5.0. As bracts enlarge with age more fluid is secreted to an apparent "fill level" . Such active fluid secretion in the bracts is indicative of a facile water transport in the pseudostem. Since cut flowers do not show great water uptake (Broschat and Donselman 1983a), a large part of the water necessary to maintain bract fluid levels, and inflorescence turgidity is probably due to root pressure.

1.

HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

19

III. HORTICULTURE In the United States, heliconias are grown for cut flowers, potted plants, and interiorscape and landscape uses in Hawaii, California, and Florida. Production statistics are available only for cut flowers. International interest in heliconias grew with the realiza tion that cut flowers for export afforded a source of dollars to the economies of the Caribbean and Central and South American nations. Additionally, Australia, Thailand, Malaysia, Taiwan, and the Philippines have begun commercial culture with an eye to exports to Japan as well as to service their own markets.

A. Cut-Flower Production 1. Cultivated heliconias. Given the diversity of wild heliconias, it is surprising that more species of this genus have not been brought into cultivation. Members of the Heliconia Society International (headquartered at the Flamingo Botanical Gardens, 3750 Flamingo Road, Fort Lauderdale, FL 33330) have been actively engaged in collecting and introducing new species and forms for many years. The Heliconia Society International has designated germplasm repositories in the United States and abroad in an effort to maintain the diversity which is being lost to forest destruction in the tropics. A list of more than 200 species and cultivars with description and color photographs is presented in Heliconia, an Identification Guide (Berry and Kress 1991). Another recent book, Exotic Tropicals of Hawaii (Kepler 1991), describes uses, history of introductions in Hawaii, and horticultural lore and also provides excellent color photographs. The January 1986 issue of the Fairchild Tropical Garden Bulletin is another source of selection and cultural information. Table 1.4 summarizes some horticultural characteristics for many commercially-grown heliconias in Hawaii. Table 1.5 provides similar information for species with prominent foliage characteristics.

2. Statistics. Statistics are mostly unavailable outside of the United States. From the Netherlands, where H. psittacorum was grown under glass in the early 1970s (Van Raalte and Van Raalte-Wichers 1973), flower auctions reported 561,000 stems of all heliconia types sold in 1988 with a value of 933,000 Dfl (P. M. G. Hendrick, personal communication), roughly a value of U.S. $0.75 per stem. Although the number of stems sold declined in 1989, the value per stem increased with hanging types (@ U.S. $1.88 per stem) and H. caribaea (@U.S. $1.30) leading the way (VBA 1989). The fast growth rate of heliconias and relatively high prices in the

I:\J

0

Table 1.4.

Heliconias grown as cut flowers in Hawaii: species and cultivars, inflorescence characteristics, seasonality, and growth habits.

Inflorescence orientation

Bract color

Flower color

Keeping Y quality SeasonalityX (days)

Height (m)

Growth habit

Nov.-Feb.

0.6-1.2

5-6

Nov.-Feb.

0.6-1.5

White

8-10

Oct.-Apr.

1.6-2.0

Slow spreading Slow spreading Spreading

Orange

Pale yellow

8-10

Dec.-Mar.

1.6-2.0

Orange Yellow rim red base Gold rim dark red base Yellow

Yellow Base white, tip green Base white, tip green Base white, tip green

5-7 4-5

Jan.-Apr. Jan.-Aug.

0.8-1.5 1.2-2.0

Slow spreading Clumping Clumping

to 10

Nov.-Apr.

2.0-3.7

Clumping

10-12

Year-round

2.5-4.0

Clumping

Species

CultivarsZ

H. angusta

Small Red Christmas Holiday (= Red Christmas) Yel. Christmas (= "Flava")

Upright

Red

White

5-6

Upright

Red

White

Upright

Yellow

Orange Christmas

Upright

Kamehameha

Upright Upright

Chocolate

Upright

Yellow

Upright

H. aurantiaca H. bihai

Z Names shown in double quotes (" ") have been used as invalid species names, but are still used in commercial trade to identify some selections. v Keeping quality estimates are from day of harvest. Estimates provided by Hawaii growers and florists. H psittacorum vaselife studies (Broschat and Donselman 1983a; Tjia and Sheehan 1984; Tjia 1985) suggest these figures may over-estimate vaselife by 4 to 5 days. Criteria for end of useful vaselife was not defined by the growers and florists consulted. xSeasons of flowering may extend a few weeks ahead of or later than the months shown due to local environments. W Many other selections of H. psittacorum have been named; omission from this table implies lack of information about their performance in Hawaii.

Table 1.4.

Species

Continued. Keeping" quality SeasonalityX (days)

Height (m)

Growth habit

July-Dec.

2.5-4.0

Clumping

to 21

Mar.-July

2.0-3.7

Clumping

Base white, tip green

10-12

Year-round

2.0-3.7

Clumping

Base white, tip green

1G-12

Year-round

2.0-3.7

Clumping

Upright

Green rim Base white, dark green band, tip green red-orange base

1G-12

Jun.-Sept.

2.0-3.7

Clumping

Nappi

Upright

Green rim wide yellow band; light orange base

Base white, tip green

Jun.-Aug.

2.0-3.7

Clumping

Kawika

Upright

Wide Yellow rim, red base

Base white, tip green

Mar.-Sept. (peak in May-June)

2.5-4.0

Clumping

CultivarsZ

In florescence orientation

Bract color

Flower color

Green

Upright

Green

Base white, tip green

6-10

Lobster Claw

Upright

Dark green rim, orange base

Base white, tip green

Claw #2

Upright

Green rim light & dark orange, no yellow below rim

Yellow Claw #2

Upright

Green rim, Yellow band, below rim red-orange base

Trinidad (Trinidad Balisier) (= Claw #3)

(= "Humilis" = Claw #1)

to 7

....N continued

~ ~

Table 1.4.

Continued. Keeping Y quality (days) Seasonality"

Cultivarsz

Inflorescence orientation

Bract color

Flower color

Manoa Sunrise

Upright

"Jacquinii" (= "Aurea") Richmond Red

Upright

Kawauchi

Upright

H. bourgeana

Rose Bourg.

Upright

Gold rim, Red base Yellow rim Red base Yellow rim Red base Yellow rim Red base Rose-pink

H. caribaea

"Purpurea" (Red)

Upright

Red

Base white, tip green Base white, tip green Base white, tip green Base white, tip green Base white, tip green Base white, tip green

Cream

Upright

Cream-yellow

Base white, tip green

to 21

Yellow

Upright

Yellow

Base white, tip green

10-14

Green (Chartreuse)

Upright

Green

Base white, tip green

to 21

Species

H. bihai X H. caribaea

Upright

Height (m)

Growth habit

8-12

Mar.-Nov.

3.0-4.5

Clumping

4-8

Mar.-Sept.

1.6-3.0

Clumping

12-14

Year-round

2.5-4.0

Clumping

8-12

May-Sept.

3.0-4.5

Clumping

4-6

Apr.-Sept.

2.5-4.5

Clumping

to 21

Year-round peaks in both winter & summer Year-round peaks in both winter & summer Year-round peaks in both winter & summer Year-round

3.0-4.5

Clumping

3.0-4.5

Clumping

3.0-4.5

Clumping

3.0-4.5

Clumping

Table 1.4.

Continued. Growth habit

Mar.-Apr.. Oct.

3.0-4.5

Clumping

to 21

May-June

2.5-3.0

Clumping

to 21

May-June

2.5-3.0

Clumping

8-10

Year-round

1.8-3.0

Clumping

Green

8-10

Year-round

1.8-3.0

Clumping

Yellow

5-8

May-Dec.

3.6-4.5

Clumping

White to pale orange

3-4

Year-round

1.0-3.0

Spreading

Yellow Yellow-green

7-10 3-4

Dec.-Mar. Year-round

2.0-2.0 1.0-1.5

Clumping Clumping

Yellow Light green to yellow

3-4 5-8

Year-round Year-round

1.0-1.5 2.5-3.0

Clumping Clumping

Flower color

Flash

Upright

Base white. tip green

8-14

Honduras (Maya Gold) Splash

Upright

Wide yellowgreen margin. rose base Yellow

Upright

H. chartacea

Sexy Pink

Pendent

Green outside white inside Green outside white inside Green

Pendent

H. collinsiana

Maroon Sexy (Marisa) "Pendula"

H. episcopalis

Spear

Upright

H. farinosa H. hirsuta

Rio var. hirsuta

Upright Upright

H. indica

Red var. indica

Upright Upright

H. champneiana

l\:)

Height (m)

Cultivarsz

Bract color

Species

c..J

Keeping Y quality SeasonalityX (days)

Inflorescence orienta tion

Pendent

Yellow. red spots Green rim, pink base Green rim, rose base Pink to reddish Yellow tips. orange basally Red Yellowgreen Red Green

continued

N

~

Table 1.4. Continued. Inflorescence orientation

Bract color

Flower color

var. rubracarpa Pink Stripe

Upright

Red to pale green

var. micholitzii

Upright

"Spectabilis"

Upright

"Striata" Yellow Stripe March Christmas

Upright Upright

Narrow red margin & rachis. green Thin pink margin. green to yellow-green at rachis Green with lighter striations Green with yellow stripes Red

H. lasiorachis

Fuchsia (=Kimi)

Upright

Pinkish

H. latispatha

Red Red & Yellow

Upright Upright

Orange

Upright

Red Yellow margin. red base Orange

Yellow

Upright

Yellow

Species

H. laneana

Cultivarsz

Keeping Y quality (days) Seasonality" 5-8

Green to pale green

Height (m)

Growth habit

Year-round

2.0-3.0

Clumping

Year-round

4.0-8.0

Clumping

Reddish-green

5-8

Year-round

2.0-5.0

Clumping

Pale green

5-8

Year-round

2.0-3.0

Clumping

White with yellow ovary Green

5-7

Jan.-Mar.

1.0-1.5

Clumping

4-7

Year-round

1.0-3.0

Yellow

3-7

Apr.-oct.

1.8-2.4

Slow spreading Spreading

Yellow YellowOrange Yellow

Table 1.4.

Continued. Keeping Y quality SeasonalityX (days)

Height (m)

Growth habit

May-Nov.

1.5-2.5

to 7

May-Nov.

1.5-2.5

Slow spreading Slow spreading

Yellow-orange Dark green, white base White

4-7 8-10

Jan.-June June-Mar.

1.2-2.0 1.6-2.1

Clumping Clumping

5-8

1.5-2.0

Clumping

White

5-8

Year-round Winter peak Mar.-June

1.5-3.0

Clumping

Yellow

3-5

July-Sept.

4.0-5.0

Clumping

Yellow

to 7

Apr.-Sept.

1.5-2.5

Slow spreading

Species

Cultivarsz

Inflorescence orientation

H. lingulata

Fan, Yellow Fan

Upright

Yellow

Yellow

to 7

Red-tipped Fan

Upright

Yellow

Hanging Orange Mini Claw

Pendent Upright

(formerly

Pendent

Yellow with reddish tips Red-orange Dark green rim Dull red Red

H. nutans H. orthotricha H. pendula

Bract color

Flower color

H. revoluta)

H. platystachys

Frosty

Pendent

Sexy Orange

Pendent

(= Red & Yellow

H. pseudo-

aemygdiana (?) H. psittocorumw

N

c:Jl

pendula) Pagoda Spiral Fan

Upright

Reddish base to pale pink margin to white tip Yellow margin, red base

Yellow

(all dark green or black tip) continued

l\:l

O'l

Table 1.4.

Species

Continued. Keeping Y quality [days) Seasonality"

Height (m)

Growth habit

Year-round

0.6-2.0

Spreading

5-7

Year-round

1.0-2.0

Spreading

7-14

1.0-2.0 1.0-2.0

3-5

Year-round Year-round Year-round

0.6-2.0

Spreading Spreading Spreading

5-10

Year-round

1.0-2.0

Speading

Green Orange White Yellow Green Light yellow

10-14

Year-round Year-round Year-round Year-round Year-round Year-round

1.0-2.0

Spreading Spreading Spreading Spreading Spreading Spreading

Yellow Yellow Orange Orange

10-14

Year-round Year-round Year-round Year-round Low in winter

1.0-2.0

Cultivarsz

Inflorescence orientation

Bract color

Flower color

Andromeda

Upright

Orange

4-20

Barbara

Upright

Yellow

Black Cherry Claire Choconiana [= Orange) Emerald

Upright Upright Upright

Light orange base to red tip Rose base green tip Dark red Rose pink Orange

Cream Orange

10-14

Green

Fuchsia Kathy Lady Di

Upright Upright Upright Upright Upright Upright

Green base to pink tip Rose pink Red Rose red Bright pink Dull pink Yellow base to pink tip Bright orange Bright pink Red Light green base. pink tip

Lizette Lucille Parakeet (= "Rhizomatosa") Petra Rosie S1. Vincent Red Sassy [= Kaleidoscope)

Upright

Upright Upright Upright Upright

7-14 3-5 10-14 7-10 5-6

10-20 10-20

1.0-2.0 0.6-1.5 1.0-2.0 1.0-2.0 1.0-2.0

1.0-2.0 1.2-2.0 1.0-2.0

Spreading Spreading Spreading Spreading

Table 1.4.

Continued.

Species

Cultivarsz

Inflorescence orientation

Bract color

Flower color

Suzy

Upright

Bright pink

Light yellow

Sybille

Upright

Light pink

Yuki

Upright

Pink

Golden Torch

Upright

Growth habit

Year-round

1.0-2.0

Spreading

Light yellow

10--20

Year-round

1.0-2.0

Spreading

Bright green

10--20

Year-round

1.0-2.0

Spreading

Gold-orange

Gold-orange

7-15

Year-round

1.0-2.0

Spreading

Upright

Orange. Red rachis

Orange

3-5

Year-round

1.0-2.0

Spreading

H. x nickeriensis Nickeriensis (= Nicky)

Upright

Gold-yellow margin. red base

Yellow

3-5

Year-round

1.0-2.0

Spreading

H.

Flexuous

Red

White with green tip

4-17

Apr.-Dec. Peak in May-June

2.0-2.5

Clumping

Upright

Yellow. red base

Yellow

3-5

May-June

1.3-1.8

Clumping

3-5

Mar.-July

1.5-2.1

Slow spreading

(= Parrot)

Double B Gold

X

rauliniana

H. richardiana H. rostrata

Parrot's Beak

Pendent

Yellow margin. red base

Yellow

H. sampaioana H. solomonensis

Alii

Upright

Red

Green

5-7

Dec.-Mar.

1.8-2.0

Clumping

Pendent

Light green

White with green tip

7-10

Year-round

5.0-7.0

Clumping

H. stricta

-..:a

Height (m)

10--14

H. psitlacorum W x H. spathocircinata

!'l

Keeping Y quality SeasonalityX (days)

(All with white flower & green band near white tip) continued

t>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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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.

HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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.

HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

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).

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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.

HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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.

HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

(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.

HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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.

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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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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

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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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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

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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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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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HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

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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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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).

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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

RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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

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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

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RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

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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HELICONIA: BOTANY AND HORTICULTURE OF A NEW FLORAL CROP

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.

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RICHARD A. CRILEY AND TIMOTHY K. BROSCHAT

___ . 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

L. KAWA AND A. DE HERTOGH

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.

ROOT PHYSIOLOGY OF ORNAMENTAL FLOWERING BULBS

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.

ROOT PHYSIOLOGY OF ORNAMENTAL FLOWERING BULBS

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

L. KAWA AND A. DE HERTOGH

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.

ROOT PHYSIOLOGY OF ORNAMENTAL FLOWERING BULBS

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 <20°C, roots can be formed before plantin g, but his is undesi rable. Nerine spp. produc e few offsets and they are also propag ated by twin scales (Bulb and Corm Produc tion 1984). The propag ation system is basical ly the same as describ ed for Narcissus. No inform ation on root format ion was reporte d. Thus, it presum ably occurr ed after plantin g. Other genera that can be propag ated by twin scales include Allium spp., Amary llis belladonna, Chionodoxa, Galanthus, Haema nthus multiflorus, Hyacin thus, Hymenocallis, Iris hollandica, Lachenalia, Leucoj um aestivu m, Muscari, Ornithogalum, Pancratium, Scilla, Sternbergia, and Veltheimia capens is (Alkema and Van Leeuw en 1977). 2. Tissue Cultur e. Tissue culture system s have been extens ively

researc hed for many flower ing bulbs (Hussey 1980a,b) (Table 2.3). The import ant aspect for rooting is that they are formed during Stage III of the tissue culture proces s (prepa ration for reestab lishme nt of plants in soil]. Generally, rooting is effecte d by transfe rring the culture d shoots to a

2.

ROOT PHYSIOLOGY OF ORNAMENTAL FLOWERING BULBS

67

medium that is either low or has no cytokinin, but contains an auxin like indolebutyric acid (IBA) at 0.1-1.0 mg/liter (Hussey 1980b). An alternative is to transplant the in vitro shots into sterile cubes of plastic foam that have been soaked in the appropriate rooting medium. A summary of the conditions reported to promote rooting of flowering bulbs is presented in Table 2.3. III. ROOT MORPHOLOGY

A. Branching Habit Although most higher plants produce branched root systems (Whittington 1969), several ornamental flowering bulbs do not (Table 2.1). Unfortunately, the presence or absence of branching roots has not been reported for many flowering bulb genera and needs to be determined for all ornamental geophytes. No branching has been observed (Table 2.1) for the following genera: Allium, Crocus, Frittilaria, Hyacinthus, Muscari, Narcissus, Scilla, and Tulipa. The selective advantage of nonbranching roots is unknown and should be investigated. Presumably, these roots have the ability to absorb water and nutrients along the entire root length as opposed to a specified region of uptake.

B. Contractile Habit The major function of contractile roots (CR) (Fig. 2.1) appears to be the positioning of the storage organ at a level in the soil for optimal growth and survival of the species. Rimbach (1929) has shown that CR are present in 450 species of 315 genera of gYmnospermous, monocotyledonous, and dicotyledonous families. The ornamental flowering bulb genera in which those roots were found are listed in Table 2.4. Root contraction can occur in tap, lateral, or adventitious roots. M. Aharoni (personal communication) indicates that CR occurs on Lillium cultivars being propagated in tissue culture. Thus, it is not necessary for the bulbs to be in soil in order to contract. In Crocus sativus, the total number of basal roots was unaffected by planting depth, but the number of CR was decreased in corms planted at 20 em compared to those at 5 em (Negbi et ai. (1989). In Gladiolus, the greatest number of CR were produced at 5 em planting depth and the number decreased in corms placed at 0, 15, and 30 em (Iziro and Hori 1983c). Halevy (1986a,b) also found that the number of CR decreased as the planting depth of Gladiolus corms increased. Moreover, he found that only small- and medium-sized corms produced CR. Large corms did not produce CR at any planting depth, but divided into two or more small

68

Table 2.4.

L. KAWA AND A. DE HERTOGH Flower bulb genera with contractile roots. Z

Taxa

Type of Storage Tissue

Family

Aconitum napellus Agapanthus umbellatus Allium ursinum Amaryllis belladonna Anemone coronaria fulgens Arisaema dracontium triphyllum Arum macula tum Begonia baumannii Belamcanda chinensis Bravoa geminiflora Brodiaea capitata Caladium bicolor marmoratum Calochortus umbellatus Calydorea nuda Camassia esculenta Canna indica Chlidanthus fragrans Commelina coelestis Convallaria majalis Crocus imperati Dahlia variabilis Eucharis amazonica Eucrosia bicolor Freesia refracta Fritillaria meleagris Galtonia candicans Gladiolus communis Habranthus andersonii Hemerocallis flava Hermodactylus tuberosus HymenocaHis calathina littoralis quitoensis Hypoxis viIlosa Ipheion uniflorum Iris germanica persica pseudacorus pumila xiphium Ixia viridiflora Leucojum vernum Lilium bulbiferum candidum martagon pardalinum

Tuberous-roots Rhizome Bulb Bulb Tuber Tuber

Ranunculaceae Amaryllidaceae Amaryllidaceae Amaryllidaceae Ranunculaceae Araceae

Tuber Tuber Tuber Tuber Corm Tuber

Araceae Begoniaceae Iridaceae Agavaceae Liliaceae Araceae

Bulb Corm Bulb Rhizome Bulb Tuber Rhizome Corm Tuberous-roots Bulb Bulb Corm Bulb Bulb Corm Bulb Tuberous-roots Tuberous-roots Bulb

Liliaceae lridaceae Liliaceae Cannaceae Amaryllidaceae Commelinaceae Liliaceae Iridaceae Compositae Amaryllidaceae Amaryllidaceae Iridaceae Liliaceae Liliaceae Iridaceae Amaryllidaceae Liliaceae Iridaceae Amaryllidaceae

Corm Bulb Rhizome Rhizome Rhizome Rhizome Bulb Corm Bulb Bulb

Hypoxidaceae Amaryllidaceae Iridaceae

Iridaceae Amaryllidaceae Lilioceae

2.

ROOT PHYSIOLOGY OF ORNAMENTAL FLOWERING BULBS

Table 2.4.

69

Continued.

Taxa

Type of Storage Tissue

Family

Muscari racemosum Narcissus jonquilla tazetta Orithogalum arabicum umbellatum Oxalis cernua Phaedranassa chloracra Polianthes tuberosa Scilla bifolia Stenomesson aurantiacum Tigridia pavonia Trillium ovatum Watsonia aletroides Zantedeschia aethiopica Zephyranthes atamasco Zygadenus fremontii

Bulb Bulb

Liliaceae Amaryllidaceae

Bulb

Liliaceae

Bulb Bulb Bulb Bulb Bulb Bulb Tuber Corm Rhizome Bulb Bulb

Oxalidaceae Amaryllidaceae Agavaceae Liliaceae Amaryllidaceae Iridaceae Liliaceae Iridaceae Araceae Amaryllidaceae Liliaceae

ZFrom Rimbach (1929).

corms that produced CR and adjusted their depth in the subsequent growth cycle (Halevy 1986b). Iziro and Hori (1983a) reported that dry weight of CR increased with growth of the leaves and it declined subsequently after contraction of the roots. Under these conditions, the number of CR was unaffected by either root or air temperature, but 12°C promoted maximum elongation and thickening of the CR (Iziro and Hori 1983b). In contrast, Halevy (1986a,b) reported that the number and weight of CR increased as the difference between the day and night temperatures increased. He demonstrated that the induction temperature was perceived at the root initiation zone. In addition to temperature, light was implicated in regulating the formation of CR (Jacoby and Halevy 1970; Halevy 1986b). When grown in darkness, CR were not formed in small corms planted shallowly under fluctuating temperatures. The light energy needed to induce CR had to be of relatively high intensity and had to be applied for a minimum of 2 weeks. Red light was found to be the most effective light in the induction of CR (Halevy 1986b). Various growth substances applied to the leaves or corms did not induce CR in dark grown plants. However, foliar sprays and soil drenches with GA 3 and IBA were effective in substituting for the fluctuating temperature requirement. GA 3 and IBA promoted the formation of CR on plants grown from small corms and on those developed from large corms that normally did not produce CR (Halevy 1986b). Oxalis bowieana is a bulb that only developed 1 CR (Iziro and Hori 1983a,b,c,d). As with Gladiolus, the dry weight of the CR increased with

L. KAWA AND A. DE HERTOGH

70

leaf growth and decreased with root contraction. In contrast to Gladiolus, growth of the CR increased with planting depth and the root contracted even when the bulb was planted at the soil surface. Air and soil temperatures did not affect the number of CR formed, which was one. The most intensive studies on the mechanisms of contraction were carried out on Hyacinthus orientalis (Fig. 2.1). Wilson and Honey (1966) grew bulbs on a hydroponic system and measured cell dimensions (Table 2.5) and osmotic pressure before and after contraction. The inner cortical cells expanded radially and contracted longitudinally during the process. The contraction occurred only in turgid tissue. They considered root contraction to be a growth process because the cells increased in volume and area and the cell wall changes were obvious. CYr et aI. (1988) and Smith-Huerta and Jernstedt (1989, 1990J examined cellular (Fig. 2.1J and subcellular changes associated with root contraction in Hyacinthus. During contraction, there was an increase in the tubulin and total protein content and the number of microtubules per cell. There were also changes in the orientation of the cortical microtubules and the cellulose microfibrils. Jernstedt (1984) showed that a brief exposure of the roots of Hyacinthus to 0.5-1.0 mg/liter of indoleactic acid (IAAJ in a hydroponic solution induced subapical swelling, root cap proliferation, and a decrease in root elongation. Following the IAA treatments, contraction did not occur in the enlarged area of the roots, but occurred above and below the region. Additional hormonal studies Table 2.5. Characteristics of cortical cells, as seen in radial view. of uncontracted and contracted portions of roots of Hyacinthus orientalis. Z Contracted roots Y

Uncontracted roots (y) (a) Cell length (}.un)

(b) Cell breadth (}.tm)

Radial wall area (a) X (b)

Pitch of helix (degrees)

(a) Cell length (}.tm)

(b) Cell breadth (}.tm)

Radial wall area (a) X (b)

Pitch of helix (degrees)

390 239 299 365 353 309 329 384 374 303

100 94 105 58 88 78 84 110 118 74

39000 22500 31200 21100 31100 24000 27500 42300 44100 22500

64 60 62 62 62 66 60 60 61 57

227 193 239 268 212 271 270 376 256 177

287 123 137 177 213 194 165 173 206 200

64900 23800 32800 47400 46300 52600 44500 64900 52900 35300

53 55 50 52 56 48 49 51 51 50

zFrom Wilson and Honey (1966). YEach figure is a mean of observations on not less than 30 cells.

2.

ROOT PHYSIOLOGY OF ORNAMENTAL FLOWERING BULBS

71

are needed to elucidate the control mechanism of cellular contraction. Anatomical studies of root contraction have been reported for Eucomis (Reyneke and Van Der Schijff 1974), Freesia (Ruzin 1979), Gladiolus (Sterling 1972), and Narcissus (Chan 1952; Chen 1969). The anatomical responses of these genera were all similar to Hyacinthus, but further elucidation of the mechanisms involved are not presented.

c.

Root Hairs

It is assumed generally that all plant roots have root hairs, but this is not true for some ornamental flower bulbs (Table 2.1). Crocus flavus and some Tulipa cultivars do not have them (De Munk and De Rooy 1971; Chilvers and Daft 1981). Botschantzeva (1982) found root hairs in only four Tulipa species. In our research (unpublished), we have not observed root hairs on Crocus vernus or Muscari armeniacum 'Early Giant' (Table 2.1).

Because these genera do not have root hairs and are herbaceous in nature, we can assume that uptake occurs over the entire root surface. P. V. Nelson (personal communication) has indicated that Tulipa roots begin absorbing N0 3 -N as soon as they emerge from the basal plate. During the winter season, Tulipa plants accumulate a large amount of N in the roots, and the N is used for the rapid shoot growth that occurs in the spring (Ohyama et a1. 1988). Ethylene in the soil atmosphere induced root hair formation in Tulipa 'Apeldoorn' (De Munk and De Rooy 1971; Chilvers and Daft 1981). This is a valuable marker to identify when there are rooting problems with Tulipa bulbs.

D. Tuberization Habit Only a limited number of ornamental geophytes have tuberous-roots as the primary storage organ (Tables 2.1; 2.4). In most cases, the primary root from the seed does not persist and the tuberous-roots that are formed are adventitious in nature. The Dahlia is a genus in which the tuberization process was studied in detail. Moser and Hess (1968) demonstrated that only 5 short-day cycles are required to induce tuberization. The critical daylength is 11-12 hand the maximum rate of tuberization occurs at 16-21 DC. Tuberization is inhibited at 10 DC or 27 DC. GA3 applications inhibit enlargement of the roots of plants grown under long or short days. When daminozide, a plant growth retardant, is applied as a spray it only promotes tuberization under long days. Similar studies with exogenous plant growth regulators (PGRs) are needed for other genera that develop tuberous-roots as the primary storage organ.

72

L. KAWA AND A. DE HERTOGH

IV. ENDOGENOUS FACTORS

Biran and Halevy (1973c) determined the endogenous levels of auxins, rooting cofactors, and inhibitor activities in easy- and difficult-to-root Dahlia cultivars. They found no differences in the levels of either extracted or diffusible auxins or in the levels of rooting cofactors. A higher inhibitor activity was obtained from the difficult-to-root 'Orpheo', while cuttings from easy-to-root cultivars had lower levels. From chromatographic analyses, they concluded that abscisic acid (ABA) was not a component of the inhibitor fraction. Grafting experiments showed that repression of root initiation and growth was transmissible. Tulipa roots contained both free and bound forms of gibberellins (Aung et al. 1969; Einert et al. 1972) and a high level of ABA (Aung and De Hertogh 1979). Roots appeared either to be biosynthetic sites of these hormones or they provided the necessary precursors for their synthesis. Le Nard (1985) studied the effects of roots on flower initiation in Tulipa 'Apeldoorn' and 'Paul Richter'. When rooted, larger bulbs were required to initiate flowers compared with bulbs without roots. This response may be related to the GAs or ABA produced in the roots and translocated to the meristem or it could reflect source/sink relationships. Photoassimilates stored in CR were translocated to the developing corms or bulbs (Iziro and Hori 1983d). Iziro and Hori (1983a) calculated that CR of Gladiolus subsequently contributed 22.4% to the enlargement of the corm, while in Oxalis bowieana it contributed 64.7%. It is obvious that more studies are needed on the endogenous control of root development and their effects on growth and development of ornamental geophytes.

V. EXOGENOUS FACTORS

A. Temperature Blaauw and his co-workers (Hartsema 1961) clearly established that temperature is a critical factor in the control of growth and development of flower bulbs. This is true not only for floral development but also for root growth. 1. Tulipa. Le Nard (1972) demonstrated that when freshly harvested 'Paul Richter' bulbs were given a short postharvest treatment of 30°C and then transferred to 15°C, rooting was earlier than in bulbs placed directly at 15°C. Following a treatment of one week at 30°C, bulbs stored at 20°C

2.

ROOT PHYSIOLOGY OF ORNAMENTAL FLOWERING BULBS

73

rooted earlier than those stored at 15°C. In contrast, when the 30°C treatment lasted 3-5 weeks, rooting of bulbs subsequently stored at 15°C was slightly enhanced over those bulbs stored at 20°C. When the bulbs were planted after a 12-week precooling treatment at 5°C, extending the prestorage treatment at 30°C led to earlier growth and increased weightofthe roots (Le Nard 1980). He concluded that the effect of 30°C applied immediately after harvest was quantitative and that there was no relationship between the earliness of stage G (formation of gynoecium) and earliness of rooting. Jennings and De Hertogh (1977) demonstrated that 17°C was the optimal temperature for root growth of planted 'Paul Richter' bulbs (Fig. 2.3). After 3 weeks the bulbs had 70 mm of root growth, which is considered an optimal length for forcing. Wilson and Peterson (1982), who used an underground rooting facility (rhizotron), showed that root growth of T. kaufmanniana did not cease when the soil temperature reached 3°C under field conditions. Presumably, root growth would cease at soil temperatures below O°C. Benschop (1980) found that roots of 9/10 em 'Apeldoorn' bulbs planted in the field always ranged from 0.400.45glbulb. This growth was about 60-70% less than roots from similarsized bulbs grown in containers. E. Fortanier (unpublished data) found similar results. Thus, the environment for root growth in pots was superior to that in the field. Benschop (1980) also found that root growth was temperature dependent and that increases in root dry weight ceased when the shoots emerged from the soil in the spring. This may reflect a significant physiological change in source/sink relationships in TuIipa. 2. Hyacinthus. Jennings and De Hertogh (1977) found that Hyacinthus had an optimal range for rooting of 17-25°C and that 70 mm of root growth was reached in 10-14 days. This high optimal range of rooting of Hyacinth us reflects the Mediterranean origin of the species (Bryan 1989).

3. Narcissus. Jennings and De Hertogh (197) found that Narcissus had a distinct optimum at 17°C and that 70 mm of root growth was achieved in 3-4 weeks. Under field conditions, Wilson and Peterson (1982) observed that N. lobularis ceased root growth at 3°C. 4. Lilium. In L. longiflorum 'Ace', De Hertogh and Blakely (1972) found the optimal rooting temperature was 21°C for non-precooled bulbs and 17-21°C for precooled bulbs. Growth of the basal and secondary roots responded similarly. This was in agreement with White's report (1940) on L. longiflorum 'Giganteum', which had a rooting optimum of 20-21°C. 5. Eranthis and Anemone. Zimmer and Girmen (1987) showed that E. hiemalis rooted quicker at 5°C than at higher temperatures. In contrast,

74

L. KAWA AND A. DE HERTOGH

/\

\

~j

D. \

100 ..

80

r" TEMPERATURE r-C)

CD

m ..J

..J

~

WATER

......

-....

iiiO

(!)

14{)

W

12.0

:I:

:::> ...... E

m

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lOX)

6 0

S,

,/

,//'

..s :I:

G z

8-0

W ..J

a::

b o

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W

a::

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BENOMYL

a

F.

ETHAZOL

16.0

140

120

100

80 60 40

20 5

9

13' 'I'f- 2''''

TEMPERATURE C·C)

Figure 2.3. Effects of preplanting treatments, temperature and sampling-time on root development of Tulipa 'Paul Richter'. (A) Fresh root weight (glbulb) of the non-dipped bulbs (control). (B) Fresh root weight (glbulb) of 3D-min. preplanting-water-dipped bulbs. (C) Fresh root weight (glbulb) of 3D-min. preplanting-benomyl-ethazol-dipped bulbs. (D) Root length (mmlbulb) of nondipped bulbs (control). (E) Root length (mmlbulb) of 3D-min. preplanting-benomyl-water-dipped bulbs. (F) Root length (mmlbulb) of 3D-min. preplanting-benomyl-ethazol dipped bulbs (from Jennings and De Hertogh 1977).

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75

A. blanda rooted quicker at 17°C than at lower temperatures. Unfortunately, the precise upper and lower temperature limits were not established for these genera. These results illustrated highly contrasting rooting temperature optima for two species that originated in the same geographical area. It appears that A. blanda will root when the soil temperatures are warmer, while E. hiemalis roots laterat lower temperatures. This response must be related to specific survival mechanisms for these species. 6. Allium, Scilla and Iphieon. Wilson and Peterson (1982) found in their rhizotron study that root growth of A. moly does not cease when the soil temperatures reaches 3°C. In contrast, root growth of S. siberiea and 1. uniflorum ceases at 3°C, but it resumes when the temperatures warm in the spring. Preplanting storage temperatures have a marked effect on root length of A. neapolitanum, with 15°C being optimal (Zimmer and Weckeck 1989). Postplanting temperatures have a significant effect on the root dry matter ofA. aflatunense andA. christophii, with 5°C and 14°C being optimal, respectively (Zimmer and co-workers 1984, 1985a,b, 1989).

B. Moisture Moisture interacts markedly with temperature and strongly influences the development of certain root and foliage diseases. The importance of these interactions depends to a large extent on the species and/or cultivar. To program bulbs of Ulium longiflorum 'Georgia' for Easter, Stuart (1954) showed that the peat used for bulb storage mustcontain 69%moisture. In contrast, when L longiflorum or L. speciosum bulbs are stored for long periods they must be held at -0.5°C in peat with 30-50% moisture and wrapped with a polyethylene liner. Prince and Cunningham (1990) used L.longiflorum 'Nellie White' and confirmed that Easter lilies require specific moisture levels during storage for proper programming. They showed that bulbs stored in sphagnum peat at 76% moisture content without a polyethylene liner produce significantly more roots when examined 2 weeks after planting. This effect was not statistically significant 4 weeks after planting, but the trend was evident. Most flower bulbs require excellent drainage either in the field or in the containers. This is related to aeration of the soil or planting medium and the moisture content (See Section V.C of this review). Blaauw (1938) showed that the level of the water table had a significant effect on root development of Tulipa, Hyacinthus, and Iris hollandica. Generally, a water level of 60 em produces the highest root dry weight.

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Bulbs such as Crocus, Hyacinthus, and Tulipa can be grown hydroponically (De Hertogh 1989). Jernstedt (1984) and Wilson and Honey (1966) used this technique to study root contraction in Hyacinthus. When bulbs are grown hydroponically, it is important that only the root plate be submerged in the solution. The scale tissue must not be covered and presumably it is this bulb tissue that requires the aeration (Van Der Valk 1971). We have observed (unpublished data) root branching and root hairs on tulips grown hydroponically at 9°C without aeration, an obvious stress condition.

C. Soils and Artificial Planting Media For bulb production, almost all flower bulb genera are planted either outdoors in fields or in greenhouses with ground beds (Bulb and Corm Production 1984; Langeslag 1988). De Haan and Van Der Valk (1971) have shown that compaction of the soils reduces root growth of Tulipa, Narcissus, and Hyacinthus. Root growth does not occur at pore-volumes of <43%. Restriction of root growth due to aeration is usually only slight if the air volume content of the soil is >10% (Van Der Valk 1971). Bulb sensitivity to reduced aeration is greater in the fall than in the spring. Presumably, this is because the bulb is fully rooted by spring (Boekel 1971). Wiersum (1971) reports similar results with only Tulipa. Although undocumented, roots of flowering bulbs are very sensitive to high salts (Fig. 2.4). Very high or low pH can cause similar injury. P. V. Nelson (personal communication) has found that the optimal pH range for tulips is 5.3-7.3. The precise pH range needs to be established for all widely used flower bulbs. Versluys (1927) compared the growth of Hyacinthus in liquid cul.ture and sand and found a root length of 51 cm in sand and on 1:- :~ ern in solution culture. De Hertogh and Tilley (1991) - _.<1.lpared nine artificial planting media on the root growth of four cultivars of Hippeastrum (Table 2.6). Regardless of the planting mledia used, 'Apple Blossom' lost the greatest amount of old basal roots. The F3B, SM4, and SPH mixes preserved the most roots of this cultivar. Based on this response and the promotion of new basal and secondary root growth of the 3 cultivars used, SM4, F2, F3B, and the SPH mixes were found to be the best planting media for Hippeastrum. In contrast, the NCB, NCS, and the ASB were found to be the poorest mixes. Similar studies and the delineation of precise physical and chemical characteristics of the planting media are needed for all forced bulbs.

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Figure 2.4. medium.

77

Example of damage to Thlipa roots caused by high salts in the planting

D. Mycorrhiza The presence and precise roles of mycorrhiza are not established for many flowering bulbs. Because mycorrhiza generally perform best under low nutrient conditions and minimal fertilizer landscape plants usually receive low fertilization, mycorrhizal research deserves enhanced investigations. Vesicular-arbuscular mycorrhiza (YAM) have been reported in Lilium longiflorum (Ames and Linderman 1977. 1978). Lilium regale and Lilium 'Burgundy' (Vanderploeg et al. 1974). They found that infections increase over time and that heavy phosphorus applications decrease the level of infection. Chilvers and Daft (1980. 1981) conducted a large study on various cultivars of Narcissus and Tulipa. Infections were more prevalent in Narcissus than Tulipa and they observed that the infections occurred through the root hairs. However, the greater the number of roots present.

ZS = Summertime, SD = Sun Dance, Apple Blossom, RL = Red Lion. YNCB = equal volumes of shredded pine bark, sand, sphagnum peat moss, and sandy loam soil; NCS = sandy loam, sand, and sphagnum peat moss (2:1:1); ASB = sphagnum peat moss, perlite, and vermiculite; F2 = sphagnum peat moss, perlite, and vermiculite; F3B = bark, sphagnum peat moss, perlite, and vermiculite; M350 = pine bark ash, sphagnum peat moss, sand and vermiculite; M360 = pine bark ash, sphagnum peat moss, sand, and vermiculite; SM4 = sphagnum peat moss and perlite; SPH = sphagnum peat moss and perlite. xNS, ., U Nonsignificant (NS) or significant at P = 0.05 (.) or 0.01 (U), respectively. Mean separation in columns is by Duncan's multiple-range test.

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79

the fewer VAM that were detected. Applications of benomyl to the bulbs before planting had no marked effects on the mycorrhiza. In Tulipa, mycorrhiza were present in the roots mainly in the spring; whereas, in Narcissus infections were recorded in the autumn. Chan (1952) also noted the presence of mycorrhiza on Narcissus roots. 'Daft's group (Chilvers and Daft 1981) found VAM on Crocus Flavus roots and also on Endymion non-scripta (Daft et a1. 1980). In the latter, mycorrhiza were present from fall to spring; this was similar to Narcissus. E. Light Studies on the direct effect of light on root growth of flower bulbs are limited. The effects of light on CR are discussed in Section III.B. As previously indicated, reduced light intensities can promote rooting of easyto-root cultivars of Dahlia (Biran and Halevy 1973a). For most geophytes, rooting takes place in the absence of light. No marked effects have been observed in which nonplanted and/or rooted bulbs of many genera were exposed to light (De Hertogh, unpublished). F. Plant Growth Regulators Halevy and Biran (1975) have reviewed most of the plant growth regulator research on tuberization of Dahlia. They concluded that daminozide and ethephon promoted the tuberization process in whole plants and inhibited it in budless cuttings. Abscisic acid (ABA) enhanced, and gibberellic acid (GA3 ) inhibited, tuberization regardless of the organs used. Short days increased the levels of endogenous ABA and endogenous ethylene. They reached a peak between the second and third week after the start of short days. Under their experimental conditions, this was 1 week before the start of the tuberization process. Rooting hormones are used with Dahlia cuttings to promote rooting, but no concentration ranges are reported (Langeslag 1988). Jernsted (1984) reported that the 0.5-1.0 mg/liter of indoleacetic acid in solution culture causes subapical swelling, root cap proliferation, and reduced root growth of Hyacinthus. Acker (1949) exposed two cultivars of L. longiflorum and L. regale bulbs to the methyl ester of naphthaleneacetic acid. After various storage periods at 1.5°C, root growth was examined. Low rates greatly stimulated root growth; whereas, a rate of 207 g/m 3 produced thickened and fasciated roots, some of which resembled tumors. De Munk (1975) showed that when bulbs are exposed to ethylene, root development was inhibited. The inhibition was observed before planting

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of the bulbs by a reduced swelling of the root zone. When planted, the exposed bulbs rooted later than nontreated bulbs. He concluded that this delay in rooting may have an influence on the flower bud abortion.

G. Diseases Flower bulbs are affected by many diseases (Bergman 1978, 1983; Gould and Byther 1979a,b,c; Moore et a1. 1979). Fungal pathogens (e.g. Botrytis, Fusarium,Phytophthora, Pythium, and Tricoderma) can affect the root systems of flower bulbs. Also, bacteria like Xanthomonas spp. can infect roots. The development of Phytophthora, Pythium, and Fusarium are enhanced by conditions that cause reduced aeration. B. cinerea and Tricoderma appear on roots in rooting rooms when the relative humidity becomes too low, while most bacterial diseases usually develop under warm, moist conditions. Raabe (1975) showed that roots of L. longiflorum bulbs infected by the necrotic fleck virus complex were more severely affected by Pythium than healthy plants. The root rot that subsequently developed affected the marketable plant quality. Thus, all possible measures must be taken to prevent root rot diseases from becoming serious problems. Jennings and De Hertogh (1977) reported that a benomyl-etridiazole (ethazol) dip and a preplant water dip stimulated root growth of Thlipa for two weeks after planting. Hyacinthus and Narcissus did not respond to these treatments. These bulbs do not, however, have a tunic that covers the basal plate and are quite different physiologically.

H. Other Factors Under certain bulb growing conditions, some Thlipa cultivars can develop a very hard tunic (Bergman 1983). When this condition occurs, the root initials have difficulty emerging through the tunic. As a result, they grow inside it. If rooting is delayed for an extended period of time, usually the flower aborts. When forcing special precooled tulips, it is advised that the tunics be removed carefully from the basal plate (De Hertogh 1989). This promotes quick rooting, which is essential for this forcing technique. M. Saniewski and L. Kawa (unpublished data) showed that removal of the roots of SPC tulips by carefully cutting the roots after each day of growth almost totally inhibited shoot growth (Fig. 2.5). However, the application of 0.2% IAA in a lanolin paste to the scape after removal of the flower bud promoted growth of the scape but not the leaves. Versluys (1927) studied the effects of root cutting on the subsequent growth of Hyacinthus and found that the effects were highly temperature

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Figure 2.5. The effects of the removal of roots and flower bud together with the upper part of scales and leaves before planting and the effect of IAA on the growth of stem and leaves of Thlipa 'Gudoshnik'. Bulbs from left to right are as follows: (A) Without roots. (B) Rooted. (C) Without roots; 0.2% IAA in a lanoline paste applied in the place of removed flower bud. (D) Rooted; 0.2% IAA in a lanoline paste applied in the place of removed flower bud (M. Saniewski and L. Kawa, unpublished).

dependent, with more roots being formed at 27°C than 16.5°C. The presence of an active cambium was critical in obtaining the response. Schuurman (1971) correlated the size and shape of tulip bulbs with root number, length, and weight. Bulb shape had no effect if the bulb weights were equal, but root number and length increased with bulb size. MacLeod (1972) demonstrated that colchicine treatment of Hyacinthus roots enhanced the rate of entry into the prophase stage of cell division. However, the roots of Hyacinthus were less sensitive to colchicine than Vicia faha and they recovered from the treatment quicker. Thinning of shoots, a normal commercial practice for Alstroemeria (De Hertogh 1989), reduced the fresh weight of the tuberous-roots (Aker and Healy 1990). Obviously, this is an effect on photosynthesis of the shoots.

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VI. CONCLUSIONS

With the exception of Tulipa, Narcissus, and Hyacinth us, comprehensive research is deficient on the physiology and root systems of ornamental geophytes. Studies are lacking on the nature of endogenous regulation, and in most instances, morphological and anatomical studies are inadequate. The general root characteristics presented in Table 2.1 need to be expanded and, in most instances, confirmed. There is a critical need to expand the information on the effects and interactions of all environmental factors. The optimal temperature ranges for rooting need to be established for all ornamental geophytes. More data is needed on the response of the bulbs to the planting medium (soil) not only for bulb production but also for forcing. The effects and potential use of plant growth regulators for rooting need to be expanded. Only limited studies have been conducted. The control of root rot diseases also needs continued efforts. The role of mycorrhiza on bulbous crops appears to be an area of very productive future research. Mycorrhiza have the potential to be genetically engineered; this could be highly important in reducing the nutrient levels required by the ornamental geophytes. Also, this could reduce bulb production costs substantially, reduce the environmental impact of the fertilizers used, and minimize fertilizer usage by gardeners. Future studies must apply all possible technologies. This includes root growth studies using containers, rhizotrons and mini-rhizotrons, the use of dyes, video camera techniques, and electron microscopy. Keen observations also remain a very important requirement. A basic data sheet to assist in the accumulation of this information is presented in Table 2.7.

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ROOT PHYSIOLOGY OF ORNAMENTAL FLOWERING BULBS

Table 2.7. A. B.

C. D.

E.

F.

Information required to evaluate root systems of ornamental geophytes.

Time and tissue of origin Morphological characteristics 1. Primary or adventitious root (origin) 2. Branching or nonbranching habit 3. Presence or absence of root hairs 4. Presence or absence of root cap 5. Presence or absence of contractile roots 6. Other distinguishing characteristics Anatomical characteristics Root number and dimensions (under field and/or container conditions) 1. Per cutting, bulb size, etc. 2. Cultivar effects 3. Diameter 4. Length Growth and development requirements 1. Temperature 2. Moisture 3. Soil requirements (pH, drainage) 4. Plant growth regulators 5. Diseases 6. Others Regions of uptake 1. Water 2. Nutrients 3. Pesticides 4. Plant growth regulators

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84

LITERATURE CITED Acker, R. M. 1949. Growth of three varieties of Lilium from bulbs stored in vapors of methyl ester of naphthaleneacetic acid. Bot. Gaz. 11:21-35. Aker, S., and W. Healy. 1990. Shoot removal affects Alstroemeria development. HortScience 25:1110. Alkema, H. Y., and C. J. M. Van Leeuwen. 1977. Vermeerdering van aantal bijgoedgewassen door middel van dubbelschubben. Bloembollencultuur 88:32-33. Ames, R. N., andR. G. Linderman. 1977. VesiculararbuscularmycorrhizaeofEasterlilyin the North-Western United States. Can. J. Bot. 23:1663-1668. _ _ . 1978. The growth of Easter lily (Lilium longiflorum) as influenced by vesiculararbuscular mycorrhizal fungi, Fusarium oxysporum and fertility level. Can. J. Bot. 56~2273-2780.

Aoba, T., S. Watanabe, and C. Saito. 1960. Studies on tuberous root formation in dahlia. 1.Periods of tuberous root formation in dahlia. J. Japan. Soc. Hort. Sci. 29:247-252. Aoba, T., S. Watanabe, and K. Soma. 1961. Studies on the formation of tuberous root in dahlia. II. Anatomical observation of primary root and tuberous root. J. Japan. Soc. Hort. Sci. 30:82-88. Aung, L. H., A. A. De Hertogh, and G. L. Staby. 1969. Gibberellin-like substances in bulb species. Can. J. Bot. 47:1817-1819. ___ . 1979. Temperature regulation of growth and endogenous abscisic acid-like content of Tulipa gesneriana L. Plant Physiol. 63:1111-1116. Bajaj, Y. P. S. 1990. Freesia. p. 413-428. In P. V. Ammirato, D. R. Evans, W. R. Sharp, Y. P. S. Bajaj (eds.) Handbook of Plant Cell Culture, Ornamental Species, Vol.5. McGraw-Hill, New York. Benschop, M. 1980. Growth and development of tulip, cv'Apeldoorn', from planting until emergence. Acta Hort. 109:189-196. Bergman, B. H. H. (Chariman). 1978. Ziekten en afwijkingen bij bolgewassen. Deel II: Amaryllidaceae, Araceae, Begoniaceae, Compositae, Iridaceae, Oxalidaceae, Ranunculaceae. N. V. Drukkerij Trio, S-Gravenhage, The Netherlands. ___ . 1983. Zietken en afwijkingen bij bolgewassen. Deel I: Liliaceae. Laboratorium voor Bloembollenonderzoek, Lisse, The Netherlands. Bertaccini, A., and F. Marani. 1986. BYMV-free clones of eight gladiolus cultivars obtained by meristem-tip culture. Acta Hort. 177:299-308. Bertaccini, A., M. G. Bellardi, and E. Rustignoli. 1989. Virus-free Freesia corms produced by meristem-tip culture. Adv. Hort. Sci. 3:133-137. Biran, 1., and A. H. Halevy. 1973a. Stock plant shading and rooting of Dahlia cuttings. Scientia Hort. 1:125-131. ___ . 1973b. The relationship between rooting of Dahlia cuttings and the presence and type of bud. Physiol. Plant. 28:244-247. ___ . 1973c. Endogenous levels of growth regulators and their relationship to the rooting of Dahlia cuttings. Physiol. Plant. 28:436-442. Blaauw. A. H. 1938. De beteekenis van den grondwaterstand voor de bloembollencultuur. Kon. Ned. Akad. Wet. (Amsterdam) Tweede Sec. 27:1-91. Blaauw, A. H., and M. C. Versluys. 1925. The results of the temperature treatment in summer for the Darwin tulip. 1. Proc. Kon. Ned. Akad. Wet. 28:717-731. Boekel, P. 1971. Soil structure problems in tulip culture. Acta Hort. 23:338-343. Botschantzeva, Z. P. 1982. Tulips: taxonomy, morphology, cytology, phytogeography and physiology. A. A. Balkema, Rotterdam, The Netherlands. Bryan, J. E. 1989. Bulbs. Timber Press. Portland, OR. Bulb and Corm Production. 1984. Ministry of Agriculture, Fisheries & Food Bulletin No. 62.

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Her Majesty's Stationery Office, London. Chan, T. T. 1952. The development of the Narcissus plant. Daffodil Tulip Yrbk. Royal Hort. Soc. 17:72-100. Chen, S. 1969. The contractile roots of Narcissus. Annu. Bot. 33:421-426. Chilvers, M. T., and M. F. J. Daft. 1980. Endomycorrhizas and root hairs of Narcissus. In: Daffodils 1980/81. Royal Hort. Soc., London. ___ . 1981. Mycorrhizas of the Liliflorae. II. Mycorrhiza formation and incidence of root hairs and field grown Narcissus L. Tulipa L. and Crocus L. cultivars. New Phytol. 89:247-261. Cumming, B. G., and D. E. Peck. 1984. Tissue culture of grape hyacinth. HortScience 19:723-724. Cyr, R. J., B. L. Lin, and J. A. Jernstedt. 1988. Root contraction in hyacinth. II. Changes in tubulin levels, microtubule number and orientation associated with differential cell expansion. Planta 174:446-452. Daft, M. J., M. T. Childers, and T. H. Nicolson. 1980. Mycorrhizas of the Liliflorae. 1. Morphogenesis of Endymion non-scriptus (L.) Garcke and its mycorrhizas in nature. New Phytol. 85:181-189. De Haan, F. A. M., and G. G. M. Van Der Valko 1971. Effect of compaction on physical properties of soil and root growth of ornamental bulbs. Acta Hort. 23:326-332. De Hertogh, A. A., and N. Blakely. 1972. The influence of temperature and storage time on growth of basal roots of nonprecooled and precooled bulbs of Lilium longiflorum Thunb. cv. Ace. HortScience 7:409-410. De Hertogh, A. A. 1989. Holland bulb forcer's guide. 4th ed. Internatl. Flower Bulb Centre, Hillegom, The Netherlands. De Hertogh, A. A., and M. Tilley. 1991. Effects of nine planting media on forcing of Swaziland and Dutch-Grown Hippeastrum hybrids. HortScience 26:1168-1170. De Munk, W. J., and M. De Rooy. 1971. The influence of ethylene on the development of 5°C-precooled 'Apeldoorn' tulips during forcing. HortScience 6:40-41. De Munk, W. J. 1975. Ethylene disorders in bulbous crops during storage and glasshouse cultivation. Acta Hort. 51:321-328. Du Plessis, N., and G. Duncan. 1989. Bulbous plants of Southern Africa-a guide to their cultivation and propagation. Tafelberg Pub. Ltd., Cape Town. Einert, A. E., G. L. Staby, and A. A. De Hertogh. 1972. Gibberellin-like activity from organs of Tulipa gesneriana L. Can. J. Bot. 50:909-914. Fakhrai, F., and P. K. Evans. 1989. Morphogenic potential of cultured explants of Crocus chrysanthus Herbert. cv. E. P. Bowels. J. Expt. Bot. 40:809-812. Feldmaier, C. 1970. Lilies. Arco Publ. Co., New York. Furmanowa, M., and H. Oledzka. 1990. Zephyr-lily. p. 800-819. In: P. V. Ammirato, D. R. Evans, W. R. Sharp, Y. P. S. Bajaj, (eds.) Handbook of Plant Cell Culture, Ornamental Species, Vol. 5. McGraw-Hill, New York. Gabryszewska, E., and M. Hempel. 1985. The influence of cytokinins and auxins on Alstroemeria in tissue culture. Acta Hort. 167:295-300. Gould, C. H., and R. S. Byther. 1979 a. Diseases of Narcissus. Washington State Univ. Ext. Bul. 709. 1979 b. Disease of bulbous iris. Washington State Univ. Ext. Bul. 710. ___ . 1979 c. Disease of tulips. Washington State Univ. Ext. Bul. 711. Griesbach, R. A. 1972. The life-structure and function in Gladiolus. p. 8-40. In: The world of the Gladiolus. N. Am. Gladiolus Coun. Edgewood Press, Edgewood, MD. Halevy, A. H., and 1. Biran. 1975. Hormonal regulation of tuberization in dahlia. Acta Hort. 47:319-329. . 1986a. The induction of contractile roots in Gladiolus grandiflorus. Planta 167:94100.

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___ . 1986b. Factors affecting the induction of contractile roots in gladiolus. Acta Hort. 177:323-330. ___ . 1990. Recent advances in control of flowering and growth habit of geophytes. Acta Hort. 266:35-42. Hanks, G. R., and A. R. Rees. 1979. Twin-scale propagation of Narcissus: a review. Scientia Hort. 10:1-14. Hartmann, H. T., D. E. Kester, and F. T. Davies, Jr. 1990. Plant propagation, principles and practices. 5th ed. Prentice Hall, Englewood Cliffs, New Jersey. Hartsema, A. M. 1961. Influence of temperatures on flower formation and flowering of bulbous and tuberous plants. p. 123-167. In: W. Ruhland (ed.) Handbuch der Pflanzenphysiologie 16. Springer-Verlag, Berlin. Havis, A. L. 1936. The morphology and anatomy of the Dahlia seedling. Proc. Am. Soc. Hort. Sci. 34:592-594. Hussey, G. 1976. Propagation of Dutch Iris by tissue culture. Scientia Hort. 4:163-165. ___ . 1980 A.Propagation of some members of the Liliaceae, Iridaceae and Amaryllidaceae by tissue culture. Petaloid monocotyledons. Linnean Society Symposium Series 8. Academic Press, London/New York, p. 33-42. _ _ . 1980b. In vitro propagation. p. 51-61. In: D. S. Ingram and J. P. Helgeson (eds.) Tissue Culture Methods for Plant Pathologists. Blackwell, Oxford, London. ___ . 1982. In vitro propagation of Narcissus. Annu. Bot. 49:707-719. Iziro, Y., and Y. Hori. 1983a. Thickening growth and contraction of contractile root(s) in relation to thickening growth of daughter corm or bulbs in Gladiolus and Oxalis bowieana Lodd. J. Japan. Soc. Hort. Sci. 51:440-458. _ _ . 1983b. Effect of ternperature on the growth of contractile root(s) and daughter corm or bulbs in Gladiolus and OXBlis bowieBna Lodd. J. Japan. Soc. Hort. Sci. 51:459-465. ___ . 1983c. Effect of planting depth on the growth of contractile root(s) and daughter corm or bulbs in Gladiolus and Oxalis bowieana Lodd. J. Japan. Soc. Hort. Sci. 52:51-55. ___ . 1983d. Retranslocation of photoassimilates accumulated in contractile root(s) to daughter corm or bulbs in Gladiolus and Oxalis bowieana Lodd. J. Japan. Soc. Hort. Sci. 52:56-64. Jacoby, B., and A. H. Halevy. 1970. Participation of light and temperature fluctuations in the induction of contractile roots of gladiolus. Bot. Gaz. 131:74-77. Jennings, N. T., and A. A. De Hertogh. 1977. The influence of preplanting dips and postplanting temperatures on root growth and development of nonprecooled tulips, daffodils and hyacinths. Scientia Hort. 6:157-166. Jernstedt, J. A. 1984. Root contraction in hyacinth. 1. Effects of IAA on differential cell expansion. Am. J. Bot. 71:1080-1089. Krikorian, A. D., R. P. Kann, and M. S. Fitter Corbin. 1990. Daylily. p. 375-412. In: P. V. Ammirato, D. R. Evans, W. R. Sharp, Y. P. S. Bajaj (eds.) Handbook of Plant Cell Culture Ornamental Species. vol. 5. McGraw-Hill, New York. Kromer, K., and K. Kukulczanka. 1985. In vitro cultures of meristem tips of Canna indica L. Acta Hort. 167:279-285. Langeslag, J. J. J. (Chairman) 1988. Teelt en Gebruiksmogelijkheden Van Bijgoedgewassen. Ministerie Landbouw Visserij en Consulentschap Algemene Dienst Bloembollenteelt, Lisse. The Netherlands. Le Nard, M. 1972. Incidence de sequence de hautes et basses temperatures sur la differenciation des bourgeons, l'enracinement et la bulbification chez la tuIipe. Annu. Amelior Plant. 22:39-59. ___ . 1980. Effects of duration of high temperature treatment on subsequent flower differentiation, rooting and flowering of tulip bulbs. Acta Hort. 109:65-72. ___ . 1985. Influence de l'activite racinaire sur l'aptitude a la floraison du bulbe de Thlipe. C.R. Acad. Sci. (Paris). 301:107-110.

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MacLeod, R. D. 1972. Colchicine induced changes in the rate of entry of cells into prophase in roots of Vicia faba and Hyacinthus orientalis. Osterr. Bot. Z. 120:15-28. Meyer Jr., M. M., L. H. Fuchigami, and A. M. Roberts. 1975. Propagation of tall bearded irises by tissue culture. HortScience 10:479-480. Mii, M., T. Mori, and N. Iwase. 1974. Organ formation from the excised bulb scales of Hippeastrum hybridum in vitro. J. Hort. Sci. 49:241-244. Moore, W. C., A. A. Brunt, D. Price, and A. R. Rees. 1979. Diseases of bulbs. 2nd ed. Ministry of Agriculture, Fisheries and Food, London. Moser, B. C., and C. E. Hess. 1968. The physiology of tuberous root development in Dahlia. J. Am. Soc. Hort. Sci. 93:595-603. Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant. 15:473-497. Negbi, M. B. Dagan, A. Dror, and D. Basker. 1989. Growth, flowering, vegetative reproduction, and dormancy in the saffron crocus (Crocus sativus L.). Israel J. Bot. 38:95-113. Ohyama, T., T. Ikarashi, A. Obata, and A. Baba. 1988. Role of nitrogen accumulated in tulip roots during winter season. Soil Sci. Plant Nutr. 34:341-350. Prince, T. A., and M. S. Cunningham. 1990. Response of Easter lily bulbs to peat moisture content and the use of peat or of polyethylene-lined cases during handling and vernalization. J. Am. Soc. Hort. Sci. 115:68-72. Raabe, R. D. 1975. Increased susceptibility of Easter lilies to Pythium root rot as a result of infection by necrotic fleck virus complex. Acta Hort. 47:91-97. Raunkiaer, C. 1934. Life forms of plants and statistical plant geography. Clarendon Press, Oxford. Rees, A. R. 1972. The growth of bulbs. Academic Press, London. Rees, A. R. 1989. Evaluation of the geophytic habit and its physiologiCal advantages. Herbertia 45:104-110. Reyneke, W. F., and H. P. Van Der Schijff. 1974. The anatomy of contractile roots in Eucomis L'Herit. Annu. Bot. 38:977-982. Rimbach, A. 1929. Die Verbreitung der Wurzelverkurzung im Pflanzenreich. Deut. Bot. Ges. 47:22-31. Ruzin, S. E. 1979. Root contraction in Freesia (Iridaceae). Am. J. Bot. 66:522-531. Sakai, A., and F. Yoshie. 1984. Freezing tolerance of ornamental bulbs and corms. J. Japan. Soc. Hort. Sci. 52:445-449. Saniewski, M., J. Nowak, and R. Rudnicki. 1974. Studies on the physiology of hyacinth bulbs (Hyacinth us orientalis L.). IV. Hormonal regulation of induction of roots and bulblets in Hyacinthus orientalis L. grown in culture. Plant Sci. Letters 2:373-376. Schuurman, J. J. 1971. Effect of size and shape of tulip bulbs on root development. Acta Hort. 23:212-217. Seabrook, J. E. A., B. G. Cumming, and L. A. Dionne. 1976. The in vitro induction of adventitious shoot and root apices on Narcissus (daffodil and narcissus) cultivar tissue. Can. J. Bot. 54:814-819. Seabrook, J. E. A.1990. Narcissus (Daffodil). p. 577-597. In: P. V. Ammirato, D. R. Evans, W. R. Sharp, andY. P. S. Bajaj(eds.) Handbook of plant cell culture, ornamental species, vol. 5. McGraw-Hill, New York. Shoub, J., and A. A. De Hertogh. 1975. Growth and development of the shoot, roots, and central bulblet of Tulipa gesneriana L. cv. Paul Richter during standard forcing. J. Am. Soc. Hort. Sci. 100:32-37. Smith-Huerta, N. L., and J. A. Jernstedt. 1989. Root contraction in hyacinth. III. Orientation of cortical microtubules visualized by immunofluorescence microscopy. Protoplasma 151:1-10. Smith-Huerta, N. L., and J. A. Jernstedt. 1990. Root contraction in hyacinth. IV. Orienta-

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tion of cellulose microfibrils in radial longitudinal and transverse cell walls. Protoplasma 154:161-171. Sterling, C. 1972. Mechanism of root contraction in Gladiolus. Annu. Bot. 36:589-598. Stuart, N. W. 1954. Moisture content of packing medium, temperature and duration of storage as factors in forcing lily bulbs. Proc. Am. Soc. Hort. Sci. 63:488-494. Van Aartrijk, J., G. J. Blom-Barnhoorn, and P. C. G. Van Der Linde. 1990. Lilies. p. 535597. In: P. V. Ammirato, D. R. Evans, W. R. Sharp, Y. P. S. Bajaj (eds.) Handbook of Plant Cell Culture, Ornamental Species, vol. 5. McGraw-Hill, New York. Vanderploeg, J. F., R. W. Lighty, and M. Sasser. 1974. Mycorrhizal association between Ulium taxa and Endogone. HortScience 9:383-384. Van Der Valk, G. G. M. 1971. Influence of short periods of restricted soil aeration on development of tulips. Acta Hort. 23:333-337. Versluys, M. C. 1927. Aanleg en groei der wortels van Hyacinthus orientalis gedurende het geheele jaaren onderverschillende omstandigheden. Verh. Kon Ned. Akad. Wet. Sect. II 25:1-100. Vijverberg, A. J. 1981. Growing Amaryllis. Grower Guide 23. Grower Books, Great Britain. White, H. E. 1940. The culture and forcing of Easter lilies. Massachusetts Agr. Expt. Stat. Bul. 376. Whittington, W. J. (ed.) 1969. Root growth. Butterworth and Co. Ltd., London. Wiersum, L. K. 1971. Tulip root behavior and aeration requirements. Acta Hort. 23:318325. Wilson, K., and J. N. Honey. 1966. Root contraction in Hyacinthus orientalis. Annu. Bot. 30:47-61. Wilson, C., and C. A. Peterson. 1982. Root growth of bulbous species during winter. Annu. Bot. 50:615-619. Zimmer, K., and M. Girmen. 1987. Temperaturabhangigkeit der Entwickling van Anemone blanda und Eranthis hiemalis. Gartenbauwissenchaft 52:263-265. Zimmer, K., and M. Renken. 1984. Untersuchungen an Allium aflatunense. Deutscher Gartenbau 38:2004-2008. Zimmer, K., M. Wallingen, and M. Renken. 1985a. Untersuchungen zur periodischen Entwicklung von Allium aflatunense. Deutscher Gartenbau 39:594-596. ___ . 1985b. Untersuchungen an Allium christophii. Deutscher Gartenbau 39:22062209. Zimmer, K., and K. Weckeck. 1989. Effect of temperature on some ornamental Alliums. Acta Hort. 246:131-134. Ziv, M., A. H. Halevy, and R. Shilo. 1970. Organs and plantlets regeneration of Gladiolus through tissue culture. Annu. Bot. 34:671-676.

3 Tuber Formation in Potato: Induction, Initiation, and Growth * E. E. Ewing Department of Fruit and Vegetable Science Cornell University Ithaca, New York 14853-5908

P. C. Struik Department of Field Crops and Grassland Science Wageningen Agricultural University Haarweg 333 6709 RZ Wageningen, The Netherlands

I. Introduction A. Definitions, Scope, and Importance B. Relation to Propagation C. Plasticity of Organ Development in the Potato II. Methods of Studying Tuberization A. Special Problems B. Techniques with Whole Plants C. Cuttings D. Isolated Buds from Whole Plants E. In Vitro Subculture of Stolons F. Growing Plantlets In Vitro G. Comparison of Methods III. Environmental Factors Affecting Tuberization A. Photoperiod and Spectral Quality B. Tern peratu re C. Irradiance D. Nitrogen E. Other Environmental Factors

·Paper Number 15, Department of Fruit and Vegetable Science, Cornell University. We are grateful to Dr. P. J. Davies and Dr. T. L. Setter, Cornell University, for helpful comments during the preparation of this review. We especially thank Dr. D. J. Hannapel, Iowa State University, Dr. R. M. Wheeler, The Bionetics Corporation, and Dr. Shmulik Wolf, The Hebrew University of Jerusalem, for critically reading the entire manuscript. Ms. M. Eames-Sheavly and Dr. K. S. Yourstone, Cornell University, helped with preparation of figures. Dr. J. Vos, H. Biemond, and Dr. K. Scholte, Wageningen Agricultural University, and G. E. L. Borm, formerly ofWageningenAgricultural University, Dr. J. J. McGrady and Dr. A. B. Rowell, formerly of Cornell University, and Dr. Esra Galun, Weizmann Institute of Science supplied original copies of figures. 89

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IV. Genetic Effects A. Variation in Critical Photoperiod B. Classifications for Response to Photoperiod C. Inheritance of Critical Photoperiod D. Usefulness for Studying Physiology V. Effects of the Mother Tuber A. Physiological Age B. Similarity of Patterns on Tubers and Cuttings C. Implications for Tuberization D. Absence of the Mother Tuber VI. Physiological Nature of Induction to Tuberize A. Effects of Induction on Overall Plant Development B. Insights from Grafting C. Dynamic Aspects D. Role of Known Hormones E. Plant Growth Regulators F. Calcium and Calmodulin G. Role of Assimilate Level in Induction VII. Changes in the Stolon Tip or Bud Associated with Tuberization A. Anatomical Changes B. Biochemical Changes C. Relation Between Changes in Shoot and Changes in Stolon Tip D. Competitive Advantage Provided by the Changes during Evolution VIII. Patterns of Stolon and Tuber Formation A. Significance B. Pattern of Stolon Formation in the Intact Plant C. Maintenance of Diagravitropic Growth D. Stolon Elongation and Branching E. Effect of Node Position on Tuberization Patterns F. Growth Rates of Individual Tubers G. Tuber Distribution in the Field IX. Resorption and Second Growth A. Resorption of Tubers B. Other Forms of Resorption C. Second Growth X. Implications for Tuber Yield XI. ConcIusion Literature Cited

I. INTRODUCTION

A. Definitions, Scope, and Importance This review is about the induction, initiation, and growth of potato tubers (Fig. 3.1). Many plants other than the potato and its relatives form tubers, but relatively little has been published about them since the over" all review of tuberization by Gregory (1965). Our emphasis will be on the cultivated potato (Solanum tuberosum L.), with information from other tuberizing Solanum species included where relevant.

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Figure 3.1. Potato tubers, stolons. and roots forming at the base of a potato stem. Plants were grown with roots in a nutrient solution and with stolons and tubers in a separate container (see Fig. 3.3).

An understanding of potato tuberization is desirable not only because the tuber is the edible portion of this important crop, but also because the time of tuber initiation in relation to other aspects of plant development plays a key role in determining potential yield. Such an understanding is also useful for cultivation of potato in the hot tropics and in other areas where tuberization is poor; for hastening maturity where the growing season is short because of climatic conditions or because of the danger of late season aphid infestations on seed crops; for reducing physiological disorders associated with a decline in the level of induction to tuberize after tubers have formed; for manipulation of tuber number, thereby controlling size distribution; and for the employment of new techniques of multiplication based upon microtubers, minitubers, seedling tubers, or cuttings. Potato tubers are often assumed to be roots because they are ordinarily formed underground. Botanically, they are greatly shortened and thickened stems that bear scale leaves (cataphylls), each with a bud in its axil. The usual site of tuber formation is a stolon tip. Potato stolons (as they are almost universally called, although some argue that the term rhizomes is more correct) are diagravitropic stems, normally arising as

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branches from underground nodes. They have long internodes and are terminated by a recurved apical portion or "hook" (Peterson et a1. 1985). Like the tubers borne upon them, stolons are characterized by the presence of scale leaves. We will consider both stolon and tuber formation in this review. To describe the status of the plant when it becomes capable of forming tubers, it is common to say that the plant has become induced to tuberize. Gregory (1956) hypothesized the existence of a tuberization stimulus that is produced in the leaves and translocated to the site of tuber initiation. In many ways this is a useful concept, except for the possible connotation that tuberization is controlled by a single chemical compound analogous to "florigen," the hypothetical flowering hormone. Some day it may be proven that in fact a single compound does control tuberization; but as we hope to make evident in the course of this review, until that day arrives it is desirable to keep an open mind to other possibilities. Our use of the term should not be construed to imply that a single compound is necessarily responsible for tuber induction: the tuberization stimulus may consist of a particular balance between two compounds-or of a balance among a group of compounds. With exceptions that will be mentioned later, tuber induction leads to tuber initiation. It is convenient to consider that initiation has occurred if the swollen portion of the stolon tip is at least twice the diameter of the stolon. We refer to these tiny swellings as tuber initials. After initiation it is common for many tubers to be resorbed; this consists of a reallocation of tuber contents to other parts of the plant and may occur even to tubers that have attained a >2-cm diameter. Tubers that are not resorbed are said to have been set. In the fullest sense tuberization is not yet completed after tuber set. After tuber set, some tubers remain small while others on the same plant continue to grow. Up to the time when tubers have matured and dormancy has become well established, conditions, such as high temperature and drought, can cause even large tubers to revert to stolon growth. Environmental factors favoring tuber induction and initiation generally favor continued tuber development and maturation. We consider in our review tuberization from induction to the establishment of developed, dormant tubers. In this review, we concentrate on literature published since Gregory's 1965 review, with some reference to earlier work, especially if needed to establish a particular point. There have been a number of other review articles covering specialized aspects of tuberization; these will be cited as we discuss the individual topics with which they are concerned. Books that contain chapters dealing with one or more facets of tuberization include Burton's The Potato (1989) and those edited by Li (1985), Vayda and Park (1990), and Harris (1992).

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93

B. Relation to Propagation The traditional mode of propagation for potato is to plant whole or cut tubers weighing 50 g or more. These produce new plants bearing the next crop of tubers. We refer to the parent tubers as seed tubers, or in some contexts as mother tubers. One or more buds in the "eyes" of the tuber sprout and develop orthotropic shoots. The axillary buds at the underground nodes of the shoots form stolons, and the apices of the stolons swell over about 12 internodes (Cutter 1992) to produce the new tuber generation. Because viruses and other disease organisms can be transmitted from one tuber generation to the next, a key objective in propagation is to start with healthy seed tubers. In many countries healthy seed is produced by selecting a sample of tubers that are proven to be free of pathogens and that are deemed to be of the desired type. The selected tubers are multiplied through successive generations by traditional methods of propagation, but with special efforts to minimize infection with diseases that might be carried over to the tuber progenies. This system is laborious, expensive, and time consuming. The rate of multiplication is often no better than tenfold per year, and even with rigorous control measures disease infections cannot be completely prevented. Fortunately, the flexibility of organ development in the potato allows for alternative methods of propagation. Tissue culture and rapid multiplication techniques have been widely adopted by potato seed improvement programs throughout the world (Bryan 1988; Jones 1988) during the last decade. These new techniques are interesting from a physiological point of view because tuber formation may occur in the absence of plant parts that ordinarily are crucial for normal functioning of the whole plant. Such tubers-commonly called microtubers when they form in vitro, and minitubers when they form on cuttings or on in vitro plantlets (small plants grown in test tubes) that have been transplanted to potshave the essential characteristics of ordinary tubers in spite of their small size. Studying their formation may teach us more about the hormonal control of the tuberization process, although as will be shown later in this review, it is important to recognize that the physiology of tuberization may change with the type of propagule used. Another alternative method of propagation that has attracted attention during the past 10 to 15 years is to plant what is commonly called true potato seed (TPS) (Le., seeds in the botanical sense of the word). In contrast to seed tubers, very few pathogens are transmitted through TPS. Stolons emerge from the cotyledonary nodes and other basal nodes of the seedlings. If the nodes from which the stolons originated are above the soil line, the stolon tips tend to grow downward, eventually burying

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themselves; and tubers may form at the buried tips. The pattern of tuberization from TPS plants may be quite different because of the absence of a mother tuber and the different sequence of stolon development. Whether dealing with TPS, cuttings, transplanted in vitro plantlets, or tiny tubers derived from one of the alternative methods of propagation, initial plant growth is often slow compared to the rates observed when ordinary seed tubers are planted. Frequently the slow growth is exacerbated by premature tuberization resulting in stunted plant development, early senescence, and extremely low yields. Because of the stunted or slow shoot growth the risks of misshapen tubers from second growth are also much greater. A better understanding of the physiology of tuberization is needed to prevent these problems.

c. Plasticity of Organ Development in the Potato Whatever the method of propagation, there can be many variations in the pattern of organ development. The potato plant is remarkable for its plasticity (Steward et al. 1981; Clowes and MacDonald 1987). Excision of the shoots at soil level may cause the stolons to turn upward and form new shoots instead of tubers (Sachs 1893), a phenomenon that can also be brought about by other factors; aboveground buds can become tubers (Vochting 1887); new tubers can form directly from the buds of the mother tuber (Wellensiek 1929); tubers can produce stolons; and even flowers (Fig. 3.2) have been known to tuberize (Werner 1954). Given the wide range of possible responses, what determines whether tubers will form? What causes the tip of a stolon or other growing point to change its course of development and turn into the very specialized stem that is a tuber? Once a tuber has been initiated, what controls whether it will continue to develop as a tuber, or revert to shoot or stolon growth, or even be resorbed by the plant? These are questions that will be considered in this review.

II. METHODS OF STUDYING TUBERIZATION

A. Special Problems Like roots, stolons and tubers are difficult to study because they are hidden in the soil. The growth and development of both stolons and tubers take place best in darkness; only under special conditions do they develop in the light. Adding to the difficulty is the fact that the exact site of tuber formation is unpredictable: under ordinary circumstances,

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Figure 3.2. Aerial tubers forming in a flower cluster. The environment in which this potato plant was grown was very conducive to tuberization; but the plant was grown from a rooted stem cutting that had no underground nodes. and hence no possibility of forming underground tubers. After the flower cluster was enclosed in a brown paper bag. the buds of the flower cluster (partially darkened) formed tubers (from Lazin 1980).

tubers are swellings in the subapical regions of underground stolons; but there is no way of predicting whether a particular stolon tip will tuberize until the swelling has already become visible. The length of the stolon before tuberizing, the location of the stolon in respect to other stolons, and the degree of branching behind the stolon tip upon which the tuber forms are all highly variable. If the entire stolon system must be dug up to examine what is happening, there is obviously so much disturbance of the accompanying root system that the examination itself interferes with normal growth. Rather than a series of observations on the status of tuberization in a single plant, it is therefore necessary to resort to sequential harvests of different plants. Many plants must be examined at each harvest to overcome the phenotypic variation observed in potato. Subtle differences in condition of mother tubers give rise to differences in number of sprouts, vigor, degree of branching, and other characteristics that can lead to considerable variability among plants supposedly grown under uniform conditions. Furthermore, differences in microclimate from plant to plant,

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or even within the stolon zone of a single plant, may strongly affect the results.

B. Techniques with Whole Plants Methods have been devised to overcome the problems in studying tuberization that are associated with its unpredictable nature and underground occurrence. In the case of whole plants, techniques have been developed (Fig. 3.3) to separate the zone of stolon and tuber formation from the soil and roots (Lugt et a1. 1964; Wurr 1977; Krauss 1985; Struik and Van Voorst 1986). The zone is best filled with an inert, light-weight material that can be removed by vacuum when examinations are to be made. The density of the medium and its moisture content have an effect on the development of both stolons and tubers. After the completion of tuber set, this medium is no longer necessary; tubers can be kept in air if they are covered to prevent exposure to light. Adventitious roots form on stolons and tubers as they do on plants grown in the conventional manner, but they die when the stolon medium is permanently removed. The apparatus makes possible frequent, nondestructive observations of stolons and tubers, permitting the collection of the kinds of data presented in Fig. 3.4. The system also permits individual control of the environments around roots, stolons, and shoots, thus facilitating study of the effects of environmental factors on the separate organs. For such studies a device has been designed (Fig. 3.5) that permits precise measurement of tuber volume, providing accurate tracking of individual tuber growth (Schnieders et a1. 1988; Struik et a1. 1988a). Figure 3.6 illustrates the kinds of growth curves that can be obtained in this way.

C. Cuttings Another approach, first used effectively by Gregory (1956) and Chapman (1958), is to take cuttings from whole plants, bury one or more buds of the cuttings, and observe whether or not tubers develop at the buried buds. The appeal of cuttings is that they represent a convenient model of the entire plant for tuberization experiments. In its simplest form the cutting consists only of a leaf, its subtended axillary bud, and a small piece of attached stem. The bud is buried in an appropriate potting mix as soon as the cutting has been taken. If the cutting is excised from a plant that was grown under strongly inducing conditions, then within 24 h the buried bud will begin to undergo detectable changes (Duncan and Ewing 1984) that will culminate in the formation of a sessile tuber a few days later (Fig. 3.7, far right). If the mother plant was grown under conditions very unfavorable to tuberization, then the buried bud on the cutting will remain dormant (Fig. 3.7, far left) or develop an orthotropic

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shoot. Intermediate levels of induction result in a diagravitropic stolon, or a stolon terminated by a tuber (Fig. 3.7). The range of responses on cuttings reflects, albeit in exaggerated form, the responses of the whole plant to increasing induction. These whole plant responses are described in subsequent parts of this review.

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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.

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Figure 3.7. Apical cuttings after 14 days in a mist chamber, illustrating the typical progression of responses to increasing levels of induction prior to cutting. Cuttings are from sibling plants in a population segregating for critical photoperiod for tuberization (J. H. van den Berg, M. W. Bonierbale, E. E. Ewing, and R. L. Plaisted, unpublished). All plants received the same photoperiods (natural daylength, 14.5 h, followed by three 10-h days) before cutting. Cuttings from left to right demonstrate changes in growth of the buried bud expected from increasingly longer critical photoperiods for tuberization of the mother plants: no growth, orthotropic shoot, diagravitropic stolon, stolon terminated by tuber, sessile tuber. Note decrease in shoot and root growth and more horizontal leaf position as tuber induction increases. The same progression of responses would be expected from exposing plants of a single genotype to increasing numbers of short days before taking cuttings. Response patterns of leaf-bud cuttings are similar, except that dormant buds are less common in the absence of a shoot apex.

1986; Wheeler et a1. 1988; McGrady and Ewing 1990; Van den Berg et a1. 1990).

D. Isolated Buds from Whole Plants Leaf-bud cuttings can be further simplified by eXCISIng the leaf (Gregory 1956; Wareing and Jennings 1980; Ewing 1985). If the remaining bud is kept under appropriate conditions, and if it has been derived from a plant that was highly induced, then the bud will tuberize. The nature of the conditions favoring tuberization will be described later in this review: for now, suffice it to say that it is best to treat the buds aseptically, implanting them on an agar or similar sterile medium. Unfortunately, most investigators who attempt the procedure encounter frequent problems with a form of microbial contamination that can be very

102

E. E. EWING AND P. C. STRUIK

1

6

7

8

9

~[bl919

WO ~~"

[P

~~ Figure 3.8. Diagrams of the scale used to rate cuttings for the intensity of induction to tuberize in response to varying induction of plants prior to cutting. Drawings depict the buried axillary bud of the cutting, which contains a strong central bud and weak ancillary buds. The scale described in Lorenzen and Ewing (1990) is: 1 = no swelling or extension growth of bud~color entirely green; 2 = orthotropic shoot from the central bud, with or without an orthotropic shoot from an ancillary bud, or the central bud barely growing with very slight swelling and white color at the base; 3 = orthotropic shoot from the central bud with a stolon from one of its axillary buds, or with a stolon from the ancillary bud of the cutting, or an orthotropic shoot with a slightly swollen base from the central bud. or central bud with definite swelling and white color at base; 4 = normal or thin stolons from the central bud, with or without stolons from the ancillary buds. or an orthotropic shoot with a moderately swollen base; 5 = orthotropic shoot from central bud and tuber at one or more ancillary buds, or shoot with slightly swollen tip. or shoot with very swollen base, or moderately thick stolon; 6 =stolon from central bud terminated by a tuber, or thick stolon from central bud; 7 = very elongated sessile tuber, verging into very thick stolons, with or without small tubers on their ancillary buds; 8 = somewhat elongated sessile tuber; 9 = shortened or round or only slightly elongated sessile tuber (drawings by M. Eames-Sheavly).

difficult to eliminate (see Gregory 1956; Ewing 1985; Grimm and Baumann 1991).

E. In Vitro Subculture of Stolons A second in vitro technique. popularized by Palmer and Smith (1969. 1970). solves the problem of microbial contamination by starting with a

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different tissue. As usually practiced, sprouts are removed from tubers, surface sterilized, and subcultured on an agar medium. A stolon-like growth is produced, and with proper manipulation tubers will form on the stolons. The technique is convenient and has yielded many interesting findings. A major drawback is that, unllke Gregory's in vitro method, this does not lend itself to study of the most clear-cut environmental factor that controls induction to tuberize: photoperiod. As will be discussed later in this chapter, the photoperiodic signal is detected in the leaf, and subcultured stolons do not have leaves. Another consideration is that the process of subculturing may deprive the stolons of compounds that would normally be present in the intact plant. Thus the fact that tuberization is dependent upon the addition of a particular compound does not necessarily imply that the compound is limiting or controlling tuberization in the intact plant.

F. Growing Plantlets In Vitro A more recent development is growing plantlets in vitro, a practice which has achieved widespread acceptance, not as a method for studying the physiology of tuberization, but because of its application to rapid propagation of disease-free material for the seed potato industry. These plantlets originate from an apical meristem and also undergo subculturing; but because they are exposed to light each day, they develop leaves, stems, and roots inside the culture vessel. Under some circumstances the plantlets will also develop tubers. Most often these are aerial tubers, usually sessile, forming directly at axillary buds without any stolon development. The relationship of such tuberization to tuberization on the whole plant is yet to be firmly established. One of the difficulties is that, unlike plants grown in soil, there is no underground portion of the plant maintained in continuous darkness where the formation of stolons and tubers would be favored. Instead the entire plantlet is exposed to the light each day, or it is all darkened (e.g., Slimmon et al. 1989).

An exception to this was recently reported (Aksenova et al. 1989). Plantlets were grown for three months in agar containing 100/0 sucrose with the entire test tube exposed to light, with the agar shaded, with only the aerial portion shaded, or in complete darkness. Tubers formed in the agar only when the aerial portion received light and the agar was darkened; all other treatments resulted in aerial tubers. Tuber formation was by far the most abundant when the aerial portion was darkened and the agar received light. It is not clear whether there was some transmittance of light to the darkened zones in these experiments, but the technique may prove to be useful.

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E. E. EWING AND P. C. STRUIK

G. Comparison of Methods All of the above methods have been used to study various aspects of tuberization. It is not surprising that the different methods sometimes lead to different conclusions. The limitations of each technique should be borne in mind when evaluating such differences. An example is the role of sugars. In the intact plant there is a change in sugar metabolism in the stolon tip before tuberization occurs (see Part VII.B.7). When tubers are produced in vitro on small nodal cuttings or on plantlets, the sugar concentration of the medium is crucial; but the sucrose may play different roles: as a substance that triggers tuberization, as an energy source necessary for the growth of the axillary bud, or as an osmoticum, necessary to prevent elongation of the structure growing from the axillary bud. This may explain why the effects of photoperiod and temperature on in vitro plantlets do not always parallel their effects on whole plants insofar as tuberization is concerned (Hussey and Stacey 1981,1984; also see Part III.A.2). One way of sorting out the effects of sucrose would be to utilize in vitro plantlets grown under sufficient irradiance and with enriched CO2 (Kozai et a1. 1988). In this manner potato plantlets can be grown in vitro autotrophically (Le., without the addition of C compounds to the medium). At 65 ,tUllol m -2 S-1 photosynthetic photon flux (PPF) outside the test tuber there was a growth response to sucrose level in the agar, but at 210 ,umol m-2 S-1 PPF and elevated CO 2 concentrations growth was equally good with or without sucrose (Kozai et a1. 1988).

III. ENVIRONMENTAL FACTORS AFFECTING TUBERIZATION

A. Photoperiod and Spectral Quality 1. Tuberization of whole plants. Induction to tuberize is promoted by

short photoperiods (or, more accurately, by long nights), and the signal is perceived in the leaves (Gregory 1965). Phytochrome is apparently involved in perceiving the signal: tuberization was diminished when long nights were interrupted by red light, and this effect was partially reversed by subsequent exposure to far-red light (Batutis and Ewing 1982). Extension of the photoperiod with high levels of irradiance from a mixture of fluorescent and incandescent lamps was much less harmful to tuberization than was extension with very low levels of irradiance from incandescent lamps only (Wheeler and Tibbitts 1986; Lorenzen and

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Ewing 1990). In part this could be explained by the effects of the extra assimilate produced (see discussion tinder Part VI.G), but light quality also played a role. Photoperiod extension with low levels of irradiance from cool white fluorescent lamps was much less inhibitory to tuberization than if incandescent lamps were used (Wheeler and Tibbitts 1986). A comparison to the flowering responses of short-day plants may be instructive in interpreting these observations. Consistent with the response previously described for potato tuberization, exposure of Chenopodium rubrum plants to red light in the middle of the dark period inhibits flowering; and the inhibition is reversed by exposure to far-red light. Yet far-red light at the end of the light period inhibited flowering of these plants, and red light at the end of the light period promoted it (King and Cumming 1972). It has been shown in other species as well that whether red light or far-red light will be more inhibitory to flowering depends upon when during the diurnal cycle the exposure to light occurs (Vince-Prue 1986). If potato tuberization is analogous, then it is not surprising if fluorescent light (low ratio of far-red to red wavelengths) at the end of the light period is less inhibitory to flowering than is light from incandescent lamps (much higher ratio of far-red to red). 2. Tuberization in vitro. The analogy to flowering in Chenopodium may

also explain a red/far-red response to tuberization in vitro. The application of five minutes of red light to potato sprouts immediately after their excision from the mother tuber accelerated in vitro tuberization when sprouts were incubated in the dark on a medium that contained 4% glucose (Blanc 1981). The acceleration was reversed by exposure to farred light after the red light. Sensitivity to red light was increased by delaying the time of treatment for at least 6 h after sprout excision, but sensitivity to far-red light was optimal at time of excision and disappeared by 6 h after excision (Blanc et al. 1986). Other responses to light quality may also be involved. In experiments by Aksenova et al. (1989) (Part II.F), red fluorescent light (600-700 nm, maximum 660 nm) and blue fluorescent light (400-580 nm, maximum 480 nm) were compared for their effects on in vitro plantlets. Both treatments were maintained at 40 W1m 2 for 16-h photoperiods. Blue light resulted in much higher tuber production, even though at the same number of watts per unit area the PPF of the blue light would have been lower than that of the red light. There is little agreement concerning the effects of photoperiod on the formation of microtubers on in vitro plantlets. In some experiments long photoperiods have promoted tuberization (Hussey and Stacey 1981) compared to 8-h photoperiods; in others no effect was obtained (Hussey and Stacey 1984); and in still others (Garner and Blake 1989) short

E. E. EWING AND P. C. STRUIK

106

photoperiods promoted the earliness of tuber formation, as might be expected from effects on whole plants. The work of Perl et al. (1991) provides exciting new insights but adds to the complexity. We will describe their procedures in some detail because of the surprising nature of the results. Shoot sections were grown in vitro on a standard agar medium containing 1% sucrose, at 25°C and under 16-h photoperiods that provided 60 }Lmol m -2 S-l PPF. After about three weeks the rooted shoots were cut into one-node sections, each containing a leaf; and these were placed on new agar media under 16-h or 8-h photoperiods. The PPF was only 20 JLffiol m-2 S-l, and the temperature was 22°C. It is important to note that the new media were supplemented with 5mg/liter kinetin and 5mg/liter ancymidol (an inhibitor of gibberellin synthesis). If the medium contained 8% sucrose, tuber initials appeared in five days, and microtubers in seven days, whether photoperiods were 8 h or 16 h. No tubers formed if the sucrose concentration was only 2%. Up to this point, the results are not unexpected: in the presence of a high level of sucrose, kinetin, and a potent inhibitor of gibberellin synthesis, tuberization occurs regardless of photoperiod. The surprise came when sections were transferred to a 2% sucrose medium after various numbers of days on the 8% sucrose (Table 3.1). Even one day on the 8% sucrose was sufficient for tuberization, but only if the photoperiod during the next two days was 16 h rather than 8 h! The authors concluded that high sucrose was obligatory for tuberization during the first day. Once this requirement was satisfied for the first day, 'Thble 3.1. Effects of sucrose concentration and photoperiod on tuberization in vitro. The agar medium, which contained kinetin and an inhibitor of gibberellin synthesis (ancymidol), was either 2% or 8% sucrose. One-node cuttings from in vitro plantIets were placed on the 8% sucrose under 8-h photoperiods for induction periods of 0,1,2.3,4,5, or 7 days. After induction they were moved to 2% sucrose medium and given either 8-h or 16-h photoperiods for the balance of the week. Tubers were present or absent on all cuttings in a given treatment by the seventh day of treatment (summarized from Fig. 3A of Perl et al. 1991). Days on 8% sucrose with 8-h daylength

Daylength after moving to 2% sucrose

Tuberization

0 0 1 1 2

8-h 16-h 8-h 16-h 8-h 16-h 8-h 16-h

absent absent absent present absent present present present

2

3 or more 3 or more

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either high sucrose or long photoperiods during the next two days would produce tuberization. Neither high sucrose nor long photoperiods were required during the four subsequent days. Photoperiod extension with dim light (4 JLIllol m-z S-l) was equally effective in promoting tuberization, indicating that the promotion of tuberization was not related to photosYnthesis (Perl et a1. 1991). Other aspects of this work, which involve molecular biology, will be considered in Part VII.B.3. There is no obvious way to reconcile the well-established promotive effects of short days on the tuberization of whole plants or cuttings with the promotive effect of long days on the in vitro tuberization of Table 3.1. In the in vitro studies, induction may have been triggered by the medium containing high sucrose, kinetin, and gibberellin-inhibitor. (It is uncertain whether the 8-h photoperiod, which was apparently present the first day in all experiments, was also a contributing factor.) It seems that exposure to long photoperiods is beneficial for in vitro tuberization once such a triggering event occurs. Although the resemblance is probably no more than coincidental, these data are reminiscent of puzzling findings with leaf extracts (Struik et a1. 1987). Leaf-bud cuttings were taken from 'Bintje' plants that had received eight cycles of 12-h days before cutting; consequently they were moderately induced to tuberize. Extracellular extracts that had been obtained by centrifuging leaves from plants exposed to different numbers of short days were applied to the cuttings. Cuttings from all treatments tuberized to some degree, but the percentage of cuttings that tuberized was improved slightly if the extract was from plants that had been exposed to continuous light rather than to 12-h days. 3. Stolon formation. Under ordinary growing conditions in the temperate

zones, stolons form on whole plants before or shortly after plant emergence. If stolon growth has already been initiated, exposure to short photoperiods will inhibit stolon growth because tuberization will be favored instead. However, when one or more factors aside from photoperiod are extremely unfavorable for tuberization (see discussion that follows on temperature, irradiance, N fertilization, plant size, status of the mother tuber, and especially genetic differences between tuberosum and andigena), then moderate shortening of the photoperiod may promote stolon production (Werner 1934; Driver and Hawkes 1943; Pohjakallio et a1. 1957; Langille and Hepler 1991). An example may be seen in Table 3.2. In this experiment plants were grown from detached sprouts about 9cm long, so there was little or no stolon induction from the mother tuber. Exposure to 10-h photoperiods with day/night temperatures of 21/11°C increased stolon number and length compared to control plants maintained under 16-h photoperiods and 28/25°C. Although the

lOB

E. E. EWING AND P. C. STRUIK

Table 3.2. Effects of growth regulator applications on growth of 'Katahdin' plants under inducing and noninducing conditions. There were two experiments, each with eight plants per treatment, and data are means per plant from both experiments. Plants were grown from detached sprouts under nonindueing conditions (16-h photoperiods, day/night temperatures of 2Bo/25°q for three weeks. Half of the plants were moved to inducing conditions (10-h photoperiods, 21 °/11 0c) for three days. Half of the plants under inducing conditions were sprayed with gibberellic acid (GAJ. 100 mglliter. Part of the plants under noninducing conditions were treated with inhibitors of gibberellin synthesis: BAS-ll1 sprayed on the foliage at 1000 mg/liter. BAS-l06 applied to the soil at 15 mg per plant in the first experiment and at 30 mg in the second experiment, or 2-ehloroethyltrimethyl ammonium chloride (Ccq sprayed on the foliage at 1000 mg/liter. The inducing and noninducing conditions were continued. and chemical applications were repeated seven days later. Measurements were made after three more days of treatment. Stolon length is the sum of the lengths of the individual stolons (from Table 3 of Langille and Hepler 1991J..

Treatment Induced Control GA Noninduced Control BAS-ll1 BAS-l06 CCC

Plant height (cmJ

No. Stolons

Stolon length (emJ

No. Tubers

59 b 64 a

7.8 a 3.4 b

15.5 a 13.5 a

4.6 a 0.0 c

64 47 51 50

1.7 2.8 2.8 2.2

a d e e

c be be c

3.0 5.2 5.4 3.0

b b b b

0.0 1.8 1.1 0.4

e b be bc

Within columns, means followed by the same letter were not significantly different by Waller-Duncan k ratio T test (k = 100J or (P = 0.05).

effects of photoperiod cannot be separated in this experiment from the effects of temperature, it is probable that both short days and cool temperatures contributed to the increased stolon growth. Eight cycles of 12-h days increased stolon formation of 'Desiree' plants that had been exposed to 18-h days within 8 days after treatment started (A. J. G. Engels and E. E. Ewing, unpublished). At the point where induction is strong enough to produce tubers, of course, further shortening of the day length will cause a switch from stolon to tuber growth. After this point is reached long days rather than short days will favor stolon growth. A further cause of confusion as to whether short days promote or retard stolon growth is the question of when the observations are made. Examinations relatively soon after the photoperiod is shortened may reveal that short photoperiods hasten stolon initiation in plants where this has not yet occurred. Later examination of plants in the same treatments may show that although stolons form earlier, their growth is also terminated earlier by tuber initiation,

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leading to more stolon production by the end of the season under long days than under short days (Pohjakallio et al. 1957). Exposure of 'Bintje' plants to 12-16 cycles of 24-h photoperiods (12-h photoperiods extended by 12 h dim light) delayed tuber initiation and stimulated stolon elongation and stolon branching if the long-day treatments were initiated soon after plant emergence (Struik et al. 1988b). This had the effect of increasing the number of tuber initials and caused a shift toward smaller tuber size. Applying the long photoperiods at later stages of growth delayed tuber set and tuber growth. The photoperiod treatments also affected the proportion of large (dry weight >8 g) tubers that developed on a stolon branch rather than at the end of the main stolon axis (Fig. 3.9). More than half the large tubers developed on stolon branches under prolonged exposure to 24-h days. When exposure was only for 16 days, the later the treatment started, the smaller the proportion of large tubers forming on stolon branches (Fig. 3.9). In spite of the marked effects on numbers and sizes of stolons and tubers from exposing plants

o

~ Stolon end

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100 ~

Vl

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t';:i:i ••••

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20

~.~~

~~ ~~

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~~

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T1 T2 T3 T4

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Figure 3.9. Proportion of tubers (>8 g, d. wt.) developed at end of stolons or on stolon branches as affected by photoperiod treatment. Treatment codes are: C = control (all 12-h days); Tl = 24-h daylength treatment (LD) from 14-30 days after planting (DAP); T2 = LD from 22-38 DAP; T3 = LD from 30-46 DAP; T4 = LD from 38-54 DAP; Tt = LD from 22-62 DAP (from Struik et al. 1988b, as summarized by Struik et al. 1991).

110

E. E. EWING AND P. C. STRUIK

to 24-h photoperiods, neither early nor late treatments of 12-16 cycles had a detectable effect on total tuber yield.

B. Temperature Like photoperiod, temperature exerts a major influence on tuberizalion, with cool temperatures favoring induction to tuberize (see reviews by Bodlaender 1963; Gregory 1965; Ewing 1981; Struik and Kerckhoffs 1991). The negative effects of high temperature on tuberization are much more pronounced under long photoperiods (e.g., Wheeler et al. 1986; Snyder and Ewing 1989). Whether day or night temperatures have more important effects on tuberization is difficult to establish from available data, but diurnal temperature fluctuations were generally beneficial (Steward et al. 1981; Bennett et al. 1991). It ~s interesting to note in this connection that alternating day and night temperatures also overcame the deleterious effects of continuous irradiance, which caused a chlorotic and stunted growth in some potato cultivars under constant temperatures (Tibbitts et al. 1990). Exposing 4-week-old plants to high soil temperatures for 3 weeks caused about the same reduction in tuber dry weight as exposing them to high air temperatures (Menzel 1983a). When plants were grown at high air temperatures and soil temperature was varied, tuberization on cuttings was poor whether soil temperatures of the mother plants were warm or cool (Reynolds and Ewing 1989b). Experiments in which the temperatures of roots were controlled independently of tubers and stolons confirmed that high temperatures of the shoots had by far the most serious inhibitory effects on induction to tuberize (Table 3.3). High root temperatures had a small negative effect; and a slight increase in temperatures around the stolons and tubers had no effect (Table 3.3). The effects of high soil temperature were ameliorated by bud removal from the shoot, although curiously the effects of high air temperature were not (Menzel 1981, 1983a). Under high temperatures buds produced very high levels of gibberellin-like compounds (Menzel 1983b), which are known to inhibit tuberization (see Part VI.D.1). When air temperatures were cool and soil temperatures were warm, tuberization was also poor; but this was not because leaves failed to produce the tuberization stimulus: leaf-bud cuttings tuberized equally well whether soil temperatures of the mother plant were warm or cool (Reynolds and Ewing 1989b). Stolons in the heated soil became orthotropic; but when they reached the cooler temperatures above the soil surface aerial tubers formed (Fig. 3.10). This indicates that the stimulus was transported through the stolons butcould not be expressed at the high soil temperature (Reynolds and Ewing 1989b). Instead of

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Table 3.3 Summary of effects of temperature on growth of stolons, tubers. and dry matter partitioning to tubers. Information was obtained through the use of equipment. like that shown in Figure 3.3. which permits separation of roots from stolons and tubers and temperature control of each compartment. Warm temperatures were 28°C and cool temperatures were 18°C for shoots and roots. Temperatures of stolons and tubers were not completely independent of shoot and root temperatures; the warm and cool temperatures differed by only about 4°C in this chamber. so effects are not strictly comparable. The summary represents what happens when the temperature of only one of the three zones was increased: heating two zones at the same time produced much more severe effects. Classification for induction to tuberize is based upon the date at which 50% of the stolons present had produced at least one tuber. Dry matter partitioning. or harvest index. refers to the percentage of total plant biomass present in tubers. [This table is a summary of results from Stroik et a1. (1989a.b.c).]

lIndicates degree of temperature increase: +. ++, +++ = minor. large. or very large positive effect; ± = no effect. or effect unclear; -, --, --- = minor, large. or very large negative effect.

forming at the tips of orthotropic stolons, aerial tubers sometimes formed on the main stem in these experiments. Aerial tubers on the main stem also were observed when temperatures of shoots, roots, and stolons were manipulated separately (P. C. Struik, J. Geertsema, and C. H. M. G. Custers, unpublished results). In these experiments the phenomenon was most common when shoots were 28°C, roots were 18°C, and stolons approximately 25-28°C. This produced profuse shoot growth with many small leaves and numerous axillary branches. Apparently with the cultivarand photoperiod employed there was more tuberization stimulus produced in the leaves than could be expressed in the moderately warm stolon chamber. Swelling just above the attachments of small branches to main stems was frequently present along with the aerial tubers. Like aerial tubers, such swelling is typical when there is either an interference in movement of the stimulus underground (e.g., stem girdling by a pathogen) or some limitation in the ability of underground buds to form tubers in the presence of the stimulus (Fig. 3.11). Effects of high temperatures around shoots, roots, or stolons and

112

E. E. EWING AND P. C. STRUIK

Figure 3.10. The effect of soil temperature on tuberization of plants grown at cool air temperatures. Plants of 'C1-8M', an early-maturing neo-tuberosum clone, were grown at air temperatures of 19° day, 17° night. The soil temperatures of the plant on the left were 20° day, 18° night. The plant on the right had soil temperatures of 32° day, 31° night. Tubers in the left-hand pot developed normally. Stolons in the right-hand pot turned upward and tuberized only after tips had emerged from the soil surface, where temperatures were cooler (reproduced from Reynolds and Ewing 1989b).

tubers are summarized in Table 3.3. Effects are indicated for season-long increases for one of the three zones; heating more than one zone at the same time was much more deleterious. High temperatures of the shoots, and to a lesser extent of the roots, delayed tuber initiation. Stolon growth was stimulated moderately whether temperatures of shoots, roots, or stolons and tubers were raised. The increased stolon growth was accompanied by more stolon branching, which permitted more sites for tuber formation (Struik et al. 1989b,c). Presumably this explains why more tubers developed under the warmer conditions. The average size of such tubers was much lower, with the result that tuber yield was severely depressed, especially with high shoot temperatures. Pot experiments showed that the number of tubers was affected more by raising the temperature during tuberization than at earlier or later stages of plant development (Struik and Kerckhoffs 1991). Experiments with reflective materials that were applied to the soil surface to moderate soil temperatures under tropical conditions showed that cooling the soil in this manner had very beneficial effects on earli-

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Figure 3.11. Aerial stolons. aerial tubers. and swollen stems on a plant grown so as to prevent underground tuberization. A rooted stem cutting was grown as described in Figure 3.2. with no underground buds. All but the youngest leaves were excised just before photographing to expose the stems. Aerial stolons grew downward but were prevented from penetrating the soil. except for one tip that was overlooked. Within a few days it produced a white tuber (farthest left). Other tubers. whether borne at the end of a stolon or sessile. were green. Stems developed nodal swellings that resembled tubers.

ness of tuberization and total tuber yield (Midmore 1984). The effects of leaf pubescence and thus ability to reflect radiation could also be of some significance in this connection (Midmore and Mendoza 1984). Tuber percent dry matter invariably declines as growing temperatures are increased (e.g., Ben Khedher and Ewing 1985). Incorporation of 14C from a labeled leaf into tuber starch was depressed at high temperatures, but incorporation into sucrose in the tuber was increased (Wolf et al. 1991).

c. Irradiance Like long photoperiods and high temperatures, low levels of irradiance during the day decrease the induction to tuberize (Bodlaender 1963; Gregory 1965; Demagante and Vander Zaag 1988). Menzel (1985) suggested that the effects of both low light intensity and high temperature are brought about by increased production of growth substances that inhibit tuber formation. Gibberellins are the most likely candidates for

114

E. E. EWING AND P. C. STRUIK

such a role (Part VI.D.1). As might be expected, a combination of high temperatures and low irradiance is especially inhibitory to tuberization (Menzel 1985). Effects of shading treatments that reduced light levels by 50% during the early part of the crop season were studied in the Netherlands and in the United States in New York State (Struik 1986). Although stolon initiation was delayed slightly by shading, no effect of shading on stolon number was detected. The lack of effect on stolon number was in agreement with earlier shading studies in Australia (Sale 1973a,b). Although the number of stolons was not affected, the period of stolon elongation was protracted (Struik 1986), consistent with the delay in tuberization described as follows. Consequently stolons in the shaded treatments were longer, they contained more potential sites for tuber initiation, stolon decay at the end of the season was delayed, and dry weight of stolons increased. In agreement with previous reports (Gray and Holmes 1970; Sale 1973a,b, 1976), the shading treatments had a pronounced effect in delaying tuberization, especially if applied shortly after the onset of tuberization (Struik 1986). Six weeks of shading at this time delayed tuberization in both New York and The Netherlands. The maximum number of tubers from six weeks of early shading was less than in the control in the Dutch experiment, and equal to the control in New York. The final number of tubers (the difference between the number initiated and the number resorbed, see Part IX.A) from shading was higher than in the control in New York; but the two treatments had equal final numbers in the Netherlands. The starting date of two-week shading periods had no effect on dry matter yields of tubers in New York, but size distribution was dependent upon the timing of treatment. Tuber size distribution in the Dutch experiment was affected by the timing of shading, too, but not in the same pattern as in New York; and total dry matter yield of tubers was depressed by late shading. The pronounced environmental and cultivar differences between the two locations complicates the interpretation of the data (Struik 1986), but the results suggest that variations in cloud cover early in the growing season might have considerable influence on tuber size distribution (Struik 1987). D. Nitrogen

A fourth environmental factor that plays a major role in induction to tuberize is N fertilization (Werner 1934; Gregory 1965). In his classical work on tuberization, Werner (1934) concludes that limiting the supply of inorganic N to plants that are growing vigorously because of exposure to

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long days and warm temperatures produces a retardation of shoot growth. This was accompanied by the accumulation of starch in the shoots, and with initiation or acceleration of tuberization. 1. Hydroponic culture. Werner's research on N in hydroponic culture

has been greatly extended in a comprehensive set of papers by Krauss and his co-workers (Krauss and Marschner 1971, 1976, 1982; Krauss 1978, 1981; Sattelmacher and Marschner 1978, 1979; Marschner et a1. 1984). Growing the plants in nutrient solutions made it possible to regulate and manipulate the N supply in defined amounts and concentrations, and frequent observations of stolon and tuber development were feasible through the use of a separate compartment. It should be noted that most of the research was with sprouts detached from the mother tuber. These experiments and other research on the control of tuberization through the regulation of N nutrition have been reviewed by Krauss (1985). We will summarize the main points. Plants of the tuberosum cultivars 'Ostara' and 'Clivia' did not tuberize under 12-h photoperiods in this system unless the N supply was temporarily withdrawn. In similar experiments, andigena plants grown under 8-h photoperiods tuberized even under continuous N, but withdrawal of the N hastened tuberization (Krauss and Marschner 1976). The interference from N was obtained whether it was supplied to the roots as ammonium or nitrate ion (Krauss and Marschner 1976); but supplying the leaf with urea was ineffective in blocking tuberization, even though the N content of the plants was thereby increased (Sattelmacher and Marschner 1979). Second growth of tubers (see Part IX. C) was promoted by changing from zero N to a high N concentration after tuber formation started (Krauss and Marschner 1976). Tuberization was not induced by N withdrawal under 18-h photoperiods or under constant temperatures of 30°C (Krauss and Marschner 1982). It may be that the ability of N to interfere with tuberization is dependent upon the level of irradiance. Repetition of the above experiments with similar equipment but at a much higher light intensity produced different results: tuberization was slightly delayed, but definitely not inhibited by high concentrations of N in the nutrient solution (p. C. Struik and G. Coster, unpublished results). 2. Field experiments. Under field conditions the effect of N application

on delaying time of tuber initiation was also small (Radley 1963) or not significant (Gunasena and Harris 1968, 1969; Dyson and Watson 1971). Clutterbuck and Simpson (1978) found a delay in tuberization associated with N application, but only in the absence of irrigation. Simpson (1962) reported more tubers in the N treatment receiving 67 kg ha -1 than in the

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control, 12 weeks after planting, whether irrigated or not. (Neither treatment tuberized at 8 weeks after planting.) In several of the above studies it was observed that once tubers were initiated, their rate of enlargement was more rapid in the treatments receiving N than in the control treatments. Thus unless harvests are frequent and treatments are very well replicated, any small delays in the time of tuberization caused by N applications within the range of normal agronomic practice are likely to be hard to detect in field experiments. It might be anticipated that the delay would be more obvious under tropical conditions where warm temperatures would tend to discourage tuberization. This was observed in one experiment when potatoes were planted at an elevation of 300 m in Peru; there was a significant linear decrease in tuber number 43 days after planting associated with increasing the rate of N from 0 to 240 kg ha-1 applied at planting. However, no difference in time of tuberization was detected in several similar experiments carried out nearby at an elevation of 800 m (Payton 1989), even though temperatures were still very warm.

3. Cutting response. Cuttings taken from a field experiment in New York one month after planting tuberized somewhat better if plants received zero N at planting than if they had received Nat 168 or 336 kg ha -1, but the effect disappeared when cuttings were taken two or three months after planting (Santeliz-Arrieche 1981; Ewing 1985). This would suggest that application of N at planting reduced slightly the level of the stimulus for tuberization in leaves early in the growing season, but that the effect disappeared as the season progressed. Increased tuberization on cuttings is usually associated with an increase in the harvest index (ratio of tuber to total biomass). As we discuss in Part VI.A.6, the harvest index consistently declines as N fertilization increases. 4. In vitro response. There are several reports that in vitro tuberization responded to N content in the medium, but little agreement as to the optimal concentrations or the effect of N source. One reason may be the variety of materials and protocols employed (Table 3.4): axillary buds removed from intact plants and incubated in darkness for two weeks on an agar medium with no other additives except sucrose (Ewing 1985); single-node segments of etiolated sprouts taken from old tubers cultured in darkness for three weeks on modified White's medium plus sucrose ranging from 2% to 8% (Koda and Okazawa 1983a); and cuttings taken from plantlets propagated in vitro under 16-h photoperiods on modified Murashige-Skoog medium containing 4% sucrose (Garner and Blake 1989). In the first of these systems tuberization on a medium containing 6%

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Table 3.4. Effects of N additions to medium on in vitro tuberization [summarized from Ewing (1985), Koda and Okazawa (1983a), and Garner and Blake (1989)}.

Propagule

Light Conditions Medium

Length of culture

Effect of N additions on tuberization

1-2 weeks

Tuberization was improved by adding NH 4 N03 , with 16-60 mM N giving best results. No effect of adding NH 4 N0 3 if (sucrose) ;::: 4%. Tuberization on 2% sucrose depressed by adding 40 mM N as NH 4 N0 3 and by even lower concns of NH4 CI or amino acids, but not by Ca(N0 3 lz up to 28 mM N. Optimal N concn was 60 mM for fresh weight yield of microtubers. Decreasing nitrate/ammonium ratio to 1:2 or less inhibited tuberization.

Axillary buds from intact plants

Darkness

One-node segments of etiolated sprouts from old tubers

Darkness

Modified White's, + 2%-8% sucrose

3 weeks

One-node segments from in vitro plantlets

16-h days

Modified MurashigeSkoog, + 4% sucrose

4 weeks

Plain agar

+ 6% sucrose

sucrose was improved by adding either ammonium or nitrate N, and concentrations of ammonium nitrate in the range of 16-60 mM N gave good results (Ewing 1985). The addition of ammonium nitrate did not affect tuberization of sprout segments if the sucrose concentration was 4% or higher, but even 40 mM N (present as 20 mM ammonium nitrate) decreased tuberization on a medium containing 2% sucrose. The decrease on 2% sucrose medium was not observed with calcium nitrate additions, but was present with additions of ammonium chloride and amino acids (Koda and Okazawa 1983a). If the 2% sucrose medium contained amino acids to give 7 mM N, then the addition of 10 mM ammonium nitrate decreased the tuberization of sprout segments (Koda and Okazawa 1988). Decreasing the ratio of nitrate to ammonium ion below 1 was detrimental to the number and fresh weights of tubers obtained by propagating nodes of plantlets on 4% sucrose under 16-h photoperiods (Garner and Blake 1989). In this system the optimal concentration of N for tuber number and size was 60 mM. In summary there is little indication that the presence of nitrate N inhibits tuberization in vitro. Combinations of ammonium and nitrate N at concentrations up to the 60 mM N present in Murashige-Skoog medium were generally not inhibitory if the ratio was near 1. An exception was that ammonium nitrate was inhibitory on 2% sucrose. It is inter-

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esting that just as the negative effects of N applications on induction to tuberize in whole plants appear to be more severe at low light intensities, so the negative effects of ammonium nitrate on tuberization in vitro appear to be more pronounced at low sucrose concentrations. It is difficult to know how much of the negative effect of the ammonium ion on tuberization was associated with direct effects on induction to tuberize versus indirect effects on overall growth. Garner and Blake (1989) noted a deleterious effect on plantlet growth in vitro, with pale green leaves and radial stem enlargement, when the ratio of nitrate to ammonium was low.

E. Other Environmental Factors Photoperiod, irradiance, temperature, and N rate are the major environmental factors that, in addition to genotype (to be considered in Part IV.) and the size and physiological age of the mother tuber (Part V.), control induction to tuberize; but numerous other environmental factors affect tuberization. Many of these appear to affect the subsequent development of stolons and tubers rather than the level of induction. Specific effects of most of these factors on tuberization are difficult to distinguish from generalized effects on the growth and development of the whole plant. 1. Carbon dioxide and oxygen. High concentrations of CO2 surrounding the roots increased the number of tubers initiated when plants were grown in nutrient solution (Arteca et a1. 1979). Stolons grown in separate chambers containing no soil or other medium developed fewer stolons when surrounded by a gas mixture containing 10% O2 and 15% CO2 than when surrounded by ambient air, both gas mixtures having a high relative humidity (Cary 1986). In similar experiments, lowering the O2 content to 5% in nitrogen produced elongated stolons and greatly reduced tuberization (Harkett and Burton 1975). Data from experiments such as these are difficult to interpret in view of the fact that stolon and tuber formation may be affected by the absence of mechanical resistance (see below). Data from experiments with tuberization on cuttings have given conflicting results. Tuberization of in vitro cuttings was promoted by increasing the CO 2 concentration to 8% or 10% (Mingo-Castel et a1. 1974, 1976a). It might be assumed that the role of the high CO2 was merely to counteract the effects of endogenous ethylene, but this did not seem to be the case. Promotion from CO2 was observed even if steps were taken to remove ethylene. On the other hand, cuttings were able to form tubers equally well whether the darkened bud at the base of the cutting was

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exposed to gas mixtures containing CO 2 concentrations as high as 12% or O2 concentrations as low as 4% (Ewing 1985). Whole-plant studies on atmospheric CO 2 effects on tuberization show increased tuber yields from CO 2 enrichment (Collins 1976); but the response can vary depending on cultivar, temperature, and irradiance (Yandell et al. 1988). The effect of two CO 2 levels in the atmosphere surrounding the shoots was tested with three cultivars, two photoperiods, and two levels of irradiance (Wheeler et al. 1991). 'Denali' showed the most tuber yield response, and 'Norland' the least, to increasing the CO2 from 350 Ilmol-1 to 1000 Ilmol mol-1 • Averaged over cultivars, there was more tuber yield response to the higher CO 2 concentration at a 12-h photoperiod than at a 24-h photoperiod; and more response at a PPF of 400 Ilmol m-2 S-1 than of 800 Ilmol m-2 S-1. 2. Drought stress. Drought stress might be expected to have a significant effect on induction to tuberize, but none was detected (E. E. Ewing and D. W. Wolfe, unpublished results) when cuttings were taken from plants grown in a line-source irrigation experiment (Wolfe et al. 1983). The wellknown effects of drought in restricting potato yields may be associated with reductions in the canopy and consequent restrictions in the ability to produce assimilate rather than with direct effects on induction. It is uncertain whether water around the stolon tip (or bud, in the case of a cutting) is needed for tuberization to occur. Struik and Van Voorst (1986) reported that the formation of tuber initials was greatly enhanced when the stolon medium was kept air dry during tuberization. This effect was more pronounced when the uptake of water by the roots was sufficient, but was also seen when drought occurred both in the root and in the stolon media. However, the large increase in the number of swollen stolon tips, caused by the dry stolon medium, did not result in more tubers. Field experiments on the effects of drought on tuber initiation and development have produced contradictory results (Taylor and Rognerud 1954; Bradley and Pratt 1955; Patzold and Stricker 1964; Cavagnaro et al. 1971; Steckel and Gray 1974). In part the confusion can be attributed to a lack of uniformity in defining the beginning of tuber initiation, and to the fact that many of the effects are probably indirect. Pot experiments showed that tuber number per stem was reduced by soil moisture stress if this occurred early in the season during tuber initiation; similar stress after tuber initiation did not affect tuber number per stem (MacKerron and Jefferies 1986). Early drought stress also reduced tuber number in long-term field studies. There were about twice as many tubers per stem when 120 mm of rain fell during the first 40 days after planting as there were when rainfall was only 18 mm, and the relationship between rainfall and tuber number

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was linear during the 12 seasons examined (Haverkort et al. 1990a). The reduction in tuber number per stem appeared to be caused mainly by a reduction in the number of stolons per stem: drought stress applied from plant emergence until stolon initiation reduced stolon and tuber numbers per stem in pot experiments, but later drought stress had no effect (Haverkort et al. 1990a). Tubers per stolon were not affected. 3. Soil saturation. Excessive soil water was reported to have deleterious effects on the number of tubers, either by restricting the number initiated or set (Patzold and Stricker 1964; Harkett and Burton 1975) or by causing too many to be initiated (Harris 1978). The nature of these effects is not understood; presumably they involve changes in soil gas exchange or soil compaction. Tuberization is generally poor in solution culture (R. M. Wheeler, personal communication); but tuber yields were very satisfactory when plants were grown by nutrient film technique where stolons were not submerged (Wheeler et al. 1990). 4. Soil tilth. As is discussed in Parts VI.D.5 and VIII.D, the density of the medium surrounding the stolons may play an important role in encouraging tuber formation on plants that are induced to tuberize. 5. Salinity. Table 3.5 presents data from a factorial experiment that test the effects of conductivity of the nutrient solution around the roots at two temperatures. The warmer nutrient solution, 20°C, was not warm enough to increase the number of tuber-bearing stolons; but the number of tubers per tuber-bearing stolon increased, mean tuber size decreased, and tuber yield was unaffected compared to nutrient solution at 15°C. Increasing the salinity of the nutrient solution through the addition of NcCI drastically lowered tuber number at both temperatures. At the higher salinity there were also fewer tuber-bearing stolons, fewer tubers Table 3.5. Effect of temperature and electrical conductivity (EC) of the nutrient solution on the development of 'Desiree'. Conductivity was increased by the addition of N aCI (Unpublished data of L. H. J. Kerckhoffs and P. C. Struik).

(0C)

EC (mS/cm)

No. tubers/plant

Tuber yield (kg/plant)

No. tuber-bearing stolons per plant

15 15 20 20

0.7 4.5 0.7 4.5

39 13 52 23

1.67 0.73 1.63 1.22

13.9 7.7 14.0 10.6

Temp.

No. tubers per tuber-bearing stolon 2.8 1.7 3.7 2.1

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per tuber-bearing stolon, and lower yields of tubers at both temperatures. It is not clear to what extent the decrease in tuberization from the saline

treatment was caused by an overall negative effect on plant growth and development as opposed to more specific effects on stolon and tuber formation.

IV. GENETIC EFFECTS A. Variation in Critical Photoperiod

The critical photoperiod for tuberization of a potato cultivar, analogous to the terminology used for flowering, is the longest photoperiod that will permit tuberization of the cultivar (Garner and Allard 1923; Kopetz and Steineck 1954; Madec and Perennec 1959). Solanum tuberosum ssp. andigena-the potato cultivated in the Andes, where the crop was first brought under cultivation-has a very short critical photoperiod for tuberization. Even when grown under cool temperatures and high light intensity, most accessions of this subspecies will fail to tuberize unless photoperiods approach 12-13 h. In contrast, S. tuberosum ssp. tuberosum, the subspecies that is grown in the temperate zones, is capable of tuberizing under much longer days; i.e., tuberosum has a much longer critical photoperiod. In fact, some tuberosum cultivars will tuberize under continuous light if other environmental conditions are favorable. Genes for the longer critical photoperiod, however, are present in some andigena germplasm. Through a process of recurrent selection, clones have been developed from andigena populations that are able to tuberize as well as tuberosum under long days (Simmonds 1966; Glendinning 1975; Cubillos and Plaisted 1976; Tarn and Tai 1977; Munoz and Plaisted 1981). It is important to stress that neither these "neo-tuberosum" clones nor the tuberosum that they resemble can be said to be truly "day neutral", i.e., unresponsive to photoperiod. Even genotypes that have a long critical photoperiod under cool temperatures will respond strongly to photoperiods at higher temperatures in terms of induction to tuberize and associated traits. To illustrate, at day/night temperatures of 20°115°C 'Norchip' was as induced to tuberize under 16-h days as under 10-h days; but when temperatures were increased to 30 0 /25°C, the longer photoperiods gave much less induction, as measured by tuberization of leaf-bud cuttings (Snyder and Ewing 1989). Likewise, the very early maturing 'Norland' grown at 16°C produced higher tuber yields under 24h photoperiods than under 12-h photoperiods; but when the temperature

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was increased to 20°C the yields were higher under the shorter photoperiods (Wheeler et a1. 1986). In addition to differences among genotypes in critical photoperiod, there appear to be genetic differences in the number of daily cycles shorter than the critical photoperiod that must be applied to evoke tuber induction (P. C. Struik, unpublished results). The overall tendency is that fewer short-day cycles are required if the photoperiods are much shorter (rather than barely shorter) than the critical photoperiod; but here, too, there is genetic variation.

B. Classifications for Response to Photoperiod A source of confusion has been the attempt to classify cultivars based upon the effect of photoperiod on tuber yields (e.g., Hackbarth 1935). Very late maturing clones from the Andes are called "short-day" genotypes in such classification systems because highest yields are obtained when grown under short days, whereas very early cultivars from northern latitudes are classified as "long day." These latter types tuberize so strongly and so early under short days that top growth is highly restricted and maturity comes too early for satisfactory yields. Genotypes intermediate between the two extremes will yield about as well under short days as under long days and are therefore called "day neutral." Unfortunately, these names often lead to the erroneous conclusion that induction to tuberize is favored by short days in the first category, by long days in the second, and by either in the third. To minimize such confusion it seems preferable to think in terms of the critical photoperiod (Kopetz and Steineck 1954; Madec and Perennec 1959; Krug 1960; Maierhofer 1963; Ewing 1978). Even using this trait no quantitative classification is possible because of the modifying effects of such factors as temperature and irradiance level. The critical photoperiod of a cultivar might be 18 h under cool temperatures, but 14 h at warm temperatures (Snyder and Ewing 1989). Another classification that seems to be related to critical photoperiod is determinate versus nondeterminate growth habit. Apical growth of the main stem of the potato plant terminates in a flower cluster, but sympodial growth of an axillary branch permits further shoot growth there. The new axillary branch will terminate in a flower cluster in the same manner, but new sympodial growth may again occur. In this manner the main axis may be extended by three or more nodes. The extent of sympodial growth is affected by how strongly the plant is induced to tuberize (see Part VLA.1). Under growing conditions such as those in the northwestern United States, 'Russet Burbank' and other late maturing cultivars are able to develop extensive sympodial growth as

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well as axillary branching at the base of the plant. They are classified as nondeterminant. High rates of nitrogen fertilizer will delay their senescence and, if the growing season is long enough to take advantage of the extra top growth, will lead to higher tuber yields. In contrast, determinate types grown in the same environment are more strongly induced to tuberize, have comparatively weak sympodial growth and basal axillary branching, and are less responsive to late N applications.

c. Inheritance of Critical Photoperiod Long critical photoperiod seems to be recessive to short. It is hypothesized that the reaction is controlled by one major gene and by several minor genes that modify its effects (Mendoza and Haynes 1977). Because the critical photoperiod is dependent upon temperature, it is not surprising that genotypes with very long critical photoperiods at cool temperatures are likely to exceed other genotypes in ability to tuberize under exceedingly high temperatures (Ewing 1981; Ben Khedher and Ewing 1985; Snyder and Ewing 1989; Midmore 1990). This is not to say that such cultivars are always the best choice for growing in hot climates; if the photoperiod is short, they may tuberize too quickly for good yields in spite of the heat; and ability to tuberize is only one of the many components of heat tolerance (Ewing et al. 1987). Cultivars with a somewhat shorter critical photoperiod tuberize a little later and are able to produce more canopy before senescing. D. Usefulness for Studying Physiology Genetic differences afford excellent opportunities for studying the physiology of tuberization. The intensity of induction to tuberize can be varied experimentally not only by environmental manipulations, but by comparing genotypes of contrasting critical photoperiods. Furthermore, there are interesting mutant plants that give the appearance of being deficient in abscisic acid (Quarrie 1982; Vreugdenhil and Struik 1990), gibberellins (Bamberg and Hanneman 1991), and perhaps cytokinins (Leue and Peloquin 1982)-all of which have been suggested as being implicated in tuberization (Part VI.D). Genetic differences of interest to physiologists extend well beyond the species, S. tuberosum, and even beyond the group of tuberizing species. A cross between S. brevidens, a non-tuber-bearing species, and S. chacoense, a weedy tuber-bearing species, produced a triploid hybrid. The hybrid possessed a 2: 1 ratio of genomes for non-tuberous to tuberous plants (Ehlenfeldt and Hanneman 1984). The triploid was crossed to S. tuberosum to produce a pentaploid that contained a 2:3 ratio of the

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respective genomes. Comparison of the abilities of the triploid and pentaploid to tuberize led the authors to conclude that the tuberization response was a dosage and/or threshold effect. The triploid, though unable to tuberize like S. chacoense, was able to form stolons, which the S. brevidens parent could not do. By contrast, the pentaploid, with a higher dose of genomes for tuberization, was able to form both stolons and tubers (Table 3.6). These responses were observed on cuttings (HannapeI1990) as well as on whole plants (Ehlenfeldt and Hanneman 1984). The stolon formation by the triploid is consistent with the observations that under noninducing conditions, exposure to shorter photoperiods may increase stolonization (Part IILA); and that stolon formation on cuttings indicates an intermediate response between orthotropic shoot growth and tuberization (Part ILC). In Part VII.B we describe experiments with the triploid and pentaploid, which further develop this theme.

v. EFFECTS OF THE MOTHER TUBER The size and physiological condition of the mother tuber exert a dominant influence on plant development, involving direct as well as indirect effects on stolon and tuber formation. An excellent review that traces the history of research on this topic is presented by Van der Zaag and Van Loon (1987). We will follow the definitions of terms agreed upon by the Section of Physiology of the European Association of Potato Research (Reust 1984).

Table 3.6. Evaluation of tuber formation on leaf-bud cuttings from Solanum spp. and hybrids (from Table 1 of Hannapel 1990). Total cuttings forming Species or Hybrid S. S. S. S.

etuberosum brevidens brevidens X S. chacoense brevidens/chacoense X S. tuberosum 'Wis AG 231' S. chacoense S. tuberosum 'Wis AG 231' S. tuberosum 'Superior'

Total cuttings

Tubers

Stolons

Shoots

18 18 24 20

0 0 0 15

0 0 3 2

18 18 21 3

18 18 10

0 17 10

15 0 0

1 0

3

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A. Physiological Age Both stolons and tubers can form directly on sprouts from mother tubers before the sprouts emerge from the soil. This occurs with Some mother tubers but not with others because of differences in the physiology of the tubers. It is customary to summarize differences in physiological condition of mother tubers by referring to their physiological age (Madec and Perennec 1959; Toosey 1963; Wurr 1978; Perennec and Madec 1980; Reust 1984; Burton 1989). The physiological age of a tuber is its stage of development, which is modified progressively by increasing chronological age, and which also depends upon storage conditions and growth history. Physiological aging occurs both in the absence and presence of a sprout. 1. Determinants of physiological age. An important determinant of the physiological age of a tuber is its chronological age (Kawakami 1962); but

the temperature at which the tuber is stored also plays a major role, with higher temperatures promoting more rapid aging (Fischnich and Krug 1963). There are a number of reports that higher temperatures during the growth of the seed crop also accelerate physiological aging (Iritani 1968; Claver 1973). The temperature after death of the tops or separation of the tubers from the tops, however, was found to be much more important than the temperatures prior to this event [Van Ittersum and Scholte 1992].

The importance of storage temperature has led to a search for a mathematical function that will enable us to predict the extent of physiological aging if we know the length of storage and the storage temperature (Reust 1983, 1984). To the degree that temperatures between tuber initiation and harvest playa role, it might be useful if they were taken into account as well. While the concept of thermal time is helpful, it proved difficult to find a simple formula that fits all the data (Van der Zaag and Van Loon 1987; Burton 1989). For example, Scholte (1987) showed that the effect on dormancy of a short period of high storage temperature depends upon when it occurs. More recently Klemke and Moll (1990) published a model to predict plant emergence. The model uses a nonlinear relation between temperature and what is termed physiological age rate. There is a major genetic component of physiological age: tubers of cultivars that sprout early in storage tend to become physiologically old faster than those that sprout later; but this rule is not without exceptions. Not only is there great genetic variation as to how long tubers can be stored at warm temperatures before visible sprouting occurs (e.g., Wright and Whiteman 1949; Bogucki and Nelson 1980; Susnoschi 1981), but there are also cultivar differences in how much time elapses between the

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beginning of sprouting and the formation of "sprout-tubers"-also called "little potatoes" [Reust 1983; Hartmans and Van Loon 1987). The first of these intervals is variously called the rest or dormant period; it is considered to begin at tuber initiation and to extend until sprouts appear. The second interval, from sprouting to sprout-tubers, is known as the incubation period (Claver 1975; Madec 1978). Comparisons of cultivars indicate that the length of the dormant period is not a reliable predictor of the length of the incubation period. The first may be long and the second short, or vice versa; and both are affected by environmental factors (Umaerus and Roslund 1979; Reust 1983; Hartmans and Van Loon 1987; Bodlaender and Marinus 1987). In addition to cultivar and storage temperature, the length of the dormant period is affected by tuber size, tuber maturity at harvest, and numerous other factors (Hemberg 1985; Burton 1989). 2. Assessing physiological age. During the period from the end of

dormancy to the formation of sprout-tubers there are significant differences in the potential value of tubers for seed. Attempts have been made to define these objectively. The sprouting capacity is measured by the fresh weight of sprouts produced by the seed tubers under standardized conditions (Hartmans and Van Loon 1987). Growth vigor is the potential of a tuber to produce sprouts and plants rapidly under conditions favorable to growth (Hartmans and Van Loon 1987; Van der Zaag and Van Loon 1987). Both the sprouting capacity and the growth vigor increase as the tuber passes from the stage of single to multiple sprout production; but the sprouting capacity typically continues to increase after the growth vigor has started to decline (Van der Zaag and Van Loon 1987). During aging the number of sprouts continues to increase; but after a certain point, indicators of vigor such as final plant height begin to decrease. Methods were developed to calculate indices of physiological aging for cultivars based upon the growth vigor shown by seed tubers stored under controlled conditions [Van Ittersum et a1. 1990). There was year-to-year variation in cultivar performance in these tests, showing that more than one season is required to characterize a cultivar. It is apparent that there is also a large interaction between storage conditions and cultivar with respect to sprouting behavior and growth vigor. Attempts have also been made to find a biochemical marker that would lead to a more precise characterization of physiological age [Apelbaum 1984; Coleman and King 1984; Reust and Aerny 1985; Van Es and Hartmans 1987; Mikitzel and Knowles 1989; Knowles and Knowles 1990), but as yet no procedure is widely accepted. 3. Modifying effects. The growth vigor of a tuber depends upon the

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condition of the tuber itself and the condition of its sprouts. Therefore, treatment of either the mother tuber or the sprouts may modify the response. For example, sprouting of seed tubers in diffuse light instead of darkness changes the nature of the sprout and increases growth vigor (Scholte 1989). Treatment of aged seed pieces with 1-naphthaleneacetic acid partially ameliorates the effects of aging compared to physiologically young seed pieces (Mikitzel and Knowles 1990). Application of calcium to sprout tips is effective in preventing tip necrosis. Sprouts with black tips are a very common sight in potato storages, depending upon cultivars and storage temperature and humidity. Sprout tip necrosis is related to an inability of the tuber to supply adequate calcium to the growing tip (Dyson and Digby 1975; Davies 1984a). When the deficiency becomes severe, the tips become necrotic; and the loss in apical dominance encourages sprout branching. This calcium deficiency is certainly one explanation, but not necessarily the only one, for loss of apical dominance of individual sprouts as tubers age physiologically. 4. Summary. Although there are other contributors to physiological age

of tubers, three key factors are chronological age, storage temperatures, and cultivar. The stages of physiological aging have been identified in terms of the morphology of developing sprouts; and the consequences for plant development of using tubers in each stage are well understood. We turn next to the sequence of sprouting patterns associated with increasing stages of physiological aging. B. Similarity of Patterns on Tubers and Cuttings As pointed out by Madec and Perennec (1962) in a somewhat different context, there is an interesting parallel between the patterns of: (a) sprout development on tubers of increasing physiological age, which are stored in the dark; and (b) growth at buried axillary buds of cuttings taken from plants that have been exposed for increasing periods of time to tuber inducing conditions before cutting. These patterns are compared in Figure 3.12 and described as follows. 1. Response of tubers to aging. Newly harvested tubers typically go

through a dormant (or rest) period when buds in all eyes fail to make visible sprout growth even if exposed to favorable temperatures (Hemberg 1985; Burton 1989). If exposed to warm temperatures (10-15°C) as soon as the dormant period is passed-Le., while still very young physiologically-the tuber tends to produce a single unbranched sprout at the apical eye cluster (Krijthe 1962). If the tuber is kept for progres-

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o Figure 3.12. Comparison of developmental patterns at underground buds of cuttings and on buds in eyes of tubers stored in darkness. The top row depicts a simplified version of the pattern shown in Figure 3.8. As mother plants become more strongly induced to tuberize, the progress of responses shown by cuttings is Oeft to right): dormant bud. orthotropic shoot, stolon, stolon terminated by a tuber, and sessile tuber. The bottom row depicts the progression of sprout development as tubers attain increasing physiological age before being moved to temperatures that permit sprouting (left to right): buds in all eyes dormant; single sprouts. mainly at the apical eye cluster. lacking in branches or with upright branches; multiple sprouts at many eyes, with stolon branches; tubers forming at the ends of some of the stolon branches; sessile tubers. Drawings are schematic representations; they are not drawn from actual specimens. Not all stages of the progression or possible types are included (from Ewing 1990).

sively longer periods at cold temperatures (less than ca. 4°C) before transfer to temperatures that permit sprouting-Le., as the tuber becomes progressively more advanced in physiological age before sprouting starts-the nature of its sprouts changes. The number of eyes that develop sprouts increases, multiple sprouts form at these eyes, and the sprouts are more likely to be branched (Krijthe 1962). With further advance in physiological age before sprouting starts, the branches assume a diagravitropic growth (Dyson and Digby 1975), becoming stolons. Tubers held at cold temperatur-es for a very long time will develop tubers at the end of the stolons once they are allowed to sprout, or after extreme aging will form sessile tubers directly on the mother tuber. Thus the pattern in response to increasing physiological age is: (1) dormancy; (2) single, upright sprouts; (3) multiple sprouts with upright branching; (4) multiple sprouts with stolon branches; (5) multiple sprouts with stolons terminated by tubers; and (6) sessile tubers (Fig. 3.12).

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2. Response of cuttings to induction. Apical cuttings taken from mother

plants that have been exposed to a range of inductive conditions display almost the same progression seen in tubers. To facilitate comparison, we simplified the stages from Figure 3.8 and presented the progression again in Figure 3.12 above the progression of sprouting types of tubers. Recapitulating from Part n.e, the buried bud of an apical cutting from a mother plant that receives no induction remains dormant. Exposure of the mother plant to a few days of photoperiod shorter than its critical photoperiod causes the buried bud to break dormancy and develop as an upright shoot. As the length of exposure to short days is prolonged, the response of the buried bud changes from an upright shoot to a diagravitropic stolon to a stolon terminated by a tuber to a sessile tuber. We do not know whether this similarity in progression of responses is coincidental or a consequence of the fact that the stimuli for upright sprouts, stolons, and tubers are common to tubers and leaves. Also to be determined is whether the progression of responses is controlled by a series of independent stimuli, each "turned on" in succession as the physiological age of tubers or the exposure of leaves to short photoperiods increases; or whether the progression reflects increasing strength of a single stimulus. (We emphasize again that even a single stimulus may derive from a particular balance among a number of different compounds.) For more discussion of these possibilities, see Ewing and Wareing (1978) and Ewing (1985).

c.

Implications for Tuberization

Differences in physiological age of the mother tuber produce the differences in sprout morphology described previously and also exert a major influence on subsequent development of the plant. Evidence that the physiological age of the mother tuber affects the induction to tuberize comes from in vitro experiments, studies with cuttings, and observations of whole plants. 1. In vitro. Single-node segments cut from etiolated sprouts tuberize

much more rapidly in vitro on a 2% sucrose medium as time after tuber harvest elapses (Koda and Okazawa 1983a). In order to use such segments in a bioassay for the presence of the tuberization stimulus from leaves (see Part VI.D.3), it is necessary to select sprouts from physiologically young tubers. Otherwise there is tuberization in the controls. Alternatively, sprouts from physiologically old tubers can be employed if 10 mM ammonium nitrate was added to the medium to discourage tuberization (Koda and Okazawa 1988; for a fuller description, see Part III.D.4). In vitro tuberization of excised sprouts is more sensi-

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sensitive to far-red light (see Part IILA.2) if tubers are physiologically older (Blanc et al. 1986). 2. Cuttings. Cuttings taken from whole plants tuberize more readily if the

mother tubers from which the plants are grown are physiologically older (Madec and Perennec 1962; Ben Khedher 1983). 3. Whole plants. Effects on development of whole plants generally

ascribed to physiological age include effects on rapidity of emergence, initial growth rate, number of stems, flowering, time of tuberization, maximum weights of stems and leaves. and time of senescence (Iritani 1968; Van der Zaag and Van Loon 1987; Reust 1990). To this list we would add number of stolons. number of tubers, size of tubers. and susceptibility of tubers to second growth. Not all of these effects apply to comparisons within the whole range of physiological ages. For some. a difference is detected only if the range is very wide; others show differences only within a narrow range of physiological ages; and some effects may be seen more or less across the entire spectrum. We will consider in more detail a few of these effects; in particular those that bear upon induction to tuberize. It is obvious that the planting of mother tubers. which are so physiologically young that they have barely started to sprout. will lead to slow plant emergence compared to somewhat older mother tubers; but there are limits to the advantage of more advanced physiological age in this respect. Extremely old tubers that have started to produce sprouttubers will give slow plant emergence or none. This led Kawakami (1962) to speak of a proper age for good sprouting. For seed tubers between the extremes of just breaking dormancy and formation of sprout-tubers. most of the reason for faster emergence of physiologically older seed can be ascribed to its longer sprouts. Breaking off the sprouts from physiologically old and young seed tubers eliminates the difference in time to plant emergence in some but not all experiments (reviewed by Van der Zaag and Van Loon 1987). Stem numbers increase with physiological age as a consequence of the weakening of dominance by the sprouts in the apical eyes (Krijthe 1962). With more stems we can expect more stolons and more tubers initiated (Iritani 1968; Allen 1978; Haverkort et al. 1990b). This is not the only cause of an increase in tuber number: seed tubers that are very old physiologically produce more tubers per stem as well as more stems per seed tuber compared to very young tubers (Van Loon 1987). The implications of the increased number of tubers initiated will be discussed in Parts VIII.F and IX.A. Stolons form on dark-grown sprouts of physiologically old tubers that

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have not been planted in the soil (Dyson and Digby 1975). Therefore it is not surprising that increasing the physiological age promotes earlier stolon formation, even to the point where stolons form before plant emergence. The implications of this will be discussed in Part VIlLe. Although there are exceptions (Van Loon 1987), it is generally accepted that tubers form earlier on plants from physiologically older tubers (e.g., Wurr 1978). This is consistent with the presence of a stronger tuberization stimulus. Also consistent with the presence of a stronger tuberization stimulus are the earlier maturity and lower final yield associated with physiologically older as compared to younger seed tubers. Plants that are more strongly induced to tuberize tend to form tubers earlier, but they also tend to mature sooner (Part VI.A). This may improve tuber yields at an early harvest, but will reduce yields at later harvests (Part X.), especially for early-maturing cultivars. Van der Zaag and Van Loon (1987) point out that in the case of late-maturing cultivars, the yield advantage may tip toward the physiologically older seed if its more rapid crop emergence outweighs the benefits of the younger seed in delaying senescence. D. Absence of the Mother Tuber An obvious way to estimate the contribution of the mother tuber to growth and development of the progeny is to see what happens when the mother tuber is either detached early, very small, or lacking entirely. Early detachment can be accomplished experimentally, but it is approximated in the field when decay organisms attack and destroy the seed tuber soon after plant emergence. Examples of very small mother tubers are the micro- and minitubers described in Part LB. Plants may be grown in the absence of mother tubers by planting TPS, by setting out in vitro plantlets, or by rooting cuttings. 1. Double induction. Madec and Perennec (1962) used rooted cuttings

and compared the ensuing plant growth with that obtained from propagation via tubers. Under short photoperiods, the reactions of the two types of plants were similar: plants from both cuttings and tubers tuberized strongly, as expected, while the physiological age of the mother tuber had little effect. Under long photoperiods, tuberization on plants from cuttings was delayed or absent, depending upon the critical photoperiod of the cultivar (Part IV.A). By contrast, plants grown from tubers were able to tuberize even under long days, but the physiological age of the mother tuber was influential. These results led Madec and Perennec (1959, 1962) to the hypothesis that there is a double induction to tuberize. That is, both the leaves and the mother tuber may contribute the tuberization stimulus,

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depending upon the photoperiod and other environmental conditions to which the leaves are exposed, and depending upon the physiological age of the mother tuber. As discussed elsewhere in this review, modifying factors, such as soil temperatures and nitrate levels, must also be considered; and it must be remembered that factors inhibitory to tuberization as well as promotive of it may be contributed by either shoots (and probably by roots and stolons) or by mother tubers. For example, physiologicallyyoung mother tubers may supply a factor inhibitory to tuberization (presumably a gibberellin-see Part VI.D.1) that partially overcomes the effects of exposing the shoots to short days, permitting more shoot growth than would occur in the absence of mother tubers. With these caveats in mind, the basic concept outlined by Madec and Perennec (1962) seems valid today: induction to tuberize results from the interplay between factors that may be supplied by the leaves alone, by the mother tuber alone, or by both together. 2. Growth characteristics. Based on the influence of the mother tuber as described previously it follows that growing plants from very small tubers or in the absence of a mother tuber may present problems. If the photoperiod is short in relation to the critical photoperiod for the genotype under the prevailing temperatures and irradiance, small tubers will be initiated before plants have attained an adequate size to support them. Growth will be stunted severely and marketable yields will be greatly reduced or nil. Even if premature tuberization is prevented, initial growth rates are slow compared to those obtained from a 50-g mother tuber, as might be expected in view of the difference in the supply of nutrients available from the propagule. Also contributing to the slow plant establishment is the fact that early root development is retarded even more than shoot development, so that shoot/root ratios are higher compared to those from plants that developed from normal mother tubers (w. J. M. Lommen and P. C. Struik, unpublished results). Much more time is required to achieve full ground cover by the canopy, with all the implications that this may have on soil temperatures and light interception as related to yield and quality (see discussions in P arts VI. A. 4, IX. C. 4 and 5, and X.). Transplants, whether obtained from rooted cuttings, in vitro plantlets, or TPS seedlings, have only one stem per propagule while micro- and minitubers tend to sprout at only one eye. A general rule is that at a given population of propagules per unit area, the fewer the main stems per propagule, the more axillary branches per main stem. For this reason, and perhaps because there is weaker apical dominance associated with their slower shoot growth, plants from transplants, directly seeded TPS,

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or tiny tubers all display a tendency for greatly increased branching at the base of the plant (Fig. 3.13]. This can partially compensate for their lower number of main stearns per unit area in terms of achieving ground cover by the canopy. 3. Remedial measures. We do not attempt a full discussion of steps that

should be taken to improve growth in the absence of normal mother tubers. Research on this question is being pursued actively at several locations. Under most circumstances a key goal is to prevent premature induction to tuberize, and to encourage rapid establishment and growth of the roots and shoots of the young plant. All the factors discussed in Part III should be considered to see whether they can be manipulated-either before or after transplanting-to help achieve this goal.

Figure 3.13. Multiple axillary branching at the base of the stem typical of plants not grown from mother tubers. The plant shown came from a true potato seed in the greenhouse and was transplanted to the field at the Cornell University research farm (courtesy A. B. Rowell).

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VI. PHYSIOLOGICAL NATURE OF INDUCTION

TO TUBERIZE

A. Effects of Induction on Overall Plant Development To understand the physiological nature of induction it is useful to consider how the growth and development of the entire plant change with induction to tuberize. Under Part III we surveyed the environmental factors that contribute to tuber induction, and we describe how each of these affects stolon and tuber development. We turn now to how the shoot and roots are affected. The effects on plant development of induction to tuberize are studied most thoroughly with respect to photoperiod. In the following sections, we describe the effects of photoperiod first, and then compare them to the effects of temperature and other variables that affect induction. 1. Photoperiod extensions with dim incandescent radiation. The long-

term effects of short photoperiods on the growth of the potato plant are well documented. The vast majority of studies, however, compared short photoperiods with long photoperiods that were obtained by extending the photoperiod with dim incandescent light or by extending it with natural light during the early morning or late evening hours. In either case the light would have been relatively high in red and far-red wavelengths. (As indicated in Part IILA, photoperiod extensions with fluorescent lamps produce different results.) Compared to long photoperiods obtained by extending with incandescent lamps or dim natural light, short photoperiods favor tuber growth relative to the growth of all other parts of the plant (Rasumov 1931; Driver and Hawkes 1943; Krug 1960; Madec and Perennec 1962; Hammes and Ne11975; Steward et a1. 1981). There are may other changes: (1) leaflets are larger (Edmundson 1941; Bodlaender 1963); (2) stems are shorter (Schick 1931; Edmundson 1941); (3) flower bud abortion increases (Edmundson 1941; Turner and Ewing 1988); (4) the angle of the leaf to the stem increases (Werner 1934; Wheeler et a1. 1991); (5) the dry weight ratio of leaves to stems increases (Driver and Hawkes 1943); (6) axillary branches at the base of the main stem and sympodial branches are suppressed (Gregory 1956; Demagante and Vander Zaag 1988; E. E. Ewing, unpublished observations); (7) root dry weight decreases (Steward et a1. 1981); (8) senescence is accelerated (Bodlaender 1963; Demagante and Vander Zaag 1988); and (9) stolon growth is replaced by tuber growth (Rasumov 1931). The extent of the above changes in response to photoperiod depends upon the cultivar and upon the other factors that affect induction. Thus if an early-maturing cultivar (capable of tuberizing under long days) is

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changed from 16-h to 12-h photoperiods, the effects on overall morphology will probably be much less striking than if a typical andigena cultivar (requiring 12-h days for tuberization under otherwise favorable conditions) is given the same treatment. Nevertheless, the general tendency is that partitioning of assimilate is shifted toward tubers as photoperiod is shortened (Wolf et al. 1990); and all cultivars seem to respond to photoperiod in this way, given the right set of environmental conditions. The early maturing 'Norchip', for example, showed little difference in plant height or leaf number whether grown under 8-h or 16-h photoperiods when the day/night temperature regime was 27°/12°C; but increasing the temperatures to 32°/22°C produces the expected responses to photoperiod. In the same experiments the later maturing 'Desiree' was sensitive to photoperiod even at the cooler temperatures (Wolf et al. 1990). 2. Photoperiod extension with fluorescent lamps. Lengthening the photoperiod with fluorescent lamps-whether or not at full light intensity-instead of with dim incandescent light gave plants more similar to those produced under short days (Wheeler and Tibbitts 1986). When fluorescent lamps were used the increased stem length was not detected and the decreased partitioning to tubers was much less pronounced (Wheeler and Tibbitts 1986). The effects of these light regimes on overall plant morphology are consistent with their effects on induction to tuberize, which are delineated in Part IILA.1. As explained in that section, fluorescent lamps give a much lower ratio of red to far-red wavelengths than incandescent lamps. Presumably this difference in spectral quality is responsible for the different effects both on overall morphology and on induction to tuberize. 3. Short-term effects of photoperiod. The short-term effects of shortening the photoperiod were somewhat different from the long-term effects just described; leaf expansion was not slowed until well after a strong tuber sink was established (Lorenzen and Ewing 1990). Eighteen days after plants were shifted from 18-h photoperiods of full irradiance to 10-h photoperiods (SD treatment), their leaf area was not significantly different from that of plants that remained under the long days of high irradiance (LD treatment). Both SD and LD plants had much greater leaf areas than plants shifted to a regime where photoperiod was extended with dim light from incandescent lamps (DE treatment). After 18 days tubers had not yet formed in the DE treatment, but they comprised more than 30% of the biomass under SD (Lorenzen and Ewing 1990). Although leaf expansion was not retarded during the first two weeks of SD treatment, other changes were manifested very early (Lorenzen and

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Ewing 1990). Four days after starting treatments, DE plants were taller than LD and SD plants and leaf-bud cuttings taken at this time showed that the SD leaves were already much more strongly induced to tuberize than LD or DE leaves. Tubers on whole plants were detected after 8 cycles of SD (Lorenzen and Ewing 1990). Thus plants under SD were able to maintain rapid shoot growth until well after a strong tuber sink was established. Compared to LD and DE plants, they produced higher dry matter per unit of photosynthetically active radiation to which plants were exposed [Lorenzen and Ewing 1990). A possible reason for their greater efficiency in utilizing photons was that the SD leaves were thinner; on both a fresh and dry basis, they had the highest specific leaf areas [cm 2 g-1) of the three treatments (Lorenzen and Ewing 1990). It might be expected that thinner leaves would show lower rates of photosynthesis per unit leaf area. For example, "shade" leaves are typically thinner, have lower specific leaf areas, and lower carbon exchange rates per unit leaf area compared to "sun" leaves [Lichenthaler 1985). However, the SD leaves had carbon exchange rates equal to LD leaves on an area basis and hence higher rates on a leaf dry weight basis (J. H. Lorenzen and E. E. Ewing [submitted)]. The higher rates of photosynthesis per unit leaf dry weight in SD leaves were accompanied by more leaf starch accumulation (per unit dry weight) during the day, evident two days after treatments started and still present after more than two weeks [Lorenzen and Ewing 1992). Leaf starch levels [per unit dry weight) at the end of the dark period under SD were intermediate between the DE and LD treatments (Lorenzen and Ewing 1992), and export of assimilate [per unit leaf dry weight) during the light was higher under SD than under LD (J. H. Lorenzen and E. E. Ewing (submitted)] . In vitro activity (per unit leaf fresh weight) of sucrose-phosphate synthase, implicated as a major control point for partitioning between sucrose and starch in the source leaf (Huber and Israel 1982), was lowest from the leaves of the SD treatment [J. H. Lorenzen and E. E. Ewing (submitted)]. This may seem surprising in that higher rates of assimilate export might be expected to accompany higher rather than lower activity of sucrose phosphate synthase. However, the effects of SD were parallel to the effects of low levels of nitrogen fertilization in this respect [see Part A.6, as follows). There is evidence from field experiments that the rate of photosynthesis rises after tuber initiation [Moorby 1970; Moll and Henniger 1978; Markowski et al. 1979). The usual explanation is that the strong activity of the tuber sink is responsible. This is probably correct [Dwelle 1990); but it is interesting that in the experiments described above the increase in net photosynthesis per unit leaf dry weight of SD leaves

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preceded tuber initiation [J. H. Lorenzen and E. E. Ewing (submitted)]. This indicates that short photoperiods have a direct effect on increasing accumulation of starch in the leaf (dry weight basis) and daytime export of assimilate from the leaf (dry weight basis), a combination that is possible because the rate of photosynthesis per unit leaf dry weight is stimulated. In summary, it would seem that the production by SD plants of thinner leaves that have the ability to carry out photosynthesis at the same rate per unit leaf area as LD leaves is an effective strategy for making maximal use of the dry matter partitioned to leaves for assimilate production. Implications of maximizing assimilate production are discussed in Section VI.G in relation to the role of assimilate in induction. The general effects on shoot morphology of photoperiod extension with incandescent lamps appears similar whether the treatment is applied early or late in plant growth. Thus 12 days of treatment increased stem height within 12 days whether the treatments were initiated 12,24, 36, or 48 days after planting (Struik et al. 1988b). The increases in stem height were caused by increased elongation of internodes, not by the production of more nodes. 4. Effects of temperature. Generally speaking, the effects on plant

morphology of raising the temperature resemble those of lengthening the photoperiod with dim incandescent light. At high temperatures (for example, night temperatures >20°C, day temperatures >30°C) plants are taller because internodes are longer and because of more sympodial growth, total leaf dry weight increases, the leaves are shorter and more narrow, leaflets are smaller, the angle of the leaf to the stem is more acute, total stem dry weight increases, the leaf/stem ratio decreases, axillary branching at the base of the main stem increases, more flowers are initiated, and flower bud abscision is reduced (Werner 1934; Borah and Milthorpe 1962; Bodlaender 1963; Petri 1963; Ewing 1981; Ben Khedher and Ewing 1985; Menzel 1985; Wheeler et al. 1986; Turner and Ewing 1988; Manrique et al. 1989; Struik et al. 1989a). Senescence may be either accelerated or delayed by increasing the temperature, depending upon the photoperiod and other conditions. Under long photoperiods, high temperatures may so shift the partitioning away from tubers toward shoot growth that plant senescence is delayed (Marinus and Bodlaender 1975; Ben Khedher and Ewing 1985; Struik et al. 1989a); but if photoperiods are short enough to permit reasonable tuberization even at the high temperatures, then the more rapid growth and development at high temperatures is likely to shorten the growing season (Manrique et al. 1989; Vander Zaag et al. 1990). Although a full picture of the effects of temperature on the life-spans of individual

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leaves has not been determined, the optimal temperatures for rate of leaf appearance and duration of individual leaves are relatively low (Burton 1972; Vos and Klemke 1992). The rate of leaf appearance shows a peak at a day temperature of 20°C, irrespective of night temperature (Borah and Milthorpe 1962). Root length was increased by raising the night temperature from 10°C to 20°C (Saha et al. 1974); but high soil temperatures inhibited root growth, especially of sensitive genotypes (Sattelmacher et al. 1990a,b; Sattelmacher and Marschner 1990). Root diameter and root hair formation were stimulated by heat treatment (Sattelmacher et al. 1990a). It is difficult to say whether day or night temperatures are more instrumental in their effects on morphology. Conventional wisdom places the emphasis on night temperatures (Went 1959); but the effect of the night temperature is by no means independent of the day temperature; and the stage of growth also appeared to affect the relative importance of day versus night temperatures (Benoit et al. 1986). Although there is considerable genetic variation in sensitivity to high temperatures (Ben Khedher and Ewing 1985; Reynolds and Ewing 1989a; Manrique et al. 1989), the overall effects appear to apply to all genotypes tested: high temperatures decrease the partitioning of assimilate to tubers and increase partitioning to other parts of the plant just as long photoperiods do (Wolf et al. 1990). Inasmuch as short photoperiods favored leaf starch accumulation during the light period, it is not surprising that cool temperatures had the same effect. Plants grown under 12-h photoperiods had higher leaf starch at the end of the day when grown at 20°C than at 30°C (J. H. Lorenzen and E. E. Ewing, unpublished results). No difference in starch levels was detected at the end of the night. 5. Effects of irradiance level. Consistent with the effects on induction to

tuberize (Part III. C), lowering the light intensity decreases the partitioning of assimilate to the tubers (Bodlaender 1963; Gray and Holmes 1970; Sale 1973a,b; Menzel 1985; P. C. Struik, unpublished results). This was true even when photosynthesis was not limiting (Menzel 1985). In several respects low levels of irradiance affect the morphology of the potato plant similarly to long photoperiods and high temperatures, and the undesirable effects are exacerbated when these factors are present in combination (Bodlaender 1963; Menzel 1985; Demagante and Vander Zaag 1988). In particular, stem elongation is increased and plant senescence is delayed at low light intensity (Demagante and Vander Zaag 1988). The delayed senescence may result in a prolonged duration of the canopy, but this tends to be at least partially offset by delays in canopy development and achievement of ground cover (Struik 1986; Struik et al.

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1990b). Leaves are thinner, but leaf area may be higher, unless the irradiance level is extremely low (Bodlaender 1963). Node number (number on main stems plus branches) was increased at low irradiance, and the dry weight ratio of leaves/stems was decreased (Menzel 1985). One effect of lowering irradiance that differed from the effects of long photoperiods and high temperatures was on flowering; in contrast to long photoperiods and high temperatures, shading the plants increased flower bud abortion (Bodlaender 1963; Demagante and Vander Zaag 1988; Turner and Ewing 1988). 6. Effects of N nutrition. Just as increasing the N fertility tends to

decrease the induction to tuberize (Part III.D), so the effects of high Non the development of the rest of the plant are consistent with the effects of the other factors that decrease the induction to tuberize. Increasing N gives taller plants, more sympodial nodes, longer internodes, higher leaf dry weights, higher stem dry weights, and lower leaf/stem ratios. Increasing N also gives higher root dry weights (at least over the normal range of N application), higher shoot/root ratios, and delayed senescence [Humphries and French 1963; Gunasena and Harris 1968; Clutterbuck and Simpson 1978; Dyson and Watson 1971; Santeliz-Arrieche 1981; Millard and MacKerron 1986; Oparka et al. 1987; Payton 1989; Biemond and Vos 1992; Vos and Biemond 1992]. The increased partitioning of dry matter to shoots rather than to tubers produces a great increase in shoot biomass (Fig. 3.14). although the response of stems to increased N is stronger than that of leaves, the leaf area for plants that receive high N fertilization is very high compared to C ro

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Figure 3.14. Effects of N fertilization on development of shoot biomass in plants grown in the greenhouse. N1 = 2.5 g/plant; N2 = 8.0 g/plant; and N3 = 16.0 g/plant. The vertical bar indicates plus or minus the standard error of the mean (S.E.); absence of a bar indicates that the S.E. was less than the width of the symbol marking the data point (from Biemond and Vos 1992).

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those that receive none. The resultant prolongation of shoot growth and the increased duration of a canopy for light interception usually produces a much higher final yield of tubers than in plots that receive no N fertilizer, although the unfertilized plants have a much higher harvest index (Fig. 3.15). This assumes that the season is long enough (with sufficient levels of irradiance at the end of the season) to take advantage of the extra canopy duration. Similar to the short-term effects of short photoperiods (see Part VI.A.3), the withholding of N fertilizer from potatoes is associated with higher starch levels in the leaf, increased percentage export of assimilate from the leaves, and decreased activity of sucrose phosphate sYnthase (Oparka et a1. 1987). 7. Genetic differences. Genotypes that are less induced to tuberize under a given set of environmental conditions display the expected differences in morphology when compared to strongly induced genotypes (Driver and Hawkes 1943). Of course there are exceptions because of other unrelated genes that modify the responses, but observations within ssp. andigena and andigena X tuberosum populations segregating for adaptation to long days have shown clear and consistent associations of morphology with tuberization response (Glendinning 1975; Lazin 1980; Rasco et a1. 1980; E. E. Ewing, unpublished observations). The genotypes that were poorly induced (as evidenced, for example, by poor tuberization on cuttings taken from the plants) were taller, more highly branched, had smaller leaflets, more acute angles between leaves and stems, higher total leaf

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Figure 3.15. Effects of N fertilization on harvest index (the proportion of total dry matter in tubers) with time after emergence. Nt = 2.5 g/plant; N2 = 8.0 g/plant; and N3 = 16.0 glplant. The vertical bar indicates plus or minus the standard error of the mean (S.E.); absence of a bar indicates that the S.E. was less than the width of the symbol marking the data point (from Biemond and Vas 1992).

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dry weights, higher stem dry weights, lower dry-weight ratios between leaves and stems, more flowering, more abundant rooting, and later senescence. Genetic differences followed the expected pattern with respect to accumulation of leaf starch during the light period: the early maturing 'Norchip' exceeded the late maturing 'LT-1' in this respect (Lorenzen and Ewing 1992). 8. Physiological age of the mother tuber. The general effects of the

status of the mother tuber on plant development are discussed in Part V.C.3. In Part V we also discussed evidence that the stimulus for tuberization present in mother tubers increases with physiological age. Does physiological age of the mother tuber affect overall plant development in a manner parallel to photoperiod and other determinants of induction to tuberize? Effects of physiological age of the mother tuber on stem number complicate the comparisons. However, the smaller plant size, earlier tuberization, and earlier senescence that are often observed when physiologically old mother tubers are compared to young ones are consistent with the contrasts observed when short-day plants are compared to long-day plants (VI.A.1). 9. Relation to assimilate deprivation. Many of the morphological

changes brought about by induction to tuberize appear to occur even if tuberization is artificially prevented. For example, to decrease flower bud abscission breeders sometimes resort to stolon pruning, grafting to tomato stocks, or other techniques to prevent tuberization. This may be of some benefit in preventing flower bud abscission under inducing conditions, but certainly it is not totally successful (Abdel-Wahab and Miller 1963; Weinheimer and Woodbury 1966; Sadik 1984). Likewise, under strong induction to tuberize it is difficult to obtain roots on cuttings, even if the buried bud is excised to prevent tuber formation (E. E. Ewing unpublished observations). Finally, shoots of plants grown at cool air temperatures under short days have a similar appearance (except for the presence of small aerial tubers) whether or not the soil is heated to prevent normal tuberization (Reynolds and Ewing 1989b). These observations suggest that growth retardation in the rest of the plant over the long-term is not caused solely by deprivation of assimilate, Le., by diversion of the assimilate to the strong tuber sink. However, the interpretation is complicated by the frequent production of alternate sinks such as aerial tubers (Dwelle 1990) or swollen stems and petioles when normal tuberization is blocked (see also Part VI. C); possibly assimilate is diverted to one of these tuber-like structures when normal tuber formation is artificially prevented. Removal of tubers from rapidly growing plants led to accumulation of dry matter in leaves and lowered

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the rates of net assimilation (Burt 1964a; Nosberger and Humphries 1965), a finding consistent with the widely accepted hypothesis of sinklimited photosynthesis. The observation that leaves continue to grow at a rapid rate well after the plant is moved to short days and strong tuber sinks have been formed (Part VLA.3) underscores the complexity of the changes that are initiated when a plant is shifted to strong inducing conditions. 10. Summary. The plant that has been grown under conditions that favor tuber induction (Le., an induced plant) has a very different appearance from a noninduced plant. The induced plant is shorter, both because of shorter internodes and because of fewer sympodial nodes. It has fewer axillary branches at the base of the plant, larger leaves, a higher dry weight ratio of leaves/stems, and flatter leaf angles to the stem. Flowering is reduced, and so is rooting. Within a few days after transfer to inducing conditions it is possible to detect physiological and biological changes in the leaves. Compared to leaves of noninduced plants, the leaves of induced plants become more efficient per unit of leaf dry weight in several attributes: photosynthesis, starch accumulation during the light period, and export of assimilate.

B. Insights from Grafting Grafts of potato to potato have demonstrated that the effects of induction can be realized across graft unions. Scions from induced plants of 'Kennebec' caused tuberization when grafted to cuttings from noninduced 'Kennebec' plants; noninduced scions produced no tubers on the induced stocks (Gregory 1956). Similar results were obtained with grafts of andigena cuttings: only scions taken from plants that had received short days produced tubers on stocks (Kumar and Wareing 1973). A related experiment addressed the transport of the stimulus: four-leaf cuttings were intergrafted between stem segments that had been exposed to long days, and cuttings were later removed from the apices. Tubers formed on these apical cuttings only if the intergraft was taken from a plant that had received short days, leading to the conclusion that the stimulus moved from the intergraft acropetally as well as basipetally (Kumar and Wareing 1973). Grafts of cuttings were used to find out whether leaves of a clone with a long critical photoperiod were able to produce the stimulus for tuberization even under long days, or whether the receptor buds of such a clone were capable of tuberizing even though the leaves were producing little or no stimulus (Ewing and Wareing 1978). If the leaf of a 2-node, grafted

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cutting was taken from a clone with a long critical photoperiod, tuberization occurred even though the buried bud was from a clone with a short critical photoperiod, and though plants were exposed to long days before and after cutting. The reciprocal crosses did not tuberize, showing that characteristics of the leaf rather than the buried bud conferred the ability to tuberize under long days (Ewing and Wareing 1978). Interspecies grafts have also been performed to study potato tuberization. Intergrafts showed that transport of the tuberization stimulus was possible through a leafless segment of tomato or eggplant, but the leaves of these species diminished tuberization whether or not such leaves were exposed to short photoperiods (Madec and Perennec 1959; Okazawa and Chapman 1963). On the other hand, a tomato leaf or a noninduced potato leaf promoted tuberization when grafted to a leafless cutting taken from an andigena plant that had received 20 short days before excision (Wareing and Jennings 1980). In the absence of a leaf, the bud of the cutting formed an orthotropic shoot unless abscisic acid was added. Abscisic acid would substitute for the requirement of a leaf in producing a tuber. It is important to note two points. The first is that neither the grafting of a noninduced leaf nor the addition of abscisic acid would cause a tuber to form if the leafless cutting was taken from a plant that had received only long days. The second point is that if the cutting was taken from a plant that had received many weeks of short days, a tuber would form even in the absence of a leaf or of abscisic acid. Thus the buds on the cuttings used in the experiments appear to have been on the verge of tuberization; the abscisic acid or other substance(s) supplied by the noninduced leaf "tipped the scale" toward tuberization. Nitsch (1965, 1966) found that when a sunflower (Helianthus annuus) requiring short days for flowering was grafted to a leafless stock of Jerusalem artichoke (H. tuberosus), tuberization on the latter occurred only if the sunflower scion was exposed to short days. Some fifteen years later, Nitsch's work inspired independent studies in the Soviet Union and France involving tobacco scions grafted to leafless potato stocks (Chailakyan et a1. 1981; Martin et a1. 1982). Both studies reported similar findings. When the scion was the usual "Mammoth" type of tobacco requiring short days to flower, tuberization occurred on the potato stocks only if the scion was exposed to short days. Still more remarkable was the effect of utilizing Nicotiana sylvestris, which requires long days for flowering, instead of the Mammoth as the scion. Now tubers formed on the stock only if the scion was exposed to long days! The number of plants in these experiments was not large, and although similar results were obtained in both studies, it would be well to have further confirmation. The data indicate that the stimulus for flowering in tobacco was graft transmissible to the potato, where it induced tuberization, and that this

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was true even if the stimulus was produced by a tobacco species that requires long photoperiods rather than short ones for flowering.

c. Dynamic Aspects Under tuber-inducing conditions, the tuberization stimulus moves throughout the plant and affects overall morphological development; but the formation of stolons and tubers takes place preferentially in underground (or otherwise darkened) parts of the plant. The pattern of such formation will be discussed in more detail in Part VIII of this chapter. There is no required "juvenile period" through which plants must pass before tubers can form, and there is no minimum age or size requirement for tuberization. For example, when TPS of very early maturing genotypes is planted under short, cool days, then it is not unusual to find tubers on seedlings that have opened only one or two leaves beyond the cotyledons. All other factors being equal, however, the larger the plant the more likely it is to tuberize. The induction to tuberize is influenced by the status of the mother tuber as this interacts with the tuberization stimulus produced in the leaves. Under inducing conditions both young and old leaves are capable of producing the stimulus (Hammes and Beyers 1973); so the greater the leaf area, the more stimulus is available for transport underground (Kahn et al. 1983). The presence of the tuberization stimulus in leaves or tubers does not necessarily lead to tuber initiation. Causes for such failure include interference with translocation from the leaves to the underground parts of the plant, as when stems are girdled by Rhizoctonia disease; excision of underground buds, or use of special techniques to separate stolons from roots and keep stolons above the soil; and the presence of unfavorable conditions at the potential sites of tuber initiation. An example of this last situation is the plant grown under cool air temperatures but in heated soil (Part III.B). As in the case of the heated soil, if expression of tuberization is completely blocked and the induction is sufficiently intense, then axillary buds on aerial portions of the plant may tuberize in spite of the inhibiting effects of light (Fig. 3.10). Although this may be prevented by excision of the axillary buds, the stems or leaf petioles may still become swollen. This is especially striking when a petiole of a leaf cutting with the axillary bud removed is taken from a strongly induced plant and inserted into soil. The base of the petiole will swell like a tuber (Knight 1816; Kupfer 1907; Isbell 1931; Ewing and Wareing 1978). The swollen tissue also resembles tubers under microscopic examination: it is packed with starch, and there is evidence of extensive cell division (E. E. Ewing, unpublished results). When the swollen petiole bases were stored in moist vermiculite to

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prevent dehydration, they eventually developed protuberances thatexcept for the absence of nodes-resembled the sprout-tubers seen after long storage of ordinary tubers (compare Fig. 3.16 with the drawing of sprout-tubers in Fig. 3.12). One might speculate that although the photoperiodic signal is perceived in the leaves, it is then transmitted to the roots, and that part or all of the tuberization stimulus is actually produced in the roots. In this connection it may be noted that it is possible to grow apical shoots of the potato for many months in the absence of visible roots by inserting the cut stem into nutrient solution and regularly excising the base of the cutting before adventitious roots can form (Wang and Wareing 1979). Every few days the portion of the stem that was in contact with the solution is cut away, and a new portion of the stem is immersed in the solution. Single node cuttings taken from apical shoots that were grown by this technique formed tubers if the shoots were exposed to short photoperiods, but not if they had been exposed to long photoperiods after the stem excisions were started (Ewing 1985). This does not rule out the possibility that

Figure 3.16. Swollen petiole base with protuberances developed during storage. Leaves were excised from a plant that was highly induced to tuberize. and the base of the petiole was inserted into a rooting medium in the mist bench. In the absence of an axillary bud. the base of each petiole developed a starchy swelling that resembled a tuber. These swollen bases were covered with moist vermiculite and placed in cool storage. After several months there were protuberances at the cut surface {courtesy of J. J. McGrady}.

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the roots contribute to the tuberization stimulus, but it does indicate that the stimulus can be formed even in the absence of visible roots.

D. Role of Known Hormones There are a number of in-depth reviews on the effects of known hormones on tuberization (Wareing and Jennings 1980; Wareing 1982; Melis and Van Staden 1984; Krauss 1985; Stallkneckt 1985; Ewing 1985, 1987; Vreugdenhil and Struik 1989). We will limit our discussion to a summary of the main points and will cite only a few representative references. 1. Gibberellins. The most clear-cut evidence concerning hormonal con-

trol is that gibberellins interfere with tuberization. Inhibition of tuberization occurs as a result of exogenous application of gibberellic acid in systems utilizing whole plants (Okazawa 1960), cuttings (Tizio 1971), in vitro plantlets (Hussey and Stacey 1984), and excised sprouts cultured in vitro (Koda and Okazawa 1983a). Environmental conditions that lower induction to tuberize (long days, high temperatures, low irradiance, high N fertilization) are associated with higher levels· of gibberellin activity (Woolley and Wareing 1972c; Railton and Wareing 1973; Krauss and Marschner 1982; Menzel 1983b), and gibberellin activity of slightly swollen stolons is less than in stolons with no swelling (Koda and Okazawa 1983b). A mutation that appears to block gibberellin synthesis is associated with increased tuberization in ssp. andigena (Bamberg and Hanneman 1991). Evidence from selected ion monitoring shows that potato sprouts contain gibberellinzo and gibberellin! (Jones et al. 1988). Is induction to tuberize merely a condition brought about by a lowering of gibberellins in the plant? The fact that the stimulus is transmissible through grafts (Part VI. B) makes this seem unlikely, but there is every reason to think that gibberellins play an important negative role in tuberization, shifting growth away from tubers toward stolons. This does not necessarily mean that under all circumstances the higher the gibberellin level in the plant, the more stolons will be produced. Especially in ssp. andigena-which even in normal environments evidently tends to have high levels of gibberellin compared to ssp. tuberosum-it may be that gibberellins can be too high not only for tuberization, but also for good stolon production under combinations of long photoperiods, high temperatures, and low irradiance. Table 3.2 shows an example of this occurring in ssp. tuberosum. We discussed in Part III.A.3 how a combination of short photoperiods and cool temperatures increased stolon formation in the experiments summarized in Table 3.2. Application of gibberellic acid to the cuttings in these experiments decreased the number

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of stolons under tuber-inducing conditions (Table 3.2). There was some indication that inhibitors of gibberellin synthesis favored stolonization under noninducing conditions, although the differences were not significant. 2. Inhibitors. As might be expected from the effects of gibberellins, treat-

ment with chemicals that block the synthesis of endogenous gibberellins are reported to promote tuberization in whole plants (Gunasena and Harris 1969; Hammes and Ne11975; Menzel 1980), cuttings (see Table 3.2), in vitro plantlets (Hussey and Stacey 1984; Estrada et al. 1986; Dodds 1990), and sprouts cultured in vitro (Tizio 1969). It is logical to hypothesize that the tuberization stimulus is a naturally occurring inhibitor or antagonist of gibberellin, and there has been much interest in finding such a compound. Abscisic acid is a candidate (Krauss and Marschner 1982), but on the whole the evidence is not convincing that abscisic acid promotes tuberization except in special circumstances (Wareing and Jennings 1980). Coumarin (Stallknecht and Farnsworth 1982) and phenolic acids (Paupardin and Tizio 1970) promoted tuberization in vitro, but again the case is not compelling that they playa significant role in the whole plant. 3. Compounds related to jasmonic acid. The most recent prospect for a natural inhibitor that counteracts the effects of gibberellin is a compound related to jasmonic acid (Van den Berg and Ewing 1991). The chemical structure of the active compound was identified as 3-oxo-2-(5'/3-D-glucopyranosyloxy-2'-z-pentenyl)-cyclopentane-1-acetic acid, and its aglycone (which can be simplified to 12-0H-jasmonic acid) was named tuberonic acid (Yoshihara et al. 1989). The emphasis was first on the glucoside as the tuber-inducing substance (Koda et al. 1988). Since then it was reported from the same laboratory that jasmonic acid, its methyl ester, tuberonic acid, and the glucoside of tuberonic acid all showed similar promotion of tuberization in the bioassays utilized (Koda et al. 1991). The closely related cucurbic acid, which differs from jasmonic acid only in having a hydroxy group instead of an oxygen at C-3, and the methyl ester of cucurbic acid were active but required higher concentrations to obtain the same rate of tuberization in the bioassay (Koda et al. 1991). Tests with other closely related compounds led to the conclusion that the partial structures necessary for tuber-indueing activity included a carboxyl group or its ester at the C-1 position, a double bond (pentenyl group) in the substituent at C-2, and an oxygen at C-3. It was pointed out that the double bond in the substituent at C-2 is not essential for growth inhibition of rice seedlings or for promoting senescence in oat leaves,

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which indicates a different mechanism of action for tuberization than for the other two processes (Koda et al. 1991). In view of the reactions of underground buds of cuttings to increasing numbers of short days (Fig. 3.8), it is interesting that in addition to their effects on tuberization, jasmonic acid and its relatives changed the growth of lateral shoots from negatively gravitropic to diagravitropic (Koda et al. 1991). Helder et al. (1991b) found more 11-0H-jasmonic acid than 12-0Hjasmonic acid in S. dernissurn leaves from short-day plants and did not find glucosides of either compound. Leaves from long-day plants in the same experiments did not contain either the two aglycones or their glucosides. All of these compounds are too recently discovered for a full assessment of their significance in controlling tuberization, but what has been published to date looks promising. The glucoside of 12-0H-jasmonic acid stimulated tuberization in vitro when concentrations as low as 3 X 10-8 M were added to the agar (Koda et al. 1988), whereas both abscisic acid and zeatin riboside were ineffective (Koda and Okazawa 1988). The new compounds are under active investigation in a number of laboratories, and more information on their role in tuberization should be available soon. 4. Cytokinins. Cytokinins are necessary for cell division, which is an

early event in tuber initiation (see Part VILA); so it is not surprising that the addition of cytokinin frequently promotes tuberization in vitro (Palmer and Smith 1969). Transfer of plants to cooler temperatures and shorter photoperiods has been associated with a temporary increase in cytokinin content of leaves (Langille and Forsline 1974), but cytokinin activity in stolon tips shows little increase until tubers are more than twice the size of attached stolons (Koda and Okazawa 1983b). The major cytokinin in potato leaves was identified as cis-zeatin riboside (Mauk and Langille 1978), but this seems questionable: although cis-zeatin riboside is a component of plant tRNA, it is trans-zeatin riboside that is considered to have biological activity in higher plants (McGaw 1987). For more discussion of the evidence as to whether zeatin riboside (or other cytokinin) is the tuberization stimulus, see the reviews by Ewing (1985, 1987, 1990) and Vreugdenhil and Struik (1989). In brief, there is much to indicate that cytokinins are involved; but not all the pieces fit. It is often suggested that a high ratio of cytokinin to gibberellin constitutes the tuberization stimulus (Melis and Van Staden 1984). The suggestion is attractive, but it would not be surprising if other compounds also playa role. 5. Ethylene. There is evidence that at least under some circumstances

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ethylene favors tuberization in dahlia (Biran et al. 1972) and radish (Vreugdenhil et al. 1984). It does not appear that ethylene plays a similar role in potato tuberization. The application of ethephon, which produces ethylene, to extremely old seed tubers causes a restoration of more normal sprout growth instead of sprout-tubers forming directly at the eye (Dimalla and Van Staden 1977). Gibberellin activity is higher in the elongated sprouts than in the sprout-tubers. Apparently the ethylene stimulates gibberellin levels, which have their usual effect of inhibiting tuberization. Applied ethylene inhibited potato tuberization in vitro in several studies, and addition of an ethylene antagonist promoted such tuberization (Vreugdenhil and Struik 1990). Although there is little evidence to support a positive effect of ethylene on potato tuberization per se, it is suggested that ethylene produced by friction between soil particles and the growing stolon tip might stop extension growth of the stolon, thereby exerting an indirect effect on tuberization (Vreugdenhil and Van Dijk 1989; Vreugdenhil and Struik 1989, 1990). This could help explain the effects of soil resistance on stolon growth and development (described under Part VIII.D).

6. Auxin. Auxin has been studied less than the other known hormones with respect to its role in tuberization (Melis and Van Staden 1984), but the data that were reported did not indicate much positive evidence for its involvement. Together with gibberellins and cytokinins, it is probable that auxin helps to control stolon orientation and growth (although the recently reported effects of jasmonic acid and its relatives in this respect should also be considered-see Part VI.D.3). More direct effects of auxin on tuberization are possible, especially in combination with other hormones; but based upon existing information this does not seem likely. E. Plant Growth Regulators Exogenous applications of a great many different types of compounds have been made to whole plants or to in vitro cultures of potatoes. For the most part, the results have been unimpressive. As already mentioned, compounds that block gibberellins sometimes improve tuberization under environments unfavorable to tuber induction, but even this is inconsistent. The gibberellin inhibitors are more useful in obtaining tuberization on plantlets in vitro (Hussey and Stacey 1984), and many reports indicate that cytokinins improve tuberization of in vitro plantlets. Application of cytokinins and a wide range of other compounds to whole plants has often produced increases in numbers of tubers set, but because the average tuber size was less, this has not usually improved

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tuber yields. Moreo ver, the degree to which tuber set increas es tends to vary from one experim ent to anothe r. In assessing such data it should be borne in mind that under conditi ons that favor tuberiz ation, a slight phytot oxicity to the leaves is often accom panied by a heavie r set of smalle r tubers whethe r herbici des or growth regulat ors are applied . This is far differe nt from obtaini ng tubers by applica tion of a chemic al to an otherw ise nonind uced plant, such as a typical ssp. andigena grown under long days. Furthe rmore, the phytot oxicity genera lly causes smalle r leaflets , the opposi te effect from that observ ed when plants becom e induce d to tuberize.

F. Calcium and Calmodulin A combin ation of chelati ng agent plus calcium ionoph ore blocke d tuberiz ation on induce d cutting s (Balamani et aI. 1986). Subseq uent treatment with CaCl2 reversed the inhibition, and antago nists of calmod ulin also preven ted tuberization. Thus it appear s that Ca++ and calmod ulin must be presen t for tuberiz ation to occur, althoug h their role in tuber initiation is unknow n.

G. Role of Assimilate Level in Induction At one time the level of nonstr uctura l carboh ydrate in the leaf was believed to be the controlling factor in inducti on of tuberiz ation. Accord ing to this hypoth esis, short photop eriods and cool temper atures slow down leaf growth causin g the accum ulation of assimil ate and a high C:N ratio, which in turn brings about tuberiz ation (Garner and Allard 1923; Wellensiek 1929; Werne r 1934; Driver and Hawke s 1943; Borah and Miltho rpe 1962; Burt 1964b). High N fertilization tends to negate the effects of the short days by decrea sing the C:N ratio. The hypoth esis fell from favor as eviden ce mounte d for the hormo nal control of tuberiz ation. It could be argued, howev er, that there is still reason to think that high assimi late level is a contrib uting factor in induction along with hormo nal effects . One bit of evidence is that in vitro tuberiz ation is highly depend ent on sucros e level (Gregory 1956; and many subseq uent studies). Increas ing the sucros e concen tration to at least 175 mM greatly increas es the freque ncy and size of tubers; and this is not simply an osmotic effect (Rocha-Sosa et aI. 1989; Wenzler et aI. 1989; Perl et aI. 1991). Anothe r reason to suspec t that high assimil ate level is involved in inducti on to tuberize will be describ ed in Part VILB: several genes that seem to be intima tely associa ted with tuberiz ation are "turned on" by high sucros e concen tration s. As noted (Part VI.A.3), howev er, leaf growth does not slow down until

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after the increased leaf starch accumulation in response to short photoperiods has already occurred (Lorenzen and Ewing 1990; 1992J. Therefore the explanation advanced by Driver and Hawkes (1943J and others (Le., that the retardation of growth caused the accumulation of starchJ is incorrect. Nevertheless, it is possible that starch accumulation in the leaves plays a significant role in tuber induction. Transfer to short photoperiods increases daytime accumulation of leaf starch in many species (e.g., Chatterton and Sylvius 1980), including those classified as long day, short day, and day neutral in flowering response. This occurs in the potato leaf as well, and it continues to happen even after a strong tuber sink is making heavy demands for assimilate (Lorenzen and Ewing 1992). In the potato it seems that the extra assimilate available for export during the night may provide part of the stimulus for tuberization. Certainly there is potential adaptive value in tuberizing plants if increased leaf starch is available for transport to the tubers. Therefore it is logical that high levels of assimilate exported from the leaf could help promote tuber initiation.

VII. CHANGES IN THE STOLON TIP OR BUD ASSOCIATED WITH TUBERIZATION A. Anatomical Changes

Anatomical changes in the stolon during tuber initiation have been thoroughly reviewed by Cutter (1992J. Additional information on in vitro tubers has also been published (Peterson et al. 1985). It is well known that increases in cell division, cell enlargement, and starch deposition all occur before visible swelling of the stolon tip (Plaisted 1957); but there is conflicting evidence in the early literature as to whether increased cell division precedes cell enlargement (Artschwager 1924; Plaisted 1957; Reeve et al. 1969) or the reverse is true (Booth 1963). A problem in approaching this question has been the impossibility of predicting whether a particular stolon tip on an intact plant will tuberize until swelling has already been observed-by which time the earliest events in the sequence will already have taken place. A labeling experiment of field plants with 14C02 underscores the problem. Twenty hours after a 2-h labeling of shoots, some stolon tips that showed no visible signs of swelling had much higher specific activities (cpm g-1 fresh weight) of 14C than did other stolon tips borne on the same stem whether or not swelling was visible (Oparka and Davies 1985a). The higher specific activities were observed in both the ethanol-soluble and ethanolinsoluble fractions; and many small stolon tips had higher ratios of the

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latter to the former. The results were interpreted as an indication that before any external sign of tuber initiation, certain stolon tips had undergone changes that produced increased accumulation of soluble carbon compounds, and increased conversion of these to insoluble compounds. No pattern was detected that would explain the differences in activity or predict which stolon tips had undergone such changes (Oparka and Davies 1985a). Thus on the intact plant there is no nondestructive way to identify the earliest stage of interest. To circumvent this uncertainty buried buds of leaf-bud cuttings from strongly induced plants were examined at daily intervals after cutting (Duncan and Ewing 1984). On such cuttings it is virtually certain that tuberization will begin immediately and that the site will be the bud. Anatomical sections through the buds (Fig. 3.17) showed that by onetlay after cutting there was a statistically significant increase in the content of starch and in the percentage of nuclei that had entered mitosis. Starch deposition increased even more dramatically over the next several days. The mitotic index increased again the second day after cutting, but cell enlargement could not be detected until the fourth day (Duncan and Ewing 1984). The conclusion was that increases in cell division and starch deposition preceded increases in cell enlargement as early events in tuberization. Observations of tubers produced in the conventional manner on stolons of intact plants led to the conclusion that radial cell expansion

Figure 3.17. Longitudinal sections through axillary buds of leaf-bud cuttings given short photoperiods for tuber induction (206 x). The apex is to the left of each section. A. Bud at time of cutting. B. Two days after cutting. There is still no significant increase in cell size. but numerous cell files indicate that a high rate of division has occurred. C. Four days after cutting. Cells are much larger and contain large vacuoles (from Duncan and Ewing 1984).

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produced swelling of the stolon before a detectable increase in cell division had occurred (Koda and Okazawa 1983b), the reverse of the order seen in cuttings. The method used to detect whether increases in cell division or radial cell enlargement occurred first was not by observation of mitotic figures over time in the zone where tuberization was expected, but by counting the number of cells across the diameter of slightly swollen stolons. The diameter of the swollen zone and the number of cells across this diameter were compared with the equivalent measurements of the stolon about 5 mm basipetal to the swelling. From these data we have calculated the mean cell diameter in each zone. At the earliest stage of swelling the diameter was 157% of the stolon diameter basipetal to the swelling. The number of cells across the swollen zone was 118% of the number basipetal to the swelling, and the mean cell width was 133% of the cell width basipetal to the swelling. Although the difference in the changes for the two processes was not large, the authors conclude that cell enlargement rather than cell division was responsible for this stage of swelling. At the next observed stage of swelling the diameterwas 295% of the diameter basipetal to the swelling. At this stage the number of cells across the diameter of the swelling was 236% of the number basipetal to the swelling, and their width was 125% of the cell width basipetal to the swelling. The authors concluded that cell division became the important factor to explain the later increase in diameter. In comparing the results of the studies by Duncan and Ewing (1984) and Koda and Okazawa (1983b) it should be noted that the timing of the observations in relation to detectable swelling was different. In the former study anatomical examinations were made three days before swelling was visible. By the time swelling was visible on buried buds of cuttings, increases in cell enlargementhad replaced increases in cell division as the dominant change. Because of the unpredictability of tuberization in whole plants, the latter study made comparisons only after visible swelling had occurred. This was too late to tell whether there was a switch from cell division to cell enlargement in whole plants like the switch observed in cuttings. The sequence of events may well be different in cuttings than in whole plants, but the difference in the timing of observations also may account for the different conclusions as to whether increases in cell division or cell enlargement occur first during tuber initiation. Data on starch accumulation during tuberization in vitro should also be mentioned. Sprouts were cultured in vitro in the absence or presence of gibberellic acid, and tips were assayed for starch (Iriuda et a1. 1983). In the presence of the hormone there was no tuberization, and starch granules were 2-5 pm in diameter; tubers formed in the absence of the hormone, and the diameter of starch granules was 10-25 pm.

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B. Biochemical Changes Whatever the exact order of their occurrence, by the time the stolon tip or other buried bud is visibly swollen there have already been dramatic increases in mitosis or cell enlargement (or both) and in starch deposition. There are other biochemical changes: endogenous activity of gibberellin-like compounds in the stolon tips decrease (Koda and Okazawa 1983b) as the stolon tips begin to develop into tubers, and a glycoprotein, patatin, increases significantly in buried buds of induced cuttings within two days after cutting (Paiva et al. 1983). Biochemical changes associated with tuberization are attracting a great deal of attention from molecular biologists, and there promises to be something of an information explosion on the topic. Recent reviews have been written by Park et al. (1985); Park (1990); Prat et al. (1990); Sanchez-Serrano et al. (1990); and Willmitzer et al. (1990). 1. Patatin accumulation. Patatin is the trivial name for a family of glycoproteins that contain 5% neutral sugar (mannose) and 1% (glucosamine + galactosamine) (Racusen and Foote 1980). The genes coding for these proteins have been divided into two classes (Park 1990). Class-I patatin genes are expressed mainly in tubers; class-II patatin genes are expressed at low levels in tubers and are also expressed in roots (Park 1990). Patatin is always observed in tubers; Racusen and Foote (1980) found it in the tubers of all 31 cultivars examined. Normally patatin is located in the vacuole (Sonnewald et al. 1990). It consists of dimers with a molecular weight of 88 ± 4 kD (Racusen and Weller 1984). There are many isotypes and every cultivar has a typical set of isotypes (Racusen and Foote 1980; Park et al. 1983). The patatin content as percentage of the total soluble protein content has been reported as low as 16% in Desiree (Racusen 1986) to as high as 45% in Superior (Paiva et al. 1983). Racusen (1983) concluded that this percentage remains constant during growth and storage of the tubers until sprouting; but Hannapel (1991a) found considerable increases, especially in some cultivars, between 70 and 120 days after planting: 'Superior' increased from 15% to 30% during this period. The specificity of expression of class-I patatin genes in tubers make them and their promoters interesting to molecular biologists (Park 1990; Willmitzer et al. 1990). Chimeric genes containing a reporter gene (e.g., the gene for fJ-glucuronidase) and a class-I patatin promoter can be induced to expression in transgenic plants by those factors that normally induce patatin expression. In transgenic potato plants, such expression for the most part has been restricted to tubers and to stolons associated

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with growing tubers. Exceptions were occasional trace amounts in other tissues and special cases described as follows (Park 1990; Willmitzer et a1. 1990).

2. Patatin in cuttings. The leaf-bud cuttings described in Part ILC have been utilized for many studies of patatin. Wheeler et a1. (1988) used the patatin accumulation in such cuttings as a tool to assess tuber induction in plants and predict harvest index. The visual rating of the development of the axillary bud was correlated with the patatin accumulation in the petiole. The protein composition of axillary bud tubers was similar to the protein composition of below-ground tubers grown on stolons (Paiva et a1. 1982). Patatin was present in underground stolons from whole plants, especially those close to a growing tuber; but the content in the stolon decreased when the tubers became larger or when their growth slowed. Whereas patatin does not normally accumulate in significant amounts in potato petioles or stems, it is interesting that such accumulation does occur (Paiva et a1. 1982, 1983) under the special conditions described in Part VI. C when petioles of highly induced leaves with excised buds are inserted into soil. It is not surprising to find that patatin accumulates along with starch in the swollen base of such a petiole (Paiva et a1. 1983), but there is more to the story. The swelling of the petiole base occurs only if the plants are strongly induced to tuberize before bud excision (Ewing and Wareing 1978); yet even leaf cuttings from plants that are not induced to tuberize and do' not show signs of cell proliferation at the base of the buried petiole accumulate both starch and patatin (Park 1990). This leads to the suggestion that the process of tuberization can be divided into at least two separate processes: (1) morphogenesis and cell proliferation and (2) starch and protein deposition (Park 1990). The same technique has been used to separate starch deposition from patatin synthesis within the genus Solanum. In the experiments described in Part IV.D, S. brevidens and S. etuberosum formed neither tubers nor stolons on cuttings (Table 3.6). Nevertheless, in both species there was accumulation of starch at the base of petioles from which the bud had been excised prior to insertion of the petiole base into the soil (HannapeI1990). In spite of their starch accumulation, patatin did not accumulate in the S. brevidens or S. etuberosum petiole bases. These species contained DNA sequences related to a patatin probe from S. tuberosum according to Southern blot analysis (Hannapel 1990); but patatin genes were not expressed in petiole bases or in leaf explants cultured on media containing 300 mM sucrose (see the following paragraphs for significance of this point). By contrast, patatin genes were expressed in petioles and leaf explants from genotypes containing some tuberizing genome. The expression of

156

E. E. EWING AND P. C. STRUIK

the patatin gene in the bases of petioles was about the same for the triploid (which formed stolons but not tubers-see Table 3.6), the pentaploid (which formed tubers), and the tuberosum parent-Le., it was unrelated to the dose of tuberizing genomes. Still other experiments with petioles of cuttings from which the buds were excised have tested the effects of gibberellic acid. The hormone reduced the accumulation of starch and even more the accumulation of patatin (Hannapel et al. 1985). 3. Inducibility in other tissues. The inducibility of the class-I patatin promoter in leaf or stem tissue depends upon the concentrations of sucrose, nitrogen, and gibberellic acid in the culture medium. Activity of the promoter could be induced by increasing the sucrose concentration in which leaf or stem tissues were immersed under axenic conditions, but the shape of the response curve depended upon the nitrogen and gibberellic acid levels in the medium. Just as high nitrogen ~nd gibberellin discourage tuberization in intact plants, so their presence in the medium required higher sucrose concentrations in order for the class-I patatin promoter to be induced in leaf or stem tissues (Park 1990). The in vitro system of Perl et al. (1991) described in Part III.A.2 was used to study the inducibility of the class-I patatin promoter in this manner. Transgenic plants were subjected to four treatments selected from those shown in Table 3.1. Cuttings in all four treatments were placed on the 8% sucrose medium and exposed to an 8-h photoperiod for one day to provide the initial trigger for tuberization. Cuttings were given either zero or two more days of this treatment, after which they were moved to a 2% sucrose medium. Photoperiod on the 2% sucrose was either 8-h or 16h. Consistent with the data of Table 3.1, tubers formed in axillary buds of cuttings that received three days on the 8% sucrose, regardless of subsequent photoperiod, and in buds of cuttings that received one day on the 8% sucrose followed by 16-h days on the 2% medium (Fig. 3.18A). No tubers formed if one day on the 8% sucrose was followed by 8-h days on the 2% sucrose. Activity of the class-I patatin promoter was assayed separately in the axillary buds and the leaves of these same single-node cuttings. The three treatments that produced tuberization showed activity in the buds, as might be expected (Fig. 3.18Aj. However, only two of these three treatments produced activity in the leaves. Exposure of the cuttings to 8-h photoperiods after their treatment with 8% sucrose was ineffective whether such treatment was for one day or three days (Fig. 3.18B). Thus it appears that tubers were induced and the patatin gene was activated in the axillary buds under conditions where the gene was not activated in leaves of the same cuttings (Perl et al. 1991). Although the gene could be

3.

157

TUBER FORMATION IN POTATO

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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

E. E. EWING AND P. C. STRUIK

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.

TUBER FORMATION IN POTATO

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

E. E. EWING AND P. C. STRUIK

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.

TUBER FORMATION IN POTATO

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

E. E. EWING AND P. C. STRUIK

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.

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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

E. E. EWING AND P. C. STRUIK

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.

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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

E. E. EWING AND P. C. STRUIK

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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.

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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

E. E. EWING AND P. C. STRUIK

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.

TUBER FORMATION IN POTATO

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

E. E. EWING AND P. C. STRUIK

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'Or
.

internal tuber )' characteristics

7

t~::~

tuber-growth rate

~

?

duration of tuber growth

/

relative tuber size

Figure 3.21.

Schematic summary of the factors influencing the competition between tubers of one potato stem. Question mark denotes that a relationship is surmised, but that proof for this is lacking (from Struik et al. 1990a).

of two plants are presented. Note that in Plant 1 the largest tuber 35 days after planting was Soon surpassed in volume by the second largest tuber. This pattern is common. The fate of a given tuber initial may be determined during a very narrow time frame, and we have little understanding of the controlling factors. Therefore it is difficult to manipulate tuber size distribution or even to predict how a given set of environmental factors will affect it under field conditions. One might assume that the largest tubers tend to be the first ones formed, but this has been questioned by many investigators (WUIT 1977; Struik and Van Voorst 1986; Struik et al. 1988b; Struik et al. 1990b, 1991). In spite of the range in final sizes of tubers on a typical plant, most of the tubers that develop to a marketable size are initiated over a relatively short time-span (Krijthe 1955; Struik et al. 1988b). This is well illustrated in Figure 3.4, which also shows that many stolons were initiated after the first tuber appeared. Because of the typically short time span over which

3.

TUBER FORMATION IN POTATO

171

tubers that grow to marketable size are initiated, there is usually little correlation between the time of initiation and the final size. The experiments of Oparka and Davies (1985a) described in Part VILA suggest that significant differences in sink strength already exist among stolon tips before visible swelling of tubers occurs. This might be related to Chapman's (1958) evidence that leaves on one side of a plant control the tuberization of stolons on that side of the plant: a tuber associated with a leaf that is shaded or otherwise impaired could be held back. The rate of initial growth may be much more important than the timing of the first swelling. Tubers with restrictions in their mitotic processes during early stages of growth grew more slowly during later stages (Reeve et a1. 1973). Differences in the activities of enzymes involved in starch synthesis might also playa role (see Part VII.B.7). The formation of an adequate number of phloem strands and associated cells can be another important criterion, even though Engels and Marschner (1987) stated that the transport system is probably not limiting the growth potential during later stages of growth. One can only speculate as to which factors eventually become dominant in controlling the observed differences in growth rates among tubers as they enlarge (see Stroik et a1. 1991). There was a clear diurnal periodicity to tuber volume increases mainly attributable to water movement into and out of the tuber (Schnieders et a1. 1988). Short-term volume increases were highest at the beginning of the night and lowest at the beginning of the day. The rate of increase was much more rapid during the night than during the day (Fig. 3.22), and the nocturnal rate of volume increase was greatest when the tubers showed their greatest overall growth rate. Daytime volume increases were most rapid during the early tuber growth stage (Schnieders et a1. 1988). G. Tuber Distribution in the Field In experiments with various combinations of planting depth and ridge size, deeper planting produced less lateral dispersion of the tuber crop both in the direction of the ridge and across the ridge (Kouwenhoven 1970). Deeper planting also caused new tubers to form slightly higher with respect to the position of the seed tuber; nevertheless the average depth of new tubers below the ridge surface was increased. The combined effects of less lateral dispersion across the ridge and more distance below the surface of the ridge meant that deeper planting gave a marked reduction in green tubers. The causes of these differences are complex and can be expected to include effects of soil moisture, soil temperature, mechanical resistance, aeration, and length of underground stem over which stolons may form. The pattern of development of tuber size over time in the field is shown

172

E. E. EWING AND P. C. STRUIK Rate of volume increase (cm 3 . h- 1)

I

0.10

0.08

0.06

0.04

0.02

0.00

49

56

67

Time (days after planting)

Figure 3.22. Comparisons of rates of volume increase of tubers during the day and night. Measurements were made using the device pictured in Figure 3.5. Standard errors of the means are shown by vertical bars and indicate the tuber-to-tuber variation of a set of six tubers from three plants. Each point represents an average of 72 measurements (from Schnieders et a1. 1988).

in Figure 3.23, where yield of dry matter in various size classes is plotted throughout the season. Note that as mean tuber size increases with time, so does the range in sizes. The coefficient of variation in tuber size is negatively correlated with the number of tubers per unit area. For further discussion of this point and a more complete review of literature on the hierarchy of tuber development, see Struik et a1. (1991).

IX. RESORPTION AND SECOND GROWfH A. Resorption of Tubers

One reason why it is difficult to predict the number of tubers that will grow to a marketable size is that many tubers decay or are

3.

TUBER FORMATION IN POTATO

173

2000 /tJ

J:::

01

== "0

a;

";;..

1600

<-

1: /tJ

E I

~

"0

1200

<-


.J:l ::J l-

800

400

o o

20

I

40 60

80

120

100

140

160 180

I

I

I

I

200 240 280 320 lowet limit 220 260 300 340 uppet limit Individual tuber size I g fresh weight I

Distributions of tuber yields in the field according to size class at five different sampling dates. Yields of tuber dry matter in various size classes are plotted for June 17, July 1, July 15, August 12, and September 16 (Struik et al. 1990).

Figure 3.23.

resorbed while still small (Fig. 3.24]. Burstall et al. (1987a] found that there was a close relation between the number of tubers >1 cm in midJuly and the final number of tubers; Le., there was little change in the number of tubers >1 cm after that time. Smaller tubers were resorbed or decayed. The number of tubers resorbed often depends on the total number of tuber initials because under a given set of conditions there is a limit as to the number of tubers than can grow to a large size. If the number of tuber initials is stimulated by cultural practices or environmental conditions that do not improve productivity after tuberization has occurred, the number of tubers that survive and attain marketable size may be constant. This phenomenon was demonstrated, for example, by the results of Struik et al. (1988b). In their experiments the number of tuber initials was strongly increased by 16 cycles of long days, whereas the number of tubers set was hardly affected. The treatment variables studied affected the number of tubers initiated but not tuber enlargement. Other researchers reported more variation in number of tubers set, but this may be explained by the fact that treatments in their experiments affected not only the number of tubers initiated but also the number that enlarged. Cho

174

E. E. EWING AND P. C. STRUIK

Figure 3.24. Resorbed tubers. These show varying degrees of resorption. Only the basal end of the tuber on the left exhibits external symptoms, whereas several of the others are already shriveled (courtesy of K. Scholte).

and Iritani (1983) counted tubers resulting from three planting dates in 1979 and 1980. Two to three times as many tubers were initiated the second year as the first. The percentage resorbed ranged from 23-44% in 1979, and from 30-40% in 1980 (our estimates based on their graphical data). Resorption in these experiments was not observed to occur in tubers weighing >10 g. Unpublished data supplied by L. C. Burstall permits a comparison of the number of tubers initiated and the number resorbed in shading experiments carried out in England during 1984 and 1985. There tended to be a linear relationship between the two, although the correlation was better the second year than the first. In 1984 the fraction of tubers resorbed per maximum number observed was higher in the shaded than in the unshaded treatment, and there was considerable scatter (Fig. 3.25A). In 1985 the shaded and unshaded data fell nicely on the same line (Fig. 3.25B). Drought also affects the relation between the maximum number of tubers and the number resorbed, depending upon the plant growth stage during which the stress occurs (Krug and Wiese 1972). In fact, water availability seems to be one of the major factors in determining the proportion of initials that grow into larger tubers. This explains why irrigation is useful in increasing tuber number. The process of resorption is not well described; again the fact that tubers are hidden from view and the difficulty of predicting which tubers or tuber initials will be resorbed have hindered progress. Although one assumes that the nonstructural dry matter in tubers is redistributed to

3.

TUBER FORMATION IN POTATO

80

175

B

A

C\J

E

"C

60

;;2//

Q)

-e0 en

f!?

40

g,..e ' 0

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OUnshaded • Shade to initiation • Shade during initiation ~ Shade from initiation

_ . Shaded - - - 0 Unshaded

, 0

i

i

70

90

110

130

150

Maximum no. of tubers m- 2

170 0

70

90

110

i

,

130

150

170

Maximum no. of tubers m- 2

Figure 3.25. Relationship between the number of tubers initiated and the number resorbed in shading experiments. (A) In 1984 there were two treatments: 50% shade throughout the season and an unshaded control. (B) In 1985 there were four treatments: no shade; 50% shade until tuber initiation; 50% shade during tuber initiation; and 50% shade after tuber initiation was completed (unpublished data from the experiments described in Burstall et al. 1987b, provided by L. Burstall.).

other tubers during resorption, the degree to which this occurs has not been well documented. However, it is clear that a tuber does not have a "valve" that eliminates backward flow of assimilate. Even rapidly growing tubers can lose carbon compounds to other tubers through redistribution (Oparka and Davies 1985b). When the balance of input and output remains negative for a sufficiently long time, the tuber is resorbed.

B. Other Forms of Resorption A special case of resorption is the formation of "little potatoes" directly on the mother tuber. It should be possible to estimate the amount of dry matter redistributed, but this has not been done. It seems likely that the formation of secondary tubers (chain tubers) and knobs after initiation of second growth also involves resorption from the primary tuber. There is evidence that the dry matter in a secondary tuber or knob was transferred out of the primary tuber (in the sense of resorption), rather than just passing through the vascular bundles of the primary tuber from other parts of the plant. Growth of the secondary tubers continues at the end of the growing season, after tops have senesced to the point where the rate of photosynthesis would be too slow to account for their growth rate as Scholte (1977) demonstrated. His study (published in Dutch) involved frequent measurements of the growth of primary and secondary tubers in an undisturbed crop. In some treatments haulms were killed on August 3, just when secondary tubers had started to grow, or on September 9, when

176

E. E. EWING AND P. C. STRUIK

second ary tubers were already much larger than primar y ones. As soon as the second ary tubers started to grow the primar y tubers stoppe d their increas e in weight . From that momen t primar y tubers served as "transport gates" to the second ary ones. Eventu ally the second ary tubers could mainta in their rate of growth only by resorbing the primar y ones. First the smalle r primar y tubers were resorbed. Quite often these were tubers withou t second ary tubers on them! Later the larger primar y tubers were resorbed, especia lly the ones that carried second ary tubers. Resorp tion was manife sted as a reducti on in dry-ma tter conten t, dry-ma tter yield, and numbe r of primar y tubers. Growth of second ary tubers continu ed after destruc tion or comple te senesc ence of the haulm. Transl ucent ends and jelly end rot are defects that involve resorpt ion from one part of the tuber to anothe r (Hiller et a1. 1985). Affect ed tissue at the basal end of the tubers becom es glassy or translu cent and, in more severe cases, soft and flaccid. The tissue is deficient in starch and high in sugars, which leads to discolo ration after processing. The conditi on is commo n in 'Russe t Burban k' and other cultiva rs with long tubers (Iritani et a1. 1973). The disorde r is usually attribu ted to stress from drough t (which leads to higher soil temper atures) , especia lly during hot periods (Lugt 1960). Thus these disorders fit the genera l pattern for second growth .

C. Second Growth Under this topic we discuss knobby tubers and variou s other deform ed tubers, chain tubers, and heat sprouti ng. The inclusi on of heat sprouti ng might be questioned; but in some of its manife station s, at least, it seems to have much in commo n with the other phenom ena. 1. Relation to induction to tuberize. Second growth may be though t of as the result of an interru ption in inducti on to tuberize once tubers have already started to form. If the interru ption is strong and long lasting, buds in the apex of the tuber, which stoppe d growin g at the time of tuberin itialion, may resume their growth , forming a stolon or sprout. Because this phenom enon is most often associa ted with hot weathe r, it is called heat sprouti ng. This descrip tion might lead one to conclu de that heat sprouti ng is physio logical ly similar to the breakin g of bud dorma ncy that occurs after tubers are harves ted and stored. Such a conclu sion is not warran ted based upon our presen t knowle dge. The physiological causes of both phenom ena are poorly unders tood, but it does not appear that heat sprouti ng arises from the same factors that normal ly cause the breakin g of bud dormancy. Heat sprouti ng seems to be more an interru ption of the dorma nt conditi on than a termin ation of it; subseq uent to heat sprouti ng the tuber buds again becom e dorman t.

3.

TUBER FORMATION IN POTATO

177

If after heat sprouting has started conditions more favorable to tuber induction ensue (e.g., cooler temperatures), the stolon tip may start to swell into a new tuber. This succession of tubers and stolons is called chain tubers. If the interruption is briefer still, it may be manifested as secondary lateral growth in one or more eyes, producing a knobby tuber. Other types of deformity include bottlenecks, dumbbells, and pointedend tubers (Hiller et al. 1985). The last of these may be related to the fact that high soil temperatures tend to produce more elongated tubers (Epstein 1966). 2. Resistance of the medium. The absence of mechanical resistance

during tuber initiation also encourages second growth of tubers (Fig. 3.26). After tuber set occurs, the lack of mechanical resistance will no longer have this effect. Wheeler et al. (1990) did not report a problem with second growth in their experiments with a continuous flowing nutrient film technique. As discussed in Part VIII.D, it may be that their procedure of changing from continuous illumination to 12-h photoperiods four weeks after planting provided such strong induction that other problems were overcome.

Figure 3.26. Second growth in the absence of mechanical resistance. The plant was grown in an apparatus similar to that illustrated in Figure 3.3. except that no medium was added to the chamber to provide mechanical resistance for the growing tubers. Second growth is shown as a chain tuber at a position where tuber set was not completed when the stolon medium was removed (courtesy of G. E. L. Borm).

178

E. E. EWING AND P. C. STRUIK

3. Genetic differences. Cultivars vary in susceptibility to second growth. Russet Burbank, the leading cultivar in North America, is notoriously susceptible. The main cultivar in western Europe, Bintje, is also quite susceptible. In recent years breeders have selected for resistance to this disorder, and most modern cultivars are tolerant. 4. Drought and heat stress. Second growth in the western United States,

where 'Russet Burbank' is the leading cultivar, is commonly attributed to irregular or inadequate applications of irrigation water. Indeed, there is evidence that drought stress is a contributing factor (Van Loon 1986). However, drought stress is commonly accompanied by higher soil temperatures, and the latter are likely to be more responsible for the second growth (Lugt et al. 1964; Ruf 1964). The higher soil temperatures probably cause an increased production of gibberellins, which can be expected to interfere with tuber growth (Part VI.D.1). Application of gibberellic acid to tuberizing plants is very effective in producing second growth (Bodlaender et al. 1964). Applications of 2,4,5trichlorophenoxyacetic acid or methylchlorophenoxyacetic acid have shown some promise in counteracting effects of the environment on second growth (Bodlaender 1982). Exposure of any part of the plant to high temperatures will promote second growth, but exposure of the tubers is most effective (Struik et al. 1989c). With a seven-day exposure period of tubers to heat, there was a severe reduction in tuber growth by the end of the high temperature treatment, but no second growth. Tuber growth resumed after a return of the tubers to cool temperatures, but second growth was observed as well. Heat treatment of the "above-ground plant showed less delay in its effects on second growth, but the effects were less severe (P. C. Struik, unpublished results). 5. Other factors. If second growth results from an interruption in induc-

tion to tuberize, then it would be expected that other factors affecting induction would be potential contributors. Indeed, sudden lengthening of the photoperiod (Madec and Perennec 1962) and sudden increases in supply of nitrogen fertilizer (Bodlaender et al. 1964; Krauss 1985) during the period of tuber growth also promote second growth. The size and physiological age of the mother tuber (Part V.) may affect second growth indirectly: mother tubers that produce a high rate of shoot development will cause an early soil cover, thereby lowering soil temperatures. Data on the effects of size of micro- or minitubers on the occurrence of second growth are shown in Table 3.8. Very tiny seed tubers gave slow emergence and slow soil cover, and second growth was more than three times as frequent as when seed tubers ofnormal size were planted.

3.

179

TUBER FORMATION IN POTATO

Table 3.8. The effects of sizeofthe mothertuberon percentage ground cover 51 days after planting (DAP) and on percentage of tubers >20 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)

<0.25 g 0.25-0.5 g 0.5-1 g 1-2 g 2-4 g 25-28 mm

19 35 33 35 84

6

27.0 23.5 22.7 15.3 16.5 7.6

In addition to the reduction in soil shading there is probably a more direct cause of increased second growth when microtubers are planted. In the absence or near absence of a mother tuber, induction to tuberize depends solely upon the shoot and its response to the environment (Madec and Perennec 1962; Perennec 1966). Tuberization on plants grown from physiologically young seed tubers tends to occur much later than tuberization on plants grown either from physiologically old seed tubers or from cuttings, TPS, and microtubers. This suggests that physiologically young mother tubers supply gibberellins (Bialek and Bieliriska-Czarnecka 1975) or other factors that counteract induction from the shoots. In the absence of such factors, fluctuations in temperature and light intensity may cause corresponding fluctuations in the induction to tuberize. As already described this would increase the incidence of second growth. In the special apparatus diagrammed in Figure 3.3, where indirect effects of canopy shading on soil temperature are not a factor, experiments have been planted at various times of the year. As planting time is delayed from early February to late August and seed tubers become progressively older, there is a pronounced trend toward increased second growth of the tuber progenies (P. C. Struik, unpublished results). 6. Abscisic acid. Aside from increased gibberellin activity, there is reason to believe that second growth is associated with a decline in abscisic acid in the developing tuber. Tubers produced on cuttings provide a convenient model system to investigate second growth (Van den Berg et al. 1990), and cutting tubers showed much lower abscisic acid contents if exposed to conditions that promoted second growth (Van den Berg et al. 1991). Similarly, second growth of tubers on hydroponically grown plants that was produced by irregular supplies of nitrogen

180

E. E. EWING AND P. C. STRUIK

fertilizer was associa ted with a lower ratio of abscisic acid to gibbere llin (Krauss 1985).

x. IMPLICATIONS FOR TUBER YIELD A knowle dge of the effects of inducti on and the factors control ling it can be put to very practic al use in potato crop manag ement. A full accoun t would require a separa te review; here we will mentio n a few examp les related to tuber yield, hoping that the reader will not overlo ok the fact that there are also implica tions for tuber quality. In view of the effects of inducti on on overall plant develo pment, it should be obvious that highes t tuber yields are not necess arily associa ted with the highes t levels of inducti on. Neithe r extrem e-very strong or very weak induct ion-is desirable. If there is no induction, there can be no yield of tubers; but very strong inducti on occurri ng early in the growth of the pl~nt will severe ly limit tuber yields becaus e of the stunted canopy and roots. An examp le of the former problem is growing a typical andige na cultiva r under long photop eriods or high temper atures. Haulm growth might be excelle nt, but the harves t index would be so depres sed that yields would be late and unacce ptably low. The opposi te problem comes when an early maturi ng tuberosum cultiva r is planted under short days and cool temper atures, as at high altitud e in the tropics or during the winter in the subtrop ics. Now the harves t index is high, but yields are low becaus e the total biomas s is so restricted. Highes t yields, thus, tend to be associa ted with interm ediate harves t indices. If there is a premiu m for early yields, it may be desirab le to sacrific e potenti al yield by aiming for fairly strong inducti on early in the season. This would mean selecti on of a cultiva r with a long critical photop eriod (Le., an early-m aturing one), choosing physio logical ly old seed tubers and using a modera te to low rate of N fertilizer. If earline ss is not a prime consid eration , then later cultiva rs with shorter critical photop eriods, physio logical ly younge r seed tubers, and high rates of N are indicated. For high tuber yields it is essenti al to mainta in good canopy cover over the soil for a long part of the growin g season (Van der Zaag 1984). Under good growin g conditi ons the tuber bulkin g curve is nearly linear as long as the canopy is essenti ally closed. This means not only achiev ing rapid closure of the canopy, but keepin g it closed late. For good light interce ption late in the season it is necess ary to have many leaves on axillar y branch es [both sympo dial and basal); but axillar y branch ing is inhibit ed by strong inducti on (an added reason why very strong inducti on can

3.

TUBER FORMATION IN POTATO

181

reduce yields). Furthermore, the effects of early senescence in shortening the period of canopy closure are exacerbated by several pests: in particular the susceptibility to leafhoppers, early blight, and Verticillium wilt increase as the plants become more senescent. If these pests are present, it is all the more important to avoid excessive induction. One way of accomplishing this in areas such as the Columbia River basin that have very long growing seasons is to make late applications of N, which serve to limit induction, delay senescence, and prolong the period of maximum light interception. Choice of planting date in regions where there is flexibility will affect the pattern of induction through the season via the effects of both photoperiod and temperature. Thus cultivar selection, seed handling, N fertilization, and time of planting provide opportunities for controlling the level of induction, thereby influencing harvest index, earliness of harvest, duration of canopy cover, and tuber yield. Indirect effects of these factors may also be important. For example, the speed of crop emergence and the size and persistence of the canopy will have significant effects on soil shading and hence on soil temperature, especially under tropical conditions (Midmore and Mendoza 1984). This may have a major impact on earliness of tuberization and yield in the tropics (Midmore 1984). Closer spacing also offers a possibility for more shading and beneficial cooling under tropical conditions (Midmore 1983; 1988; Vander Zaag et al. 1990). Other ways to provide cooling include intercropping (Midmore 1990), mulching (Midmore et al. 1988a,b), and irrigation. Irrigation can provide not only direct effects of cooling, but also indirect effects through increased canopy development and consequent shading (Lugt et al. 1964; Van Loon 1986). Effects of soil cooling are most critical in tropical potato production, but they may produce beneficial effects under temperate conditions as well.

XI. CONCLUSION The physiology of tuber formation in potato is a challenging research area for the scientist, and a matter of practical importance for the agriculturist. Studies in physiology and biochemistry of the changes that accompany tuberization are producing fascinating results. Such studies promise to yield new insights that may have great significance for potato producers. In order to take advantage of the new findings as well as the information already available, agriculturists who work with potatoes need to understand the principles of tuber induction, initiation, and growth. We hope that this review will contribute to such understanding.

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E. E. EWING AND P. C. STRUIK

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development. with special reference to the control of potato tuberization: A review. Am. Potato J. 68:781-794. Van den Berg. J. H., P. C. Struik, and E. E. Ewing. 1990. One leaf cuttings as a model to study second growth in the potato (Solanum tuberosum) plant. Annu. Bot. 66:273-280; note Erratum in Annu. Bot. 67:191. Van den Berg. J. H., D. Vreugdenhil. P. M. Ludford, L. L. Hillman. and E. E. Ewing. 1991. Changes in starch, sugar, and abscisic acid contents associated with second growth in tubers of potato (Solanum tuberosum L.) one-leaf cuttings. J. Plant Physiol.139:66-89. Van der Zaag. D. E. 1984. Reliability and significance of a simple method of estimating the potential yield of the potato crop. Potato Res. 27:51-73. Van der Zaag. D. E., and C. D. van Loon. 1987. Effect of physiological age on growth vigour of seed potatoes of two cultivars. 5. Review of literature and integration of some experimental results. Potato Res. 30:451-472. Vander Zaag. P.• A. L. Demagante. and E. E. Ewing. 1990. Influence of plant spacing on potato (Solanum tuberosum L.) morphology, growth and yield under two contrasting environments. Potato Res. 33:313-324. Van Es, A.• and K. J. Hartmans. 1967. Effect of physiological age on growth vigour of seed potatoes of two cultivars. 2. Influence of storage period and storage temperature on dry matter content and peroxidase activity of sprouts. Potato Res. 30:411-421. Van Ittersum. M. K., and K. Scholte. 1992. Relation between growth conditions and dormancy of seed potatoes. 2. Effects of temperature. Potato Res. (in press). Van Ittersum. M. K., K. Scholte. and L. J. P. Kupers.1990. A method to assess cultivardifferences in rate of physiological ageing of seed tubers. Am. Potato J. 67:603-613. Van Loon, C. D. 1986. Drought. a major constraint in potato production and possibilities for screening for drought resistance. p. 5-16. Beekman, K. M. Louwes, L. M. W. Dellaert, and A. E. F. Neele (eds.).• Potato research of tomorrow. Proceed Int. Sem., Wageningen, Neth. Pudoc. Wageningen. Van Loon, C. D. 1967. Effect of physiological age on growth vigour of seed potatoes of two cultivars. 4. Influence of storage period and storage temperature on growth and yield in the field. Potato Res. 30:441-450. Vayda, M. E.• and W. D. Park (eds.), 1990. The molecular and cellular biology of the potato. C.A.B. International, Redwood Press Ltd.• Melksham, UK. Vince-Prue. D. 1986. The duration of light and photoperiodic responses. p. 269-337. In: R. E. Kendrick and G. H. M. Kronenberg (eds.), Photomorphogenesis in Plants. Martinus Nijhoff Publishers, Dordrecht. Vachting. H. 1887. Uber die Bildung der Knollen. Bib!. Bot. 4:1-55. Vos, J.• and H. Biemond. 1992a. Effects of nitrogen on the development and growth of the potato plant. 1. Leaf appearance. expansion growth. life spans of leaves and stem branching. Ann. Bot. (in press). Vos. J.• and T. Klemke. 1992. The foliar development of the potato plant and modulations by environmental factors. In: B. van den Broek, P. Kabat. H. van Keulen, B. Marshall. and J. Vos (eds.), Concepts and Models For the Simulation of Potato Production. Simulation Monographs, Pudoc, Wageningen, The Netherlands. (in press). Vreugdenhil. D.• and H. Helder. 1992. Hormonal and metabolic control of tuber formation. p. 393-400. In: C. M. Karssen, L. C. van Loon, and D. Vreugdenhil (eds.), Progress In Plant Growth Regulation. Kluwer Academic Press. Dordrecht. The Netherlands. Vreugdenhil, D., A. P. C. Oerlemans. and M. H. G. Steeghs. 1984. Hormonal regulation of tuber induction in radish (Raphanus sativus). The role of ethylene in tuber induction of radish. Physiol. Plant. 62:175-180. Vreugdenhil, D., and P. C. Struik. 1989. An integrated view of the hormonal regulation of tuber formation in potato (Solanum tuberosum). Physio!. Plant. 75:525-531.

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Vreugdenhil, D., and P. C. Struik. 1990. Hormonal regulation of tuber formation. p. 37-38. In: EAPR Abstracts of Conference Papers and Posters, 11th Triennial Conferene of the Eur. Assoc. Potato Res., Edinburgh. Vreugdenhil, D., and W. van Dijk. 1989. Effects of ethylene on the tuberization of potato (Solanum tuberosum) cuttings. Plant Growth Reg. 8:31-40. Wang, T. L., And P. F. Wareing. 1979. Cytokinins and apical dominance in Solanum andigena: lateral shoot growth and endogenous cytokinin levels in the absence of roots. New Phytol. 2:19-28. Wareing, P. F. 1982. The control of development of the potato plant by endogenous and exogenous growth regulators. p. 129-138. In: J. S. MacLaren (ed.), Chemical Manipulation of Crop Growth and Development. Butterworths, London. Wareing, P. F., and A. M. V. Jennings. 1980. The hormonal control of tuberisation in potato. p. 293-300. In: F. Skoog (ed.), Plant Growth Substances. Spring-Verlag, Berlin. Weinheimer, W. H., and B. W. Woodbury. 1966. Effects of grafting and Solanum understocks on flower formation and abscission of flowers and fruits in the Russet Burbank potato. Am. Potato J. 43:453-457. Wellensiek, S. J. 1929. The physiology of tuber formation in Solanum tuberosum L. Mededelingen Land. Wageningen. 33:6-42. Went, F. W. 1959. Effects of environment of parent and grandparent generations of tuber production by potato. Am. J. Bot. 46:277-282. Wenzler, H., G. Mignery, G. May, and W. Park. 1989. A rapid and efficienttransformation method for the production of large numbers of transgenic potato plants. Plant. Sci. 63:79-86. Werner, H. O. 1934. The effect of a controlled nitrogen supply with different temperatures and photoperiods upon the development of the potato plant. Nebr. Agr. Expt. Sta. Res. Bui. 75. Werner, H. O. 1954. Anomalous tuberization of Solanum tuberosum. Am. Potato J. 31:375. Wheeler, R. M., D. J. Hannapel, and T. W. Tibbitts. 1988. Comparison of axillary bud growth and patatin accumulation in potato leaf cuttings as assays for tuber induction. Annu. Bot. 62:25-30. Wheeler. R. M.. C. L. Mackowiak, J. C. Sager, W. M. Knott, and C. R. Hinkle. 1990. Potato growth and yield using nutrient film technique (NFT). Am. Potato J. 67:177-187. Wheeler. R. M., K. L. Steffen, T. W. Tibbitts, and J. P. PaIta. 1986. Utilization of potatoes for life support in space. II. The effects of temperature under 24-h and 12-h photoperiods. Am. Potato J. 63:639-647. Wheeler, R. M., and T. W. Tibbitts. 1986. Growth and tuberization of potato (Solanum tuberosum L.) under continuous light. Plant Physioi. 80:801-804. Wheeler, R. M., T. W. Tibbitts, and A. H. Fitzpatrick. 1991. Carbon dioxide effects on potato growth under different photoperiods and irradiance. Crop Sci. 31:1209-1213. Willmitzer, L. 1991. Molecular approaches to sink-source relations. J. Cell. Biochem. Suppi. 15A:20. Willmitzer, L., A. Basner, W. Frommer, R. HOfgen, X. J. Liu, M. Koster, S. Prato M. RochaSosa, U. Sonnenwald, and G. Vancanneyt. 1990. 'lUber-specific gene expression in transgenic potato plants. p. 105-114. In: G. W. Lycett & D. Grierson (eds.). Genetic Engineering of Crop Plants. Butterworths, London. Wolf, S., A. Marani, and J. Rudich. 1990. Effects of temperature and photoperiod on assimilate partitioning in potato plants. Annu. Bot. 66:515-520. ___ .1991. Effect of temperature on carbohydrate metabolism in potato plants. J. Expt. Bot. 42:619-625. Wolfe, D. W., E. Fereres, and R. E. Voss. 1983. Growth and yield response of two potato cultivars to various levels of applied water. Irrig. Sci. 3:211-222.

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Woolley, D. J., and P. F. Wareing. 1972a. The role of roots, cytokinins and apical dominance in the control of lateral shoot formation. Planta 105:33-42. ___ . 1972b. The interaction between growth promoters in apical dominance. New Phytol. 71:781-793. ___ . 1972c. Environmental effects on endogenous cytokinins and gibberellin levels in Solanum tuberosum. New Phytol. 71:1015-1025. Wright, R. C., and T. M. Whiteman. 1949. The comparative length of dormant periods of 35 varieties of potatoes at different storage temperatures. Am. Potato J. 26:330-335. Wurr. D. C. E. 1977. Some observations on patterns of tuber formation and growth in the potato. Potato Res. 20:63-75. ___ .1978. "Seed" tuber production and management. p. 327-352. In: P. M. Harris (ed.), The Potato Crop. Halsted Press, Wiley, New York. Yandell, B. S., A. Najar, R. M. Wheeler, and T. W. Tibbitts. 1988. Modeling the effects of light, carbon dioxide, and temperature on the growth of the potato. Crop Sci. 28:811818. Yoshihara, T., E. A. Orner, H. Koshino, S. Sakamura, Y. Kikuta, and Y. Koda. 1989. Structure of a tuber-inducing stimulus from potato leaves (Solanum tuberosum L.). Agr. BioI. Chern. 53:2835-2837.

4 The Biology, Epidemiology, and Control of Turnip Mosaic Virus V. 1. Shattuck Department of Horticultural Science University of Guelph Guelph, Ontario Canada N1G 2Wl

1. Introduction II. History III. Characteristics of the Virus A. Nomenclature B. Virus Structure C. Inclusions D. Maintenance and Preservation IV. Strains and Isolates A. Classification B. Geographical Distribution C. Virus Interactions V. Purification A. Methods of Extraction B. Molecular Properties VI. Effects of Infection A. Symptomology B. Metabolism VII. Detection A. Indicator Plants B. Serological Methods VIII. Epidemiology A. Inoculum Sources B. Aphids IX. Control Methods A. Cultural B. Chemical C. Preventative and Therapeutic D. Immunity and Resistance X. Conclusion Literature Cited

I. INTRODUCTION Turnip mosaic virus (TuMV) occurs worldwide in the temperate and tropical regions of Africa, Asia, Australia, Europe, India, and North and 199

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South America. This pathogen is included in the potato virus Y group (Potyvirus) and is one of the most important viruses that attacks the Brassicaceae. The host range of TuMV is broad and includes 39 dicotyledonous families (Boswell and Gibbs 1983; Edwardson and Christie 1986). Since the 1920s, outbreaks of TuMV have occurred throughout the world resulting in substantial damage to both plants and plant products. Today, this pathogen is well established in many regions of the world and seriously plagues annual crops. For example, Feng et a1. (1990) revealed that 69% of the crucifer crops sampled around Beijing, China were infected with this virus. In a recent survey of plant virologists from 28 countries and regions, TuMV was ranked second to cucumber mosaic as the most important virus infecting field-grown vegetables (Tomlinson 1987). To date, the scientific literature on TuMV has not been summarized. This review was written to provide horticulturists an overview of this destructive plant pathogen.

II. HISTORY Flower-breaking symptoms on annual stock (Matthiola incana R. Br), a common sign of TuMV infection for this plant, was noted in France as early as 1862 (Tompkins 1939a). During the fall of 1919, possibly in Washington, D.C., E. S. Schultz and W. A. Orton observed mottling symptoms on field-grown Chinese cabbage (Brassica pekinensis Rupr.), mustard (Brassica spp.), and turnip (Brassica rapa L.) plants. In 1920 in South Bend, Indiana, M. W. Gardner and J. B. Kendrick also observed a mosaic disease on plants in a small turnip field. Their findings, Gardner and Kendrick (1921) and Schultz (1921), were published independently in the same journal, and all were given credit for first describing TuMV. However, it is likely that this virus was widely distributed but went unnoticed in the United States prior to being first described. In the 1920s and 1930s, reports of mosaic-like diseases began appearing in various parts of the world (Tompkins 1939b; LeBeau and Walker 1945). In 1927, a devastating mosaic disease occurred on cabbage, mustard, and turnip in Fukuoka, Japan (Takimoto 1930). Subsequent references to this mosaic disease were also made by Fukushi (1932) and Hino (1933). Fajardo (1934) reported that 30-50% of the Chinese cabbage plants in the market gardens of Manila, Philippine Islands were commonly infected with a mosaic disease. A mosaic disease causing considerable production losses in rape (Brassica napus L.) and other cultivated crucifers was reported in China in the 1930s (Ling and Yang 1940). TuMV was first recorded in New

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201

Zealand in 1932, and quickly became a serious disease of rape and other crucifers (Chamberlain 1936). In the 1930s and 1940s, TuMV became an economically important pathogen on cabbage (Brassica oleracea var. capitata L.) in Wisconsin (Blank 1935), cauliflower (Brassica oleracea var. botrytis 1.) in California (Tompkins 1934), and horseradish (Armoracia rusticana Gaertn. Mey., and Scherb.) in Oregon (Dana and McWhorter 1932), Wisconsin, Illinois, Missouri, and Washington (Pound 1948). In the early 1940s, epidemics involving TuMV with cauliflower mosaic virus (CaMV) resulted in severe damage to the cabbage seed crops in Puget Sound, Washington (Walker 1953). TuMV was first reported in the province of Ontario, Canada in 1946 on rutabaga (Brassica napobrassica Mill.) (Berkeley and Weintraub 1952) and quickly became endemic to this crop. Severe. outbreaks of TuMV on Ontario-grown rutabaga in the early 1950s, mid-1960s, 1971, and 1985 resulted in severe production losses. Recent TuMV epidemics have occurred periodically in many regions of the world causing serious crop losses: staticeILimonium perezii Mill.) in California (Niblett et a1. 1969); lettuce (Lactuca sativa L.) in California (Zink and Duffus 1970); annual stock in Argentina (Pontis 1973); endive and escarole (Cichorium endivia L.) in New Jersey and New York (Citir and Varney 1974b; Provvidenti et al. 1979); collards (Brassica oleracea var. acephala DC.) in Georgia and South Carolina (Khan and Demski 1982); cruciferous crops in New Zealand (Palmer 1983); stock in the Middle East (Bahar et a1. 1985); turnips in Alabama (Wilson and Stevens 1986); cruciferous crops in Great Britain (Pink and Walkey 1986, 1988); rutabaga in Ontario, Canada (Shattuck and Stobbs 1987); and horseradish in Sweden (G. Engqvist, personal communication). It should be noted that crop losses inflicted by the TuMV disease are not confined to commercial plantings. This disease has been known to infect plants destined for scientific investigations (Ayotte et al. 1985), stunt or destroy important crucifer germplasm within breeding nurseries in the United States and Canada, and limit the kinds of plants grown in home gardens.

III. CHARACTERISTICS OF THE VIRUS A. Nomenclature

Early TuMV isolates were named according to the host they infected and symptoms they produced. This practice resulted in numerous designations for this virus. Some of the synonyms that were used for TuMV and the TuMV disease include: black-ringspot, brassica nigra virus, brassica virus 1, brussel sprouts mosaic, cabbage A virus mosaic,

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cabbage black ringspot virus, cabbage black ring virus, cabbage mottle virus, crucifer mosaic virus, daikon mosaic virus, horseradish mosaic virus, kwuting, mustard mosaic, radish P virus, radish R virus, rape mosaic, ring necrosis of petsai, ring necrosis virus, ring spot virus, rutabaga virus, turnip virus 1, and watercress mottle (Walker 1952; Chiu et a1. 1957; Yoshii 1963; Tomlinson 1970; Smith 1972). The cabbage mosaic disease refers to the combined infection of the cabbage virus A TuMV strain and cauliflower mosaic virus (Pound et a1. 1965).

B. Virus Structure 1. Morphology. TuMV particles exceed the length of tobacco mosaic virus, ranging from 650-760 nm, with a diameter from 15-20 nm (Shepherd and Pound 1960; Tomlinson 1970; Halliwell et a1. 1978J. Although particle lengths from 150 to 840 nm were reported (Edwardson and Christie 1986J, it is possible that differences in the preparative techniques prior to measurement, measuring techniques, and the shearing of TuMV particles prior to measurement may have contributed to this range. The long flexuous rod-shaped virus particles (Fig. 4.1J can fragment during isolation from the host, or through sonication and alkaline degradation (Purcifull and Shepherd 1964J. The infectivity of TuMV appears to depend on the intactness of the virus particle. Tomlinson and Walkey (1967) reported that when TuMV particles were fragmented through sonication, a marked reduction in infectivity was noted. In their study, the principal antigens of TuMV had particle lengths of 25-200 nm. 2. Composition TuMV consists of a single strand of RNA enclosed in a protein coat. Based on the content of phosphorus, Hill and Shepherd (1972) calculated that the virus contained approximately 4-5% RNA, and the nucleic acid fraction contained 34.5% adenine, 22.4% guanine, 22.3% cytidylic acid, and 20.8% uridylic acid. Choi et a1. (1979b) estimated that the RNA fraction possessed 31.7% adenine, 22.0% guanine, 27.2% cytidylic acid, and 19.1% uridylic acid. Hyperchromicity measurements of purified TuMVRNA indicated that about 50% of the bases were involved in pairing (Hill and Benner 1976; Choi et a1. 1979b). The molecular weight of TuMVRNA, determined through sedimentation analysis and electrophoresis, has been reported to vary from 3000 to 3500 kD (Hill and Shepherd 1972; Hill and Benner 1976; Choi et a1. 1979a; Stobbs and Van Schagen 1987; Zhu and Chiu 1989).

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Examination of a few TuMV isolates has shown that the virus protein subunit has a high content of glutamic acid and a low content of cysteine, along with a high degree of amino acid sequence homology to other members of the potyviruses (Hill and Shepherd 1972; Choi and Wakimoto 1979; Kong et a1. 1990). Recently, Kong et a1. (1990) and Tremblay et a1. (1990) sequenced the coat protein genes of TuMV isolates from radish (Raphanus sativus L.) and rutabaga, respectively, and found that each consisted of 288 amino acids and had a molecular weight of 33.2 kD. Earlier studies reported low molecular weight values for the protein coat ranging from 26 to 28 kD (Hill and Shepherd 1972; Choi and Wakimoto 1979). It is possible that these studies underestimated the weight of the protein coat, and heterogeneity of virus protein (Michelin-Lausarot and Papa 1975; Moghal and Francki 1976; Choi and Wakimoto 1979; Hill and Benner 1980), coisolation of virus and host proteins (Stobbs and Van Schagen 1987; Thompson et a1. 1988) and the disruption of the capsid protein in host tissues (Choi and Wakimoto 1979) may have occurred during molecular weight determination and influenced interpretations.

c. Inclusions Potyviruses produce aggregates of nonstructural proteins in host cells called cylindrical inclusions (McDonald and Hiebert 1974). Inclusion bodies can be easily detected in light microscopic examination following staining (Christie and Edwardson 1977). Evidence exists that the inclusion proteins are coded by the genome of the virus (Purcifull et a1. 1973; Edwardson 1974; Dougherty and Hiebert 1980). TuMV induces cylindrical inclusions, pinwheels, bundles, scrolls, and laminated aggregates (Edwardson 1974; Edwardson and Christie 1986). The morphology of inclusion bodies has been studied by electron microscopy (Edwardson and Purcifull1970; McDonald and Hiebert 1975). Inclusions occur in the cytoplasms of the epidermal, mesophyll, and phloem cells (Edwardson and Purcifull1970) and in systemically infected plants as well as those showing only local lesions (Milicic et a1. 1967). The size of certain inclusions is dependent on the host plant (Stefanac and Wrischer 1989). Inclusions, once present in the cells, may disappear in time (Milicic et a1. 1967).

D. Maintenance and Preservation TuMV is maintained on rapidly growing cruciferous plants (e.g., Chinese cabbage, mustard or turnip) that exhibit systemic symptoms. Systemically infected plants are preferred, since the concentration of the virus is higher in these plants than in those displaying only necrotic lesions. To safeguard against strain contamination, single lesion

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isolations may be performed prior to maintaining the virus in isolated hosts. If the host range of the TuMV isolate is unknown, the virus should be maintained on the infected host plant. TuMV can be successfully preserved in host tissues, plant extracts, or following isolation and purification. Young leaf tissues high in TuMV titre can be preserved indefinitely by cutting them into small pieces, sealing them in a petri dish with a desiccant, such as calcium chloride, and allowing them to dry out at a reduced temperature, such as 5°C for 4-5 days (R. Provvidenti, personal communication). Alternatively, infected tissue can be easily stored for several months in sealed bags, with or without a desiccant, at temperatures below freezing (Stobbs and Shattuck 1989). Yamaguchi (1964) stored infected leaf tissues at -20°C for up to 12 months with little loss in infectivity. It has been reported that infectious plant extracts can be successfully stored at 2°C for several months (Tomlinson 1970). When freeze dried, crude plant extracts can retain infectivity for many months (Hollings and Lelliott 1960; Fukumoto and Tochihara 1983). Various additives, such as glycerol, peptone, sodium azide, and sucrose, may have potential in the long-term preservation of purified TuMV (McDonald and Hiebert 1975; Fukumoto and Tochihara 1983). Purified TuMV is stable when freeze dried and stored at -20°C (Fukumoto and Tochihara 1983).

IV. STRAINS AND ISOLATES

A. Classification The International Committee on Nomenclature of Viruses and the International Committee and Taxonomy of Viruses established internationally accepted guidelines on the taxonomy of viruses (Matthews 1982). TuMV is included in the Potyvirusgroup, which is the largest and one of the most economically important group of the plant viruses (Francki et al. 1985). Its members include: bean common mosaic virus, beet mosaic virus, papaya ringspot virus, parsnip mosaic virus, potato virus A, soybean mosaic virus, sugarcane mosaic virus, and watermelon mosaic virus. General characteristics of the potyviruses include: long, rodshaped virus morphology (Fig. 4.1); infection of hosts resulting in mosaic symptoms and the production of inclusion bodies; and nonpersistent spread of the virus by aphids (Hollings and Brunt 1981; Walkey 1985). Interestingly, unlike most potyviruses, TuMV has a considerably wide host range. Several schemes have been proposed for classifying TuMV. Yoshii

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Figure 4.1. Electron micrograph of the filamentous morphology of TuMV particles isolated from rutabaga.

(1963) separated TuMV isolates into two strains based on their reported reactions on cabbage and Nicotiana glutinosa L. Forty-one isolates were grouped into the ordinary strain that produced mild symptoms on cabbage and N. glutinosa, while 18 other isolates were classified as the cabbage strain and caused severe necrotic ring spots on these host plants. McDonald and Hiebert (1975) used host range to classify TuMV isolates into two groups: Type I isolates were those that could infect all Brassica species while Type II isolates could infect only a limited number of Brassica species. It is possible to separate TuMV strains using cytology: (1) those inducing type-3 cylindrical cytoplasmic inclusions (pinwheels, scrolls and long laminated aggregates); (2) those inducing type 4 cylindrical inclusions (pinwheels, scrolls and short curved laminated aggregates). Early researchers were aware of TuMV strains from the host range and symptomatology of infected pla.nts (Pound and Walker 1945a; Pound 1948). LeBeau and Walker (1945) distinguished four strains of TuMV isolated from turnip. Today, it is well known that numerous strains of TuMV exist. Eight strains of TuMV have been classified using the Chinese cabbage differentials of Provvidenti (1980) and Green and Deng (1985) (Table 4.1). Walsh (1989) used oilseed rape and rutabaga differentials to separate eight European and Canadian isolates into four groups (Table 4.2). Group four in Walsh's classification is identical to TuMV-C 3 (Tables 4.1 and 4.2). Recently, Fujisawa (1990) used various cabbage, Chinese cabbage, and Japanese radish (Raphanus sativus L.) cultivars to differentiate 47 TuMV isolates from the major cruciferous vegetable growing

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Table 4.1. China.

Classification of TuMV strains from the United States, Taiwan, Canada, and

Suggested strain Strain nomenclature nomenclature Host

Country

Reference

TuMV-C1 TuMV-C2 TuMV-C3 TuMV-C4 TuMV-C5

TuMV-C1 TuMV-C2 TuMV-C3 TuMV-C4 TuMV-C5

United States United States United States United States Taiwan

TuMV-C6

TuMV-S4

Cabbage Chinese cabbage Turnip Chinese cabbage Chinese cabbage and mustard Garlic mustard

TuMV-C7 TuMV-C8

C3-2 C6

Various crucifers Various crucifers

China China

Provvidenti 1980 Provvidenti 1980 Provvidenti 1980 Provvidenti 1980 Green and Deng 1985 Stobbs and Shattuck 1989 Liu et a1. 1990 Liu et a1. 1990

Table 4.2.

Canada

Classification of TuMV isolates from Europe and Canada (Walsh 1989)z.

Group nomenclature

Country and isolate

Host

1

England 1 Germany 1 Germany 2 Greek 1 England 3 Denmark 1 Canada 1

Rutabaga Cabbage Cabbage Stock Watercress Chinese cabbage Rutabaga

2 3 4Y

ZDutch white cabbage isolate (UK2) not classified due to insufficient data. YSynonomous with TuMV-C3 in Table 4.1.

areas in Japan, into 9 TuMV strains (Table 4.3). Prochazkova (1980) found differences in the host species range and host symptomology in 24 TuMV isolates from the cruciferae weed Sisymbrium Loeselii L. in Czechoslovakia, which suggests the possibility of additionial TuMV strains. There have been many reports on TuMV isolates. Yoshii (1963) lists likely TuMV isolates reported prior to 1961. Some recent reports on TuMV isolates include: Stace-Smith and Jacoli (1967) in British Columbia, Canada; Purcifull (1968) in Florida; Niblett et al. (1969) in California; Pontis (1973) in Argentina; Horvath and Besada (1975) in Hungary; Mamula and Ljubesic (1975) in Yugoslavia; Zink and Duffus (1975) in California; Fischer and Lockhart (1976) in Morocco; Rao et al. (1977) in Alberta, Canada; Halliwell et al. (1978) in Texas; Lee et al. (1978) in Korea; Provvidenti (1978) in New York State; Twardowicz-Jakusz and

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Table 4.3.

207

Classification of TuMV strains from Japan (Fujisawa 1990).

Strain

Host

A B C D E F G H I

Japanese radish and turnip Chinese cabbage and Japanese radish Broccoli, cabbage, and Chinese cabbage Brassica X napus (Hart.), broccoli, cabbage, and Chinese cabbage Japanese radish and turnip Japanese radish Japanese radish Chinese cabbage, horseradish, and turnip Japanese radish

Zielinska (1979) in Poland; Kittipakom and Sutabutra (1982) in Thailand; Klisiewicz (1983) in California; Ahlawat and Chenulu (1984) in India; Bahar et al. (1985) in the Middle East; Lesemann and Vetten (1985) in Germany; Xu and Cockbain (1987) in China; Ford et al. (1988) in British Columbia, Canada; Hammond and Chastagner (1988) in Washington State; Zhu and Chiu (1989) in China; and Stobbs and Shattuck (1989) in Ontario, Canada. Unfortunately, information is lacking on the relationships of these isolates from those presented in Tables 4.1,4.2, and 4.3. It is possible that some of these isolates may represent additional strains of TuMV. There are indications that TuMV may be capable of mutating at a high frequency to produce new strains. During the course of two investigations, TuMV strains were observed to arise spontaneously (San Juan and Pound 1963; Provvidenti 1980).

B. Geographical Distribution Different strains will predominate in certain areas from the interaction of the virus strain and air temperature (Pound and Walker 1945a), mutual exclusion among TuMV strains from cross-protection, and from the abundance or scarcity of host plants. Based on geographic distribution, Yoshii (1963) speculated that the ordinary TuMV strain developed in the Orient, while the cabbage TuMV strain differentiated from the ordinary strain in Western Europe. In Ontario, Canada the TuMV-C3 strain appears to predominate. The TuMV-C4 strain, though rare in New York State (Provvidenti 1980), is the most common strain in Taiwan (Green and Deng 1985). In China, TuMV-C4 and TuMV-C s are the most common strains (Liu et al. 1990; Feng et al. 1990).

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C. Virus Interactions Cross protection, a common feature among virus strains, occurs when plants that are infected with a mild virus are prevented from becoming infected by different strains of the same or a similar virus. TuMV strains have been shown to cross protect against one another (Pound and Walker 1945a; Pound 1948; San Juan and Pound 1963; Eversten 1974). Tobacco mosaic virus has been shown to offer some cross protection against TuMV (Sherwood 1987). In field situations, mixed infections of TuMV and other viruses are often encountered. Arabis mosaic virus (Walkey et a1. 1981), CaMV (Walker et a1. 1945; Brusse 1965; Khan and Demski 1982), cucumber mosaic virus (Berkley and Tremaine 1954; Tomlinson and Ward 1978), ribgrass mosaic virus (Ford et a1. 1988) and tobacco mosaic virus (Chiu et a1. 1962; Chiu and Chang 1982) have been found in association with TuMV. In China, TuMV has been found in mixed infections with cucumber mosaic virus and tobacco mosaic virus on Chinese cabbage. TuMV infection has been shown to induce the accumulation of cucumber mosaic virus in Japanese radish plants through the enhancement of longdistance systemic spread of this virus in co-infected plants (Sano and Kojima 1989; Ishimoto et al. 1990). The concentration of TuMV is not affected by co-infection with cucumber mosaic virus. TuMV and CaMV can coexist together within the same host cell under certain conditions. If CaMV becomes established in the host and reaches the maturation phase of multiplication, it can interfere with the subsequent infection and multiplication of TuMV; interference does not occur before this critical time (Kamei et a1. 1969).

V. PURIFICATION A. Methods of Extraction When TuMV of sufficient quantity and purity is required, the virus is isolated, purified, and concentrated from host tissues. The isolation of TuMV can be difficult because the virus particles may aggregate and become lost during extraction and purification. Aggregation can arise from interactions between the virus particles and/or interactions between the virus particles and cellular components of the host plant (Damirdagh and Shepherd 1970). Detailed extraction and purification procedures for TuMV were presented by Shepherd and Pound (1960), Hill and Shepherd (1972), and Choi et a1. (1977), but may not be successful in all cases (Moghal and Francki 1976; Shields and Wilson 1987). Plant tissues are disrupted either by grinding with a mortar and pestle

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or through homogenization in a blender. Phosphate (Provvidenti, 1980; Klisiewicz 1983; Niu et al. 1983; Shields and Wilson 1987); borate (Tomlinson 1964; Lim et al. 1978); citrate (Thompson et al. 1988); and glycine-NaOH (Niblett et al. 1969) buffers have been recommended or successfully used to extract TuMV from plant tissues. Shepherd and Pound (1960) evaluated various buffers at different pH and molarities on TuMV yields and infectivity and concluded that a 0.5M potassium phosphate buffer (pH 7.5) gave the best results. Choi et al. (1977) compared the efficiencies of a potassium phosphate buffer (pH 7.5) and a borate buffer (pH 8.0) and also found that the phosphate buffer was better and that the extraction efficiency of this buffer could be increased with the addition of 0.1% thioglycolic acid and O.OlM Na-EDTA. For routine inoculations, the ratio of the tissue weight to the amount of extraction buffer should be monitored; generally, a ratio from 1:1 to 1:10 is satisfactory. Various agents have been used during the extraction and purification of TuMV. For example, reducing agents such as 2-mercaptoethanol (Damirdagh and Shepherd 1970; Hill and Shepherd 1972), sodium sulfite (Knight et al. 1984; Walkey and Pink 1988); chelating agents, such as EDTA (Choi et al. 1977), and clarifying agents, such as butanol (Shepherd and Pound 1960) have also been helpful. Urea and various detergents may also be used during extraction to reduce virus aggregation; in certain hosts possessing a high tannin concentration, 1% nicotine may be included in the extraction buffer to safeguard against virus deactivation. A low concentration of the protease trypsin has been used to eliminate host proteins, copurifying with TuMV, which cause virus aggregation (Thompson et al. 1988). Polyethylene glycol (Choi et al. 1977), isoelectric precipitation (Shepherd and Pound 1960), ammonium sulphate precipitation (Berkeley and Weintraub 1952), conventional ultracentrifugation, and rate-zonal density gradient centrifugation have all been used to concentrate TuMV during purification. The purity of TuMV preparations can be assessed using electron microscopy and/or conventional polyacrylamide electrophoresis.

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V. I. SHATTUCK

B. Molecular Properties The stability of TuMV strains in vitro varies. The thennal inactivation point is below 70°C; the dilution end point is typically between 10-3 and 10-5 ; and the longevity in vitro of TuMV infectivity ranges from 1 to 10 days (Tomlinson 1970; Green and Deng 1985; Edwardson and Christie 1986). Purified TuMV particles exhibit an ultraviolet absorption spectra with maximum and minimum absorbances at 260-262 nm and 244-247 nm, respectively, and a slight shoulder at 288-290 nm from the amino acids, tyrosine and tryptophane, which are located in the coat protein (Hill and Shepherd 1972; Choi et al. 1977). The buoyant density of purified TuMV was estimated at 1.336 glcc (Damirdagh and Shepherd 1970). Berkeley and Weintraub (1952) noted that TuMV had an isoelectric point of pH 4.5, while Shepherd and Pound (1960) recorded a pH of 5.3. Purified TuMV-RNA has been translated in vitro in rabbit reticulocyte lysate and wheat genn extract (Shields and Wilson 1987).

VI. EFFECTS OF INFECTION

A. Symptomology The sYmptomology of TuMV infected plants is dependent on the interaction among the host, TuMV strain, and environment. In many instances, this interaction is complex and poorly understood. Generally, a close correlation exists between the severity of infected hosts' sYmPtoms and virus concentration in tissues (Pound 1952; Horwitz et al. 1985). However, the plant growth stage may have an attenuating effect on host sYmptoms, and sYmptomology may be complicated by mixed infections with other viruses or pathogens (Reyes and Chadha 1972). Infected plants may develop variations of systemic vein clearing, chlorotic mottling, and black necrotic ringspots. In certain ornamentals, such as stocks and wallflowers (Cheiranthus Cheiri L.). infection can induce the alteration of anthocyanin pigments of petal tissues creating abnormal sectors, streaks, and flecks (Kruckelmann and Seyffert 1970; Pontis 1973). General symptoms of TuMV infection on various dicotyledonous and diagnostic hosts have been reported (Pound 1948; Berkeley and Weintraub 1952; Tomlinson 1970; Pontis 1973; Fischer and Lockhart 1976; Boswell and Gibbs 1983; Green and Deng 1985). Selected references that describe and/or illustrate TuMV sYmptoms are presented in Table 4.4.

4.

THE TURNIP MOSAIC VIRUS

Table 4.4. crops.

211

Selected references describing and/or illustrating TuMV symptoms on various

Crop

Reference

Brussel sprout (Brassica oleracea var.

Tomlinson and Ward 1981

gemmifera, Zenker.)

Carrrot (Daucus carota 1. var. sativa, DC.) Chicory (Cichorium intybus L.) Collard (Brassica oleracea var. acephala DC.) Horseradish (Armoracia rusticana Gaertn., Mey., & Scherb.) Japanese radish (Raphanus sativus L. var. longipinnatus, Baily) Kohlrabi (Brassica oleracea var. gongylodes L.) Lettuce (Lactuca sativa L.) Pea (Pisum sativum L.) Rape (Brassica napus L.) Rhubarb (Rheum rhabarbarum L.) Safflower (Carthamus tinctorius 1.)

Twardowicz-Jakusz and Zielinska 1979 Provvidenti et a1. 1979 Khan and Demski 1982 Pound 1948 Sano and Kojima 1989 Shukla and Schmelzer 1972 Zink and Duffus 1969 Fischer and Lockhart 1976; Provvidenti 1978 Rao et a1. 1977; Walsh and Tomlinson 1985 Stace-Smith and Jacoli 1967; Walkey et a1. 1981 Klisiewicz 1983

TuMV infection can be broadly grouped into systemic mosaic or systemic necrotic reactions. Tomlinson and Ward (1978) further partitioned each class into three subclasses based on the symptoms produced on inoculated and noninoculated host leaves. The mosaic and necrotic reactions are not mutually exclusive. In certain genotypes, both reactions may occur simultaneously or at different periods during plant development (Tomlinson and Ward 1978; Walsh and Tomlinson 1985). For example, garden balsam (lady's slipper) (Impatiens balsamina L.) plants may simultaneously exhibit systemic mosaic and necrotic symptoms when infected with TuMV (Provvidenti 1982). Also, localized necrotic hypersensitivity lesions are possible on the inoculated leaves of resistant genotypes (Tomlinson and Ward 1978). Systemic mosaic symptoms usually develop rapidly on young or older plants following infection; delayed symptom expression has been noted (Doucett et a1. 1990). Systemic necrotic symptoms may appear at any plant developmental stage. Walkey and Pink (1988) showed that in cabbage the rate of development of necrotic symptoms differed in various host/strain combinations. Plants exhibiting systemic mosaic symptoms may produce seeds, although their yields and size are typically decreased

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V. I. SHATTUCK

in comparison to healthy plants. The mosaic reaction is not typically lethaL but the viral infection may stress plants and allow secondary infection by destructive pathogens. Systemic necrotic reactions in certain Brassica crops, such as rape and rutabaga, result in plant death. Reproduction, although severely impaired, is possible in other crops displaying systemic necrotic symptoms (Zink and Duffus 1969). 1. Cabbage. TuMV infected cabbage and Chinese cabbage develop

systemic vein clearing, chlorotic mottling, mosaics, black necrotic lesions, and complete or broken ringspot symptoms on foliage (Tompkins and Thomas 1938; Walkey 1982; Knight et a1. 1984; Walkey and Pink 1988). Plant foliage may become stunted and distorted and head weight may be reduced. By harvest, cabbage heads may develop external or internal necrotic lesions with the environment contributing greatly in the development of internal symptoms (Pink and Walkey 1990). Symptomless cabbage heads may also develop necrotic ringspots on wrapper leaves or deep within head tissues after 2-5 months of cold storage; this drastically reduces quality. The spots will vary in size and may be larger than 10 mm or smaller than 1 mm. The necrotic ringspots of infected heads should not be confused with pepper-spot necrosis, which appears to be a physiologically induced storage disorder (Zitter and Provvidenti 1984; Walkey and Pink 1988). 2. Rutabaga. Diverse TuMV symptoms are possible on rutabaga,

including systemic symptoms of mottling, mosaic, chlorotic and black spotting, and black necrotic ringspots. The earliest symptoms of the systemic mosaic reaction consist of leaf rugosity (distortion) in the form of a backward curvature of the leaf blade and downward tip roll of the youngest leaves, which may occur prior to leaf discoloration. A general stunting or death of infected plants occurs, but depends largely on the genotype/strain association and the stage of plant development at the time of infection. Plants infected during early stages of development showing severe systemic mosaic reactions will typically develop stunted, torpedo shaped roots with extended necks (Le., goosenecks) (Stobbs et al. 1991). Systemic necrotic reactions in rutabaga leads to plant death. Necrotic spotting of the foliage may also be accompanied by vascular discoloration of storage root tissue. The necrotic spotting appears in the parenchyma cells in the secondary xylem region of the root, are similar in appearance to black rot symptoms caused by Xanthomonas campestris pv. campestris (pammel) Dowson, and are probably the result of the accumulation of polyphenolic compounds. Microscopic evaluation of infected root tissues has revealed abnormal callus formation and tracheid differentiation in and around these discoloured areas (R. L. Peterson,

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THE TURNIP MOSAIC VIRUS

213

personal communication). In advanced stages the spots may enlarge and coalesce, and the root decomposes. Pound (1948) similarly reported the occurrence of necrotic flecking in TuMV infected horseradish roots. 3. Factors influencing symptomology. Differences in virulence for TuMV isolates exist, which effect the extent and severity of host symptoms (Tomlinson and Ward 1978; Provvidenti 1982; Stobbs and Shattuck 1989). Early studies revealed that air temperature influenced TuMV disease expression, and that the optimal temperature for expression varied for different host and strain combinations. Pound and Walker (1945a,b) noted that mottle symptoms of cabbage plants infected with TuMV were more pronounced and virus titre was higher in plants grown at 28°C than those grown at 16°C. When Nicotiana glutinosa was infected with TuMV, overall symptom expression was greater at 16° than at 28°C, and disease symptoms varied with temperature. In another study, TuMV symptoms and virus titre were more severe on horseradish plants grown at 16°C than at 28°C; at 28°C symptoms were essentially absent (Pound 1948). Later studies by Chiu et a1. (1957) revealed that when air temperature was held constant, variations in soil temperature influenced TuMV symptoms of Chinese cabbage plants. Necrotic black ringspot symptoms may be completely suppressed or occur less frequently when infected cruciferous plants are grown at higher (24-28°C) than lower (1B-20°C) mean temperatures (Pound and Walker 1945a; Pound 1948). TuMV symptomology response to temperature is not restricted to cruciferous hosts, because temperature effects have been noted for pea (Pisum sativum 1.) (Provvidenti 1978), a legume, as well as the solanaceous host (N. glutinosa) previously mentioned. When close associations exist among the air temperature, virus multiplication rate, and host symptomology, the masking of symptoms from air temperature is a possibility. Pound and Walker (1945a) first d~scribed this phenomenon, which occurs when the air temperature deviates from an optimum for virus activity and host symptomology in a host/strain association, resulting in the visual reduction or loss of infection symptoms in new plant growth. This masking effect may partly explain the instability of TuMV symptoms reported for cabbage byWalkey (1982) and for Japanese radish by Sano and Kojima (1989). Optimal temperatures for TuMV symptomology and infectivity may not necessarily coincide. In endive (Cichorium endivia L.), escarole and chicory (Cichorium intybus L.) optimal temperatures for TuMV infectivity and symptom expression are 30°C and 15°C, respectively (Provvidenti et a1. 1979). It is possible that in certain host/strain associations, the developmental stage of the host could interact with temperature to influence

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V. I. SHATTUCK

symptomology. Liu and Liu (1986) showed that TuMV symptoms in Nicotiana glutinosa are dependent on seedling age and environmental temperature. Walker (1952) reported that TuMV titre was greater in mature rape leaves at 16°C than at higher temperatures. whereas in young leaves the virus titre was greater at 28° than at 16°C; symptomology was not indicated. The severity of TuMV induced symptoms is usually greatest in hosts simultaneously infected with other plant viruses (Pound 1945a.b; Walkey et aI. 1976; Ford et aI. 1988; Sano and Kojima 1989). although exceptions have been noted (Khan and Demski 1982). Although early research showed that day length did not influence TuMV concentration in cabbage (Pound and Walker 1945b). other work showed that virus multiplication in rape plants was favoured by long photoperiods (Pound and GarcesOrejuela 1959). Similiarities in TuMV symptomology between greenhouse and fieldgrown hosts have been observed (Tomlinson and Ward 1981). but in other studies. distinct differences have been noted (Khan and Demski 1982; Pink et aI. 1986; Pink and Walkey 1988). Thus. greenhouse rating schemes used for predicting TuMV reactions of field-grown plants should be approached with caution.

B. Metabolism 1. Productivity. Virus induced disturbances in carbohydrate metabolism

and photosynthesis rates are well known (Matthews 1991). Besides reducing the overall chlorophyll content of leaves, TuMV infection may also impair the photosynthetic efficiency of chloroplasts. Aggregations of chlorotic chloroplasts containing few starch grains has been recorded in the local lesions of Chenopodium quinoa Willd. (Kitajima and Costa 1973). Yield and seed losses are common for TuMV infected cabbage, cauliflower. mustard. and turnip (Shukla and Schmelzer 1972). In host reactions involving systemic mosaic. plants infected early in development generally suffer greater losses in productivity than plants infected at a later stage. Once plants achieve near-maximum growth, the effects of TuMV infection on productivity may be negligible. For example, Natti (1956) observed a reduction in cabbage yield from 50 to 75% when plants were infected during early development, but yields were virtually unaffected for plants infected during late development. In rutabaga plants, a linear relationship was observed between time of plant infection and dry matter accumulation in the storage root. Starting with 6week-old susceptible 'Laurentian' plants. each weekly delay in infection

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THE TURNIP MOSAIC VIRUS

215

with TuMV-C3 results in an average increase in root dry weight of 7% at harvest (V. 1. Shattuck, unpublished data). In certain host/strain associations, TuMV infection may result in the reduction in the quality of the plant product. In cabbage, rutabaga, and horseradish the appearance of black necrotic lesions on or within plant products at harvest or arising during storage may result in inferior produce. TuMV-infected cabbage heads with external necrosis are more prone to infection by Botrytis cinerea ssp. than noninfected heads (Pink and Walkey 1990). 2. Mineral nutrition and glucosinolates. Viral infections can alter the elemental content of plants (Huber 1978; Kaplan and Bergman 1985).

TuMV infection can induce altered mineral content of rutabaga tissues, with the severity dependent on the growth stage at the time of infection (Shattuck 1987; Shattuck et a1. 1989). Nitrogen, Mg, and Zn are typically enhanced in both leaf and storage root tissues, presumably at nontoxic concentrations for plant growth (Shattuck 1987; V. 1. Shattuck, unpublished data). It is not clear whether this altered nutrient composition reflects a less efficient uptake, translocation, and/or utilization of minerals in virus infected plants. A few investigations have focused on the effects of TuMV infection on the glucosinolates in cruciferous crops. When the glucosinolate sinigrin, a natural toxic insect feeding deterrent, was added to an aqueous plant extract containing TuMV, infectivity was reduced (Spak 1988). Despite this finding, limited research has failed to establish a relationship between the concentration of glucosinolate in cruciferous plants and TuMV resistance (Spak et a1. 1987). Recent work reveals that TuMV infection can alter the glucosinolate concentration of rutabaga roots (Stobbs et a1. 1991). Preliminary studies with the leafy vegetables choy sum (Brassica parachinensis Bailey) and pak-choy (B. chinensis L.) also show that the concentration of glucosinolates in leaves are altered following TuMV infection (V. 1. Shattuck, unpublished data). In view of the quality and health implications of glucosinolates in both fresh and processed foodstuffs (Fenwick et a1. 1983), additional research into this area seems warranted.

VII. DETECTION Although visual inspection for disease symptoms by the experienced eye is the simplest and quickest way to diagnose TuMV infection, at times more precise quantitative methods are desirable. Typically, a combination of several procedures such as host range evaluation, reaction on indi-

216

V. I. SHArrUCK

cator plants, electron microscopy, the presence of inclusion bodies within host cells, transmission studies, and various serological procedures (Agrios 1978; Martin 1985J have been used to confirm the presence of TuMV infection. In routine analyses the use of host indicator plants and enzyme-linked immunosorbent assays (ELISA) testing are sufficient. A. Indicator Plants Indicator or test plants develop characteristic symptoms when inoculated with specific viruses and have proven reliable in the detection of virus-induced diseases. Indicator plants are also useful in the measurement of virus dilution end points and for single lesion virus transfers. Chenopodium quinoa Willd., tobacco (Nicotiana tabacum L.), and Chenopodium amaranticolarCoste and Reyn. are commonly used as indicator plants for TuMV. We routinely use C.. quinoa as an indicator plant, provided that the plants are free of the chenopodium latent virus (sow bane mosaic virusJ. When present in C. quinoa plants, this virus can interfere with the TuMV bioassay (L. W. Stobbs, personal communication). Leaves of C. quinoa plants at the 4- to 6-leaf stage are manually inoculated with tissue extracts from suspected TuMV-infected plants. If the tissue extract contains the virus, chlorotic local lesions and/or limited systemic mottling becomes visually apparent on inoculated leaves of plants after 5-7 days at ZD-Z5°C. Chenopodium quinoa can be used to differentiate pure isolates of TuMV from cauliflower mosaic virus (CaMV), since the latter virus will not induce a visual reaction on this indicator plant. However, it should be noted that TuMV and CaMV are easily distinguished by light or electron microscopy in a much shorter time than required on diagnostic plants (Christie and Edwardson 1977). The use of indicator plants has been discussed by Hollings (1966) and a standardized bioassay for TuMV using C. amaranticolor has been described by Chenulu and Thornberry (1964).

B. Serological Methods Serological techniques play an important role in detecting viruses in infected tissues and identifying virus isolates. The serological relationships among potyviruses (Hollings and Brunt 1981; Shukla and Ward 1989) and between potyviruses and other viruses (Van Regenmortel1978) have been discussed. Differences in the antigenic reactions gf the capsid proteins of TuMV strains may have diagnostic potential (McDonald and Hiebert 1975). Rapid electron microscope serology decoration tests have been used to aid in the identification of TuMV and to study the serological relationships of various potyviruses (Walkey and Webb 1984).

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THE TURNIP MOSAIC VIRUS

217

Two common serological techniques are gel immunodiffusion tests and ELISA. These procedures are based on the affinity between the virus and appropriate antibody. The sensitivity of several other serological methods for detecting TuMV is discussed by Choi et al. (1978). The ELISA assay involving the double antibody sandwich (Clark and Adams 1977), can utilize either monoclonal or polyclonal antisera. This technique is a rapid and sensitive diagnostic tool for detecting TuMV (Martin 1985). ELISA is useful in detecting TuMV when plant symptoms are not distinctive enough for diagnosis and is particularly suited for assaying plants on a large scale. In addition, ELISA can be used to estimate virus titre in infected tissues, which can be very useful when classifying plants as resistant or tolerant to TuMV. However, certain factors can greatly reduce its effectiveness. The sensitivity of ELISA may be lowered in tissues that have been frozen prior to analysis. Horwitz et al. (1985) noted that sap samples from the mature leaves of TuMVinfected horseradish plants displaying systemic necrosis were not always positive when analyzed by ELISA, and attributed this to low virus concentration or uneven distribution of virus in these tissues. In addition, these researchers reported that virus titre, which fluctuated with air temperature and plant part sampled, could drop below the level of detection of this assay. A broad spectrum antisera capable of detecting a large number of potyviruses, including TuMV, is now sold in the United States in a diagnostic kit. However, this antisera is not specific for TuMV. These kits, although useful in locations where laboratory services are not readily available, should be used only by qualified personnel. TuMV antisera and six TuMV isolates are available to interested researchers from the American Type Culture Collection (American Type Culture Collection 1990).

VIII. EPIDEMIOLOGY The constant threat of TuMV infection prevails in areas where weather conditions are favorable and host plants, virus inoculum sources, and aphids are present throughout most of the year. In Taiwan, where intensive and continuous multicropping is practiced, TuMV is abundant and the potential for infection is high. In temperate areas, the amount of infection through the spring and fall months may vary from year to year, with biennial or perennial hosts serving as green bridges between seasons. TuMV may be widespread in certain areas, but may go virtually unnoticed due to a low infection frequency (Demski 1973; Rao et al. 1977). Unfortunately, in these instances, the potential may exist for

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V. I. SHATTUCK

TuMV outbreaks that can result in devastating economical consequences. There have been few studies on the epidemiology of TuMV in North America (Laird and Dickson 1972; Evertsen 1974; Lowery 1987). From these limited studies it appears that the location and abundance of TuMV inoculum sources and the population and activity of aphids are the main factors contributing to the epidemiology of TuMV. A. Inoculum Sources

A primary TuMV infection source in production fields is infected transplants. Transplants become infected during plant development in the greenhouse or nursery beds, because TuMV is not transmitted in the seed (Zink and Duffus 1969; Stobbs and Van Schagen 1987; Ford et al. 1988). Where continuous monoculture is practiced, and overlapping of planting dates occurs, a crop once infected, can serve to perpetuate this virus. Volunteer cultivated plants, arising from the regrowth of virus-infected storage roots or bulbs, can also be important inoculum and overwintering sources for the virus. Often overlooked in small settings is that infected annual or perennial ornamentals, such as stocks, wallflowers, Zinnia ssp., and Petunia ssp., may serve as important virus reservoirs. TuMV has a wide weed host range (Table 4.5). Infected weeds growing adjacent to production fields or between rows of plantings can serve as inoculum sources for the virus (Larson and Walker 1938; Arnold and Bald 1960; Provvidenti and Schroeder 1972; Horvath and Besada 1975; Provvidenti 1978; Provvidenti 1979; Polak and Chlumska 1983; Sherf and MacNab 1986; Stobbs and Van Schagen 1987). The weed, Cerastium glomera tum Thuill., is an important source of TuMV for Japanese radish fields (Sakai and Kono 1978). Certain weeds, such as chickweed, pennycress, and shepherd's purse, may serve as natural overwintering hosts for TuMV (Larson and Walker 1938; Citir and Varney 1974a; Sakai and Kono 1978). Infected weed hosts may possess mild and hardly perceptible TuMV symptoms (Sakai and Kono 1978) and in natural settings the virus may be more prevalent in certain weeds than others (Polak and Chlumska 1983). Recently Stobbs and Stirling (1990) examined 169 weed species in southern Ontario and found 19 susceptible to TuMV-C 3 ; in no instance was the virus seed transmitted. Changes in agricultural practices can have a substantial effect on the epidemiology of TuMV (Tomlinson 1987). In England and Ontario, Canada. the rapid expansion of oilseed rape and canola (Brassica Napus L..) both of which can serve as reservoirs for the virus, presented a production threat to other susceptible hosts grown in close proximity (Walsh and Tomlinson 1985; Lowery 1987).

4.

THE TURNIP MOSAIC VIRUS

Table 4.5. Family

219

Some weed hosts of TuMV. Species

AMARANTHACEAE Gomphrena globosa L. BALSAMINACEAE Impatiens parviflora DC. BORAGINACEAE Symphytum tuberosum L. CARYOPHYLLACEAE Arenaria serphyllifolia L. Cerastium glomeratum Thuill. Spergula arvensis L. Steliaria media (L.) ViII. CHENOPODIACEAE Chenopodium album L. C. murale L. COMPOSITAE Senecio vulgaris L. Sonchus apser (L.) Hill CRUCIFEREAE Alliaria petiolata (M. Bieb) (Cavara and Grande) Brassica campestris L. B. geniculata L. B. Napus L. B. nigra (L.) Koch Cardamine flexuosa With C. scutata Thunb. Cardaria draba (L.) Desv. Capsella bursa-pastoris (L.) Medicus Diplotaxis tenuifoIia (L.) DC. Erucastum gallicum (WiIld.) O. E. Schulz Erysimum cheiranthoides L. Hesperis matronaIis L. Lunaria annua L. Nasturtium officinale R.Br. Sinapis arvensis L. Sisymbrium irio L. Thlaspi arvense L. LABIATAE Lamium album L. LEGUMINOSAE Trifolium hybridum L. MARTYNIACEAE Proboscidea jussieui Keller

Common name(s)

Globe amaranth Touch me not; jewel-weed Tuberous comfrey Thyme-leaved sandwort Corn spurry Chickweed Lamb's quarter

Common groundsel Spiny annual sow-thistle Garlic mustard Bird rape and wild yellow mustard Short-pod mustard Rape Black mustard

Heart-podded hoary cress Shepherd's purse Narrow-leaved wall rocket Dog mustard Wormseed mustard Dame's violet; dame's rocket Honesty; money-plant Water-cress Wild mustard; charlock Stinkweed; pennycress White dead nettle

Alsike clover Unicorn-plant; martynia continued

V. I. SHATTUCK

220

Table 4.5.

Continued.

Family

Species

PAPAVERACEAE Chelidonium majus L. Papaver nudicaule L. P. orientale L. P. rhoeas L. P. somniferum L. PHYTOLACCACEAE Phytolacca americana L. SCROPHULARIACEAE Scrophularia nodosa L. VALERIANACEAE Valerianella olitoria Poll.

Common name(s)

Greater celandine Iceland poppy Oriental poppy Corn poppy; flander's poppy Opium poppy Pokeweed Figwort Corn-salad; lamb's lettuce

B. Aphids It is known that TuMV is spread primarily by aphids. However, it is possible that flea beetles (Phyllotreta spp.) (Carden and Gladders 1984) and various thrip species such as the cabbage thrip (Thrips angusticeps Uzel) (Tompkins 1939a) can also disseminate this virus. Eighty-nine species of aphids have been identified as vectors (Edwardson and Christie 1986). Depending on location, a few aphid species may dominate in the spread of the virus. For example, in Ontario, the corn leaf aphid (Rhopalosiphum maidis Fitch) and green peach aphid (Myzus persicae Sulzer) are the main vectors of TuMV (Lowery 1988). TuMV is transmitted in a nonpersistent (stylet-borneJ manner; there is no evidence to indicate a latent period between the acquisition and transmission of the virus by aphids. Virus retention is important in the dispersal of virus in the field. Aphids typically remain infective for a short period after acquiring TuMV. Green peach aphids retained TuMV for 3-5 h, although aphids lost infectivity rapidly when feeding on healthy plants (Sylvester 1954; Nishi 1969). At room temperature, the cowpea or groundnut aphid (Aphis craccivora Koch) remained infectious for 14-16 h following shortterm feeding (Evertsen 1974J. Interestingly, Hienze (1959) reported that aphids remained viruliferous with TuMV for 6 days when kept at -1°C. It was postulated that the nonpersistence of TuMV in aphids was due to an inhibitory substance present in aphid saliva (Nishi 1969). The probability of aphid transmission of TuMV with single aphids is high (Sylvester 1954). However, aphids are not equally effective in transmitting TuMV, and transmission efficiency depends on interactions between the host plant and aphid species (Sylvester and Simons 1951; Zink and Duffus 1969). Simultaneous aphid transmission of TuMV with

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THE TURNIP MOSAIC VIRUS

221

other viruses also depends on the aphid species (Fujisawa 1985). Research by Sako (1980) showed that a host component in the soluble fraction of infected leaves might be involved in the aphid transmission of this virus. Weather conditions and temperature influence aphid activity and migration patterns, which in turn affect the dissemination of TuMV. In Ontario, a mild winter followed by a dry and warm' spring usually facilitates a grea ter number and activity of aphids in early summer, which is usually associated with the early spread of the TuMV-C3 virus strain. Cool, moist, and windy summers usually impede the movement of aphids, which reduces and delays the overall spread ofTuMV. Similarly, Polak and Chlumska (1983) showed in their survey in Czechoslovakia that the greatest occurrence of TuMV infection was in areas with the highest average temperature and lowest average rainfall. The concentration and distribution of this virus within infected plants and its relationship to aphid feeding influences the aphids' efficiency in spreading TuMV (Broadbent 1954). It should also be noted that the developmental stage of the host may influence aphid transmission of this virus (Lowery 1987).

Once the primary infection sites are established in the field, TuMV may spread relatively quickly from plant to plant if aphids are not controlled. However, in some instances the natural spread of TuMV may be surprisingly low (Evertsen 1974). Thus, infection may be sparse or evenly distributed throughout fields. IX. CONTROL METHODS

A. Cultural Sanitation practices in and around production fields, such as the removal of TuMV-infected plant debris and the eradication of infected plants can help to reduce virus inoculum and spread of the virus. The scheduling of planting dates to avoid peak aphid migration periods and allowing susceptible plants to attain as much development as possible before they become infected with TuMV have been practiced when no other options are available. In China, an integrated approach involving tolerant cultivars, the application of insecticides, and delayed planting dates are used to reduce the effects of TuMV infection on crop productivity (Chiu and Chang 1982). In Ontario, the late rutabaga crop is usually planted prior to mid-June, and production practices are optimized in order to promote the rapid development of plants before the peak aphid activity in July and August. The effectiveness of the latter approach depends on keeping the susceptible plants free of TuMV infection during the critical early stages of development when the disease can have the greatest

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V. 1. SHATTUCK

impact on the plant. At later stages of rutabaga growth, the effects of infection are less pronounced and a crop of marketable size is possible. Although crop isolation was recommended as a control method for TuMV infection years ago (Chamberlain 1936; Walker 1953), it still remains as a viable option in certain situations. The isolation of susceptible transplants in nursery beds from TuMV infection sources is practiced to ensure the production of virus-free seedlings. For statice, it has been recommended that the complete removal of infectious volunteer plants along with the dispersal and isolation of field plantings will greatly reduce TuMV infection, even in years when the aphid populations are high (Laird and Dickson 1972). Although aphids can spread viral diseases over long distances, critical isolation distances are usually difficult to determine because they depend on a number of interacting factors, including the abundance and characteristics of the inoculum source, aphid population, virus retention time in the aphid, aphid activity, wind direction, and alternative aphid food sources between the isolated fields. For TuMV, an isolation distance of 3 km may not be sufficient to ensure adequate protection (Laird and Dickson 1972). The use of whitewash sprays on plants to repel infectious aphids has met with some research success in Ontario. It has been reported that whitewash sprays on field-grown rutabaga plants reduced TuMV infection from 42% to 69% over control plants (Lowery et a1. 1990). B. Chemical Chemical sprays will not protect plants against TuMV infection. Infectious aphids can migrate into a production field and transmit the virus before the insecticides take effect. However, effective aphid control through the proper use of aphicides is desirable. An overall reduction in the aphid population within a given area may help to lower the secondary spread of TuMV. Oil sprays have been used on many crops with varying effectiveness to protect plants against aphid-transmitted viruses (Bradley et a1. 1966; Mowat and Woodford 1976; Simons 1982). The inhibitory effect of the oil appears to work at the virus-vector level (Vanderveken 1977) and the mode of action is discussed by Loebenstein and Raccah (1980). In greenhouse experiments, Walkeyand Dance (1979) showed that a 1% mineral oil spray reduced the transmission of TuMV to mustard seedlings by the green peach aphid and cabbage aphid (Brevicoryne brassicae L.), and was nonphytotoxic. However, in field experiments, weekly sprays of 1% mineral oil failed to protect cabbage plants from viruliferous aphids and appeared to be phytotoxic. Experiences in Ontario have shown that weekly applications of 1-2% 70 Superior oil to rutabaga production fields

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reduces, but does not eliminate, TuMV infection. Lowery et a1. (1990) observed in field studies that weekly application of cypermethrin (Cymbush) with 1% mineral oil reduced TuMV infection of rutabaga from 92 to 37%. The success of oil applications is highly dependent on the timing of oil sprays relative to aphid activity and proper spray coverage. Also, oil sprays appear to be more effective when aphid infection pressure is low. The estimated expense of six field applications of oil sprays to a hectare of cole crops in Ontario, ranges from $55 to $93 for 1% concentration and $68 to $106 (Can.) for 2% concentration. Thus, the cost of this control method must be carefully weighed against the value of the crop being protected.

c.

Preventative and Therapeutic

Plant extracts are known to contain certain inhibitory substances that can interfere with virus infection and replication (Hirai 1977). Tamura (1969) noted that the leaf, cone, and bark juice of Japanese black pine (Pinus thunbergii (Pari.) when sprayed on radish and Chenopodium amaranticolor plants could suppress TuMV infection. Pandey and Mohan (1986) believed that plant extracts from bottle-brush (Callistemon lanceolatus DC.), Acacia arabica, and jambolan-plum (Syzygium cuminii Skeels) contained a TuMV resistance-inducing substance. When the extracts from these plants were applied to Chenopodium amaranticolor and C. album plants, followed by a lapse of time and a TuMV challenge inoculation, a significant reduction in TuMV-induced lesion number resulted. The application of various metabolites from Actinomyces before or shortly after TuMV infection has been reported to protect plants against infection, and provides a therapeutic effect (Chiu and Chang 1982). A polysaccharide isolated from the mycelium of Phytophthora infestans (Mont.) de Bary was also shown to have an inhibitory effect on TuMV infection, and could lessen host symptoms when used at a concentration of 1000-ppm or above (Singh et a1. 1970). Heat treatments with water or air have been used to rid plants of virus infection. Holmes (1965) showed that TuMV could be eliminated from horseradish root by placing tissues in a 40-ppm solution of malachitegreen dye at 37°C for 3 weeks. Meristem tissue techniques have been used to provide TuMV-free plants of cauliflower, horseradish, rhubarb (Rheum rhaponticum L.), and watercress (Nasturtium officinale R. Br.) (Walkey 1980). A 1% CO 2 level administered during a 8 or 16 hour photoperiod inhibited TuMV-induced local lesion production in N. tabacum and N. glutinosa (Purohit et a1. 1975).

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D. Immunity and Resistance The use of immune or highly resistant cultivars remains the cheapest and most effective way of protecting plants against losses in productivity from TuMV infection. Unfortunately, cultivars that are immune or resistant to TuMV may not be identified, or are unavailable when required (Horwitz et al. 1985). Immunity or resistance to TuMV is usually preferred over tolerance because tolerant plants can still serve as reservoirs for infection for other crops. There are indications that mature plant resistance (Matthews 1991) may occur for TuMV (Lowery 1987), but the mechanism for this is unclear. 1. Sources. Immunity, resistance, and tolerance to TuMV exist in host plants (Zink and Duffus 1969; Chen 1980; Shattuck and Stobbs 1987).

These reactions depend on the host/strain relationship and may be expressed at different stages of plant development (Tomlinson and Ward 1978; Doucet et al. 1990). The virus fails to replicate in immune plants, whereas in susceptible plants the virus titre is high and plant productivity is reduced. Typically plants in which TuMV infection is confined only to the innoculated leaves (Le., nonsystemic) are classified as resistant. However, hosts that allowed low levels of virus multiplication (Pound et al. 1965) or delayed or limited systemic movement of the virus (Doucet et al. 1990) have also been classified as resistant. In tolerant reactions, hosts contain relatively high amounts of virus and slight to moderate visual symptoms are usually displayed. Such hosts often suffer little loss in productivity. An extreme case of tolerance was described in horseradish where plants display marked infection symptoms but did not appear weakened (Horwitz et al. 1985). Sources of TuMV immunity, resistance, and tolerance in various crops are shown in Table 4.6. TuMV immunity and resistance sources in other dicotyledonous species have been presented (Fischer and Lockhart 1976; Provvidenti 1978; Green and Deng 1985). Multiple resistance to TuMV-C 1 , C2 , C3 , and C4 was first recorded in Chinese cabbage line PI 418957 by Provvidenti (1980). Immunity to all five TuMV strains (TuMV-C1 , C2 , C3 , C4 , and Cs ) in Taiwan was noted in several Chinese cabbage lines (Green and Deng 1985; Asian Vegetable Research and Development Center 1987). 2. Gene Action. In several studies, maternal effects have not been

observed for TuMV immunity or resistance, which suggests that only nuclear genes are involved (Pink et al. 1986; Shattuck and Stobbs 1987; Souza-Machado et al. 1988). However, Wei et al (1991) recently reported that the cytoplasms of Chinese cabbage influenced TuMV resistance. It appears that major genes determine TuMV immunity and resistance in

4.

THE TURNIP MOSAIC VIRUS

Table 4.6. of TuMV.

Sources of plant immunity. resistance. and tolerance to various strains/isolates

TuMV strain/ isolate TuMV-Ct

TuMV-Cz

TuMV-bJ

Crop

Reference

Country

Chinese cabbage Cabbage Cauliflower Chinese cabbage Chinese cabbage

Provvidenti 1980 Green and Deng 1985

United States Taiwan

Asian Vegetable Research and Development Center 1987 Provvidenti 1980 Green and Deng 1985

Taiwan

Chinese cabbage Cabbage Cauliflower Chinese cabbage Chinese cabbage

Garden balsam Chinese cabbage Cabbage Cauliflower Chinese cabbage Chinese cabbage

Rutabaga

TuMV-c:.

TuMV-Cs

ZZ5

Canola Rutabaga Turnip Chinese cabbage Cabbage Cauliflower Chinese cabbage Chinese cabbage

Cauliflower Chinese cabbage Chinese cabbage

Asian Vegetable Research and Development Center 1987 Provvidenti 198Z Provvidenti 1980 Green and Deng 1985

United States Taiwan

Taiwan

United States United States Taiwan

Asian Vegetable Research and Development Center 1987 Souza-Machado et a1. 1988 Stobbs et aI. 1989 Doucet et aI. 1990 Shattuck 199Z Provvidenti 1980 Green and Deng 1985

Taiwan

Asian Vegetable Research and Development Center 1987 Green and Deng 1985

Taiwan

Asian Vegetable Research and Development Center 1987

Taiwan

Canada Canada Canada Canada United States Taiwan

Taiwan

zImmune. resistant. or tolerant host reactions evaluated following natural field infection or manual innoculation. continued

V. I. SHAITUCK

226

Table 4.6.

Continued.

ThMV strain/ isolate

Crop

Reference

Country

ThMV-Ce

Numerous families

Canada

A, B, C. D.

Cabbage, Chinese cabbage. and Japanese radish Chinese cabbage

Stobbs and Van Schagen 1987 Fujisawa 1990

Japan

Niu et a1. 1983

United States

Provvidenti et a1. 1979 Walsh and Tomlinson 1985 Tomlinson and Ward 1978; Tomlinson and Ward 1982 Walkey 1982; Knight et a1. 1984; Walkey and Pink 1988 Walsh 1988; Walsh 1989

United States

E. F. G. H,

and I PHW 645 Chinese cabbage isolate from China NY 77-51 and Chicory and Chicory X Endive NJ-Esc-8 Oilseed rape UK1 UK1 and various rutabaga isolates

Rutabaga

UK2, DK1, GK1. and (FRD1 or FRD2)

Cabbage

UK1. UK2, UK3, FRD1, FRD2. GK1, and DK1 UK2

Oilseed rape and Rutabaga

UK2 and Brussel sprout isolates

Brussel sprouts

Cauliflower

Pink and Walkey 1988 Tomlinson and Ward 1981; Pink et ai. 1986

Unclassified isolates/strains Z Annual stock Johnson and Barnhart 1956 San Juan and Pound 1963 Cabbage, Cauliflower, Pound et a1. 1965 and Radish Shukla and Schmelzer 1972 Chinese cabbage Lim et a1. 1978 Rutabaga and Thrnips Chamberlain 1948 Palmer 1983 Lammerink and Hart 1985 Shattuck and Stobbs 1987 Doucet et ai. 1990

England England England England England England England England England England England

United States United States United States Germany Taiwan New Zealand New Zealand New Zealand Canada Canada

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THE TURNIP MOSAIC VIRUS

227

plants. Tomlinson and Ward (1978, 1982) found discrete TuMV symptom classes that could be quickly fixed through inbreeding, which supports the view that major genes are probably involved. Furthermore, gene action may differ depending on the host/strain relationship (Table 4.7), that is, host reactions may be strain specific (Provvidenti 1980; Stobbs and Shattuck 1989; Walsh 1989). Although a few genes appear to determine immunity or resistance, a nonspecific multigenic TuMV resistance response was reported in tobacco (Troutman and Fulton 1958). Tomlinson and Ward (1978) speculated that the mosaic and necrotic reactions in rutabaga were controlled by different genes. We have noted in diverse rutabaga germplasm that these reactions are separately inherited, but plants possessing both reactions may exist. It is possible that minor genes, the genetic background of the host, and genotype X environment interaction effects may also contribute to the Table 4.7.

Crop Cabbage

Stocks Lettuce Chinese cabbage Brussel sprout Rutabaga Chinese cabbage

Oilseed rape Chinese cabbage

Inheritance of TuMV immunity or resistance in various crops. Strain/isolate used and origin

Number of genes, gene action, and gene symbol

Reference

Co-infection with TuMVand cauliflower mosaic virus isolates; United States Field TuMV infection; United States turnip isolate(s); United States PHW 645 Chinese cabbage isolate; China Dutch white cabbage isolate (UK2); England Rutabaga isolate; Canada TuMV-C 4 and TuMVCs ; possible Taiwan

Polygenic, incomplete dominance

Pound and Walker 1951

One recessive gene, rm

Johnson and Barnhart 1956 Zink and Duffus 1973 Niu et a1. 1983

UK1 isolate; England Liaoning 1; China

One dominant gene, Th Two dominant genes

Four genes, additive and non-additive effects important One dominant gene, Thm Two recessive genes

One dominant gene More than five genes, incomplete recessive, additive and non-additive effects important

Pink et a1. 1986

Shattuck and Stobb 1987 Asian Vegetable Research and Development Center 1989 Walsh 1989 Wei et a1. 1991

228

V. I. SHATTUCK

overall resistance response. These factors can explain the variability in resistance/tolerance expressions occasionally encountered (Tomlinson and Ward 1982; Knight et a1. 1984; Walkey and Pink 1988). The genes for TuMV and downey mildew (Bremia lactucae Regel) reactions in lettuce are linked 12.0 ± 2.9 centimorgans apart (Zink and Duffus 1973). It appears that the TuMV immunity response has no pleiotropic effects on CaMV or powdery mildew sensitivity in rape and rutabaga (Tomlinson and Ward 1982; Walsh 1989). Thus, selecting for TuMV immunity in these crops is not expected to simultaneously alter the expression of plants to these diseases. The mechanism of TuMV immunity and resistance is poorly understood. Sherwood (1985) showed that pathogenesis related proteins in Nicotiana sylvestris are not associated with TuMV resistance, but are the result of induced necrosis. Chinese cabbage cultivars with dark green color and relatively higher tannin content have been observed to be less prone to TuMV infection than plants lacking these features (Chen 1980). Temperature influences the stability of the TuMV immunity and resistant responses in plants. At elevated temperatures, immunity in rape and resistance in cabbage have been shown to break down (Pound 1952; Walsh 1989). 3. Breeding. The long establishment of TuMV in certain parts of the world, along with experiences of epidemics in these areas gave impetus to the identifying and/or breeding of TuMV-immune and TuMV-resistant cultivars. Breeding progress was made in early North American programs for cabbage (Pound and Walker1951; Poundetal. 1965)and annual stock (Johnson and Barnhart 1956). In China, the current 5-year national agriculture research program emphasizes breeding improvements for TuMV resistance in cabbage, Chinese cabbage, and pak-choy (S. Hao, personal communication). Screening and breeding procedures for incorporating TuMV immunity and resistance into plants are discussed elsewhere and therefore, will not be presented in this review (Pound et a1. 1965; Walker and Williams 1973; Walkey 1982; Crucifer Genetics Cooperative Resource Book, 1985; Walsh and Tomlinson 1985; Pink and Walkey 1988, 1990.

X. CONCLUSION

TuMV epidemics will continue to occur in areas where the widespread cultivation of susceptible hosts is practiced and when optimal conditions for virus buildup and dissemination coincide. When immune or resistant cultivars are not available, growers should be encouraged to initiate integrated control measures, such as proper sanitation practices, reduction of aphid populations, and eradication of inoculum sources, to minimize the

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229

conditions favorable for TuMV epidemics. To achieve this, reliable TuMV detection methods and local knowledge of aphid activity and TuMV hosts are required, along with grower collaboration. Where immune or resistant cultivars are used, the possibility of mutant TuMV strains that can overcome this protection should be of concern and closely monitored. Research activities on TuMV will continue to be important in Asia and Great Britain where ThMV is endemic to crop production and grower awareness and concern for this disease prevails. In other areas, TuMV research and plant breeding objectives will be prioritized only when the benefits outweigh the cost of such programs. However, the recent experiences involving the geminiviruses, squash leaf curl virus, and lettuce infections yellow virus on horticultural crops in the United States should forewarn growers, scientists, and administrators of the anticipated expense and duration of programs required to eradicate diseases once they become economically important and intolerable issues. Long-term control strategies should be initiated for potentially devastating pathogens, such as TuMV, prior to outbreaks; the potential returns from this action can be enormous.

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Lee, S. H., K. W. Lee, and B. J. Chung. 1978. Investigations on the virus diseases in spinach (Spinacia oleracea L.). I. Identification of turnip mosaic virus occuring spinach. Kor. J. Plant Prot. 17:33-35. Lesemann, D. E., and H. J. Vetten. 1985. The occurrence of tobacco rattle and turnip mosaic viruses in orchis ssp., and of an unidentified potyvirus in Cypripedium calceolus. p.45-54. In: R. K. Horst (ed.) Acta Hort. 164, Int. Symp. Virus Dis. Ornam. Plants, Ithaca, NY. Lim, W. L., S. H. Wang, and O. C. Ng. 1978. Resistance in Chinese cabbage to turnip mosaic virus. Plant Dis. Rptr. 62:660-662. Ling, L., and J. Y. Yang. 1940. A mosaic disease of rape and other cultivated crucifers in China. Phytopathology 30:338-342. Liu, Y., and X. Liu. 1986. The seedling age and temperature in relation to the symptom expression of TuMV inoculated on Nicotiana glutinosa. Acta PhytopathoL Siniea 16:175-178. Liu, X., W. Lu, and B. Lin. 1990. A study of TuMV strain differentiation on cruciferous vegetables from ten regions of China. I. Identification results with Green's methods. Virologica Siniea 5:82-87. Loebenstein, G., and B. Raccah. 1980. Control of non-persistently transmitted aphid-borne viruses. Phytoparasitica 8:221-235. Lowery, D. T., M. K. Sears, and C. S. Harmer. 1990. Control of turnip mosaic virus of rutabaga with applications of oil, whitewash and insecticides. J. Econ. Entomol. 83:2352-2356. Lowery, T. 1987. Summary of the 1986 and 1987 field monitoring and research for the turnip mosaic virus programs. Rpt. Ont. Min. Agr. Food, Guelph Agr. Centre, Guelph, Ontario, Canada. _ _ . 1988. Turnip mosaic virus (TuMV) of rutabaga. Ont. Min. Agr. Food Factsheet 88091. Ontario, Canada. Mamula, D., and N. Ljubesic. 1975. Identification of turnip mosaic virus in Tropaeolum majus L. Acta Bot. Croat. 34:33-42. Martin, R. R. 1985. Recent advances in virus detection. HortScience 20:837-845. Matthews, R. E. F. 1982. Classification and nomenclature of viruses. S. Karger Pub., Sidney. _ _ . 1991. Plant virology (3rd ed.). Academic Press, San Diego. McDonald, J. G., and E. Hiebert. 1974. Ultrastructure of cylindrical inclusIons induced by viruses of the potato Y group as visualized by frellze-etching. Virology 58:200-208. ___ . 1975. Characterization of the capsid and cylindrical inclusion proteins of three strains of turnip mosaic virus. Virology 63:295-303. Michelin-Lausarot, P., and G. Papa. 1975. The coat protein of the Alliaria strain of turnip mosaic virus: molecular weight and degradation products formed during purification and upon storage. J. Gen. Virol. 29:121-126. Milicic, D., Z. Stefanac, and D. Mamula. 1967. Turnip mosaic virus. p. 54-56. In: Proceed. Sixth Conf. Czechoslovak Plant ViroL, Olomouc, 1967. Acad. Pub. House, Czechoslovak Acad. ScL, Prague, 1969. Moghal, S. M., and R. I. B. Francki. 1976. Towards a system for the identification and classification of potyviruses. 1. Serology and amino acid composition of six distinct viruses. Virology 73:350-362. Mowat, W. P., and J. A. T. Woodford. 1976. Control of the spread of two non-persistent aphid-borne viruses in lilies. Acta Hort. 59:27-28. Natti, J. J. 1956. Influence of cauliflower mosaic and turnip mosaic viruses on yields of cabbage in New York State. Plant Dis. Rptr. 40:591-595. Niblett, C. L., A. O. Paulus, and J. S. Semancik. 1969. A mosaic disease of statice caused

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5 Thin Cell Layer Morphogenesis Michael E. Compton· and Richard E. Veilleux Department of Horticulture Virginia Polytechnic Institute and State University Blacksburg, VA 24061

I. II.

III.

IV. V.

Introduction Flower Bud Production A. Endogenous Factors B. Exogenous Factors C. Genetic Transformation of Flowering Shoots Vegetative Shoot Morphogenesis A. Growth Regulator Requirements of Various Species B. Genetic Transformation of Vegetative Shoots Somatic Embryogenesis Conclusions Literature Cited

I. INTRODUCTION Thin cell layer (TCL) explants are thin strips of tissue composed of the epidermis plus 1-10 layers of subepidermal parenchyma cells. Because of their small size (typically 2 X 10 mm) and the lack of preformed meristems or buds, TCLs are thought to have lower levels of endogenous plant growth regulators (PGR) and other regulating substances than larger explants (Tran Thanh Van and Trinh 1986). Desired morphogenic patterns can be obtained by manipulating the concentration of exogenous PGRs. Other desirable qualities ofTCL explants include: (a) physiological and genetic homogeneity, (b) uniform and rapid response under inductive conditions, (c) direct response of the target cells to exogenously applied PGRs without interference from other plant organs, (d) direct organ formation on the explant without an intermediate callus phase, and (e) the applicability of the TCL response to whole plants (Tran

·Present address: Central Florida Research and Education Counter, Institute of Food and Agricultural Science, University of Florida, 5336 University Ave., Leesburg, FL 34748-8203 239

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Thanh Van 1977, 1981; Tran Thanh Van and Cousson 1982; Tran Thanh Van and Trinh 1978, 1986). TCL technology is utilized to study physiological, histological, and genetic aspects of flower, shoot, and somatic embryo morphogenesis. By manipulating the concentration of PGRs, TCL explants of competent plant species can be programmed to differentiate somatic embryos (Pelissier et aI. 1990), flower buds, vegetative shoots, and roots or callus (Tran Thanh Van and Drira 1970; Chlyah 1973; Tran Thanh Van 1973a,b). For tobacco (Nicotiana tabacum L.), equimolar concentrations of auxin and cytokinin induce flower formation (Tran Thanh Van 1973a), a phenomenon that has been exploited to study flower initiation and development. High cytokinin concentrations promote shoot morphogenesis useful for micropropagation or genetic transformation. A majority of the TCL literature has focused on flower bud morphogenesis in Cichorium intybus L. (Nguyen 1975; Tran Thanh Van 1977) Nautilocalyx lynchei L. (Tran Thanh Van and Drira 1970), Nicotiana (Tran Thanh Van 1973a), Petunia hybrida Hort. (Mulin and Tran Thanh Van 1989b), and Torenia fournieri Lind. (Chlyah 1973). In vitro flowering has also been also reported on preformed meristems, hypocotyl segments, leaf discs, root sections, and entire plantlets (Scorza 1982). The purpose of this review is to present recent advances toward identifying factors that control de novo flower, vegetative shoot, and somatic embryo morphogenesis on TCL explants. II. FLORAL BUD PRODUCTION

De novo flower buds originate from differentiated epidermal and subepidermal parenchyma cells (Tran Thanh Van 1977) that dedifferentiate to form meristematic centers (Chlyah 1973; Thorpe and Biondi 1981). The meristematic centers, in turn, differentiate floral buds (Tran Thanh Van 1973a). Twenty to 50 flowers may be produced on tobacco TCL explants (Tran Thanh Van 1981), but flowers are small and vary in anther and pistil number (Bridgen and Veilleux 1988). Flower buds on tobacco TCLs form either directly on the explant surface (Fig. 5.1a) in full inflorescences (Fig. 5.1b) or on flowering shoots as in N. plumbaginifolia (Tran Thanh Van and Trinh 1986). Flower buds are visible from 12-21 days after culture initiation and form well-developed anthers with fertile pollen (Tran Thanh Van 1977; Trinh et a1. 1987). Pollination of de novo flowers resulted in fruit with viable seed (Trinh et a1. 1987). In tomato, pedicel explants form full inflorescences (Fig. 5.2a) on elongated shoots with several leaves (Compton and Veilleux 1991a). These flowers form welldeveloped anthers and pistils; and they give rise to parthenocarpic fruit (Fig. 5.2b).

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Figure 5.1. Flower bud formation on tobacco TCL explants. Individual flowers form either directly on the explant surface (Xl00) (a) or are borne on small inflorescences (X20) (b).

A combination of endogenous and exogenous factors influence de novo flower morphogenesis. Internal factors include the position of the explant on the intact plant at the time of excision, the plant genotype, and the genes expressed during flower morphogenesis. External factors include PGRs and other growth promoters, light, and carbon source. A. Endogenous Factors 1. Explant position. In vivo, tobacco flowers are borne on inflorescences with individual flowers arranged in racemes, panicles, or thyrses.

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Figure 5.2. Flower bud (X32) (a) and fruit (X4) (b) formation on tomato pedicel explants (from Compton and Veilleux 1991a).

TCL explants have been taken from pedicels (i.e., the individual flower stalk), peduncles (Le., stalk bearing the entire inflorescence), and the base of flowering branches (subfloral zone). Figure 5.3 shows the position of these organs on the tobacco inflorescence. The ability of tissue to form flowers decreases basipetally from the apical end of a flowering plant. Aghion-Prat (1965) demonstrated the floral morphogenic gradient by injecting tobacco (Nicotiana tabacum L.) plants with Agrobacterium tumefaciens. Tumors that formed near the apex developed flower buds, whereas tumors that occurred near the base formed vegetative buds. A similar morphogenic response was later demonstrated utilizing TCL explants. Explants taken from the base of greenhouse-grown 'Wisconsin 38' plants formed only vegetative shoots, whereas those explants excised from the inflorescence formed floral buds (Tran Thanh Van 1973a). Explants from intermediate regions along the main axis formed both vegetative and floral buds in culture. The capacity of plant tissue to regenerate flower buds may be determined by the age of the explant tissue at the moment of excision. Croes et al. (1985) observed that TCL explants produced more flower buds when taken from pedicels when the attached flower was at anthesis (± 1 day). The position of the explant on the intact plant is extremely important

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Figure 5.3. Tobacco inflorescence at the green fruit stage (X4). Flowering TCLs have been obtained from pedicels (Pl), peduncles (P2), and the subfloral zone (SFZ).

when attempting to obtain de novo flowers on TCL explants of photoperiodically sensitive tobacco cultivars. Flower formation for the short-day (SO) cultivar Maryland Mammoth was partially (Rajeevan and Lang 1987) or completely (Kamate et al. 1981; Bridgen and Veilleux 1985) inhibited when TeL explants were. taken from peduncles. De novo flowers were obtained on 'Maryland Mammoth' (Rajeevan and Lang 1987) and 'White Burley' (Altamura et al. 1989) TCLs taken from pedicels, but the number of flowers per explant was reduced (7.1) compared to the day neutral (DN) cultivar 'Samsun' (22.3). Rajeevan and Lang (1987) suggest that the reduced flowering observed for SO cultivars may result from a reduction in the number of cells competent for de novo flower morphogenesis. The increase in flower response among explants derived from tobacco inflorescences has been correlated with an increase in nuclear ONA content in that region (Wardell and Skoog 1973). Altamura et al. (1987) isolated ONA from individual nuclei of cells derived from the inflorescence, apical meristem, and base of flowering 'Samsun' tobacco. Using cytophotometric analysis, they determined that nuclei originating from inflorescences contained about 28% more DNA than nuclei from 2C apical meristems. Nuclei from cells isolated from the base of flowering plants contained 38% less ONA than those of the apical meristems. The effect of extra ONA on explant morphogenesis is uncertain. Some

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researchers speculate that increased DNA among nuclei of cells comprising the inflorescence may increase the ability of TCLs to regenerate floral buds (Altamura et al. 1987); however, more research is required to identify DNA sequences and gene products specific to floral bud morphogenesis. The tissue composition of TCL explants plays an important role in morphogenic competence. Torenia fournieri (Chlyah 1974) and tobacco (Tran Thanh Van 1973a) TCLs form buds directly, with no apparent callus formation, whereas more complex explants that contain xylem, phloem, and cambial tissue (Le., stem segments) only form buds after callus growth (Wardell and Skoog 1969; Chlyah 1974). A prolonged callus phase may lead to a change in ploidy (Karp et a1. 1985; Owen et a1. 1988) or structural chromosomal changes (Larkin and Scowcroft 1981; Evans and Sharp 1983; Koomneef et al. 1989) that may result in abnormal flower morphology (e.g., missing flower parts), reduced fertility, or loss of the ability of explants to regenerate. 2. Plant genotype. The flowering response of different Nicotiana TCLs is

summarized in Table 5.1. Not all Nicotiana genotypes or species are capable of de novo flower morphogenesis; however, 25 of 30 genetic lines surveyed have produced in vitro flowers. Competence for de novo flower morphogenesis has been transferred to F1 and F2 hybrids between responsive and nonresponsive tobacco genotypes (Kamate et al. 1981; Tran Thanh Van and Cousson 1982; Bridgen 1984). Competence for in vitro flower formation was transferred to intergeneric hybrid plants via somatic hybridization. TCLs from partial somatic hybrids between N. plumbaginifolia Vivo (responsive) and Petunia hybrida (nonresponsive) produced flowers after 2-3 months in vitro (Mulin and Tran Thanh Van 1989a). The transfer of competence for de novo flower morphogenesis to sexual and somatic hybrid plants Table 5.1. Nicotiana genotypes capable of flower formation on thin cell layer explants. Chromosome number Genotype

[2n)

Percent TCLs with Photoperiod 2 flowers Reference

N. tabacum cvs

Lacerata McNair 944 NC82 Samsun

48 48 48 48

DN ON ON ON

82 100 100 100

Samsun [haploid)

24

ON

100

Kamate et aI. 1981 Bridgen 1984 Bridgen 1984 Tran Thanh Van & Cousson 1982 Trinh & Tran Thanh Van 1981

5.

245

THIN CELL LAYER MORPHOGENESIS

Table 5.1.

Continued. Chromosome Percent number TCLs with (2n) Photoperiod z flowers Reference

Genotype Wisconsin 38

48

DN

100

Tran Thanh Van & Cousson 1982 Tran Thanh Van & Cousson 1982 Bridgen & Veilleux 1985 Rajeevan & Lang 1987 Bridgen 1984 Altamura et al. 1989

Xanthi

48

DN

100

Maryland Mammoth

48

SD

0

NC22NF White Burley Other Nicotiana species N. rustica N. debneyi N. plumbaginifolia N. plumbaginifolia (haploid) N. sylvestris

48 48

SD SD

100 0 89

48 48 20 10

DN DN DN DN

25-35 100 100 80-100

24

LD

0

N. tomentosiformis

24

SD

0

N. alata

18

DN

0

48 48 48 48 48

DN DN DN DN Variable

100 100 100 100 66.8

48

Variable

44.2

Bridgen 1984

36

DN

60

Kamate et a1. 1981

Kamate et aI. 1981 Kamate et a1. 1981 Kamate et al. 1981 Trinh & Tran Thanh Van 1981 Tran Thanh Van & Cousson 1982 Tran Thanh Van & Cousson 1982 Kamate et aI. 1981

N. tabacum hybrid plants McNair 944 X NC22NF NC22NF X McNair 944 NC 82 X NC22NF NC22NF X NC82 F2 (McNair 944 X NC22NF) F2 (NC82 X NC22NF) Interspecific hybrid plants N. tabacum cv. Samsun X N. sylvestris N. tabacum cv. Samsun X N. tomentosiformis N. sylvestris X N.

36

DN

75

Kamate et a1. 1981

24

LD

30

Kamate et a1. 1981

tomentosiformis N. plumbaginifolia X N.

22

LD

10

Kamate et a1. 1981

48

DN

100

Kamate et al. 1981

48

DN

89

Kamate et a1. 1981

22

LD

0

Kamate et a1. 1981

Bridgen Bridgen Bridgen Bridgen Bridgen

1984 1984 1984 1984 1984

sylvestris (N. debneyi X N.

N. tabacum (N. suaveolens X N. tabacum) X N. tabacum N. plumbaginifolia X N. tabacum)

X

tomentosiformis

zDN

= day neutral;

SD

= short day;

LD

= long day.

246

MICHAEL E. COMPTON AND RICHARD E. VEILLEUX

between responsive and nonresponsive genotypes indicates that this trait is dominant. Genome complementation may also confer competence for de novo flower morphogenesis suggesting a more complex genetic control. In one instance, 30% of the TCL explants from F1 hybrids between N. sylvestris (LD) and N. tomentosiformis (SD), two nonresponsive genotypes, formed flower buds (Kamate et al. 1981). The morphology and type of flowers produced in vitro may also depend on the nuclear and cytoplasmic genome interaction (Trinh and Tran Thanh Van 1983). TCLs from F1 hybrids between N. tabacum cv. Samsun (female) and N. plumbaginifolia (male) formed flowers directly on the explant (typical of N. tabacum cv. Samsun), whereas TCLs from reciprocal hybrids only formed flowers on elongated shoots (characteristic of N. plumbaginifolia). TCLs from haploid N. tabacum cv. Samsun (2n = 4x = 48) and N. plumbaginifolia (2n = 2x = 20) formed more and/or larger flowers than diploids forms (Trinh and Tran Thanh Van 1981; Bridgen and Veilleux 1988). Altamura et al. (1986) observed that TCLs from doubled haploid (DH) plants of N. tabacum cv. White Burley (2n = 4x = 48) formed almost twice as many flowers in less time than TCLs from amphidiploid controls. However, it should be noted that Altamura et al. (1986) selected DH plants with the greatest flowering potential. Increased flower production in haploids and DH plants may have resulted from a genetic changers) that occurred during haploidization or a meiotic segregation favoring de novo flower morphogenesis. 3. Genes expressed during flower morphogenesis. Studies on de novo flower formation of tobacco TCLs led to the identification of six gene families (FB7-1, FB7-2, FB7-3, FB7-4, FB7-5, and FB7-6), which are expressed during flower morphogenesis (Meeks-Wagner et al. 1989). These gene families were identified by differentially screening a 32p_ labeled cDNA library created from mRNA isolated from TCL explants grown either on floral bud-induction medium (FB7) or vegetative shoot medium for 7 days. Meeks-Wagner et al. (1989) observed that five of the six families (FB7-1, FB7-2, FB7-4, FB7-5, and FB7-6) were associated with flower initiation. FB7-3 was found to be a kinetin-induced gene not associated with flower bud formation. FB7-1, FB7-2, and FB7-5 were identified as single or low copy number genes that were expressed at the highest levels among day 7 (premeristem) explants and explants with visible flower buds (days 25-33). FB7 transcripts were expressed in the roots, internodes, leaves, and subapical pith cells of immature inflorescences and unopened flowers of tobacco plants grown from seed. The highest activity of FB7-1, FB7-2, and FB7-5 was detected in the roots of plants with immature inflorescences. The lack of FB7 gene expression in

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247

floral meristems grown from seed may correspond to the time and cell types from which the eDNA library was constructed. Their cDNA library originated from cells undergoing rapid division during a time in TCL morphogenesis that precedes floral meristem formation, and the expression of FB7 genes in the subapical pith cells of prefloral and floral meristems of plants grown from seed may be related to patterns of cell division and elongation correlated with floral initiation (Meeks-Wagner et a1. 1989). However, this does not explain the high activity of these gene families in the roots of the same plants. Recently, Neale et a1. (1990) reported that the gene sequences of the FB7 eDNA clones encoded four pathogen-related proteins: chitinase, 131,3-glucanase, osmotin, and extensin. FB7-1 was found to be homologous to a basic chitinase sequence, FB7-5 cDNA was homologous to a basic f3-1,3-glucanase, and FB7-2 encoded for osmotin (a protein that acculI?-ulated in plant cells under water and salt stress). RNA gel blot analyses indicated that chitinase, osmotin, and f3-1,3-glucanase were expressed primarily in the roots of flowering 'Samsun' tobacco plants and, to a lesser degree, in the basal leaves and internodes. Only chitinase and osmotin were expressed in floral organs. FB7-3, which was expressed among TCL explants cultured both on vegetative shoot and floral bud medium containing kinetin, was homologous to the 3'noncoding region of tobacco extensin eDNA isolated from tobacco shoots transformed with A. tumefaciens. A search of the nucleotide sequences and the deduced amino acid sequences in the Genebank Genetic Sequence Data Bank and the NBRF protein identification resource failed to identify a nucleotide sequence or an amino acid sequence homologous to the FB7-4 sequence. FB7-4 was expressed at moderate levels in the basal internodes and at low levels in the roots of flowering plants. The elevated expression of these genes during floral organ development in tissue other than the developing meristem indicates that they may be indirectly involved in flower morphogenesis (Neale et a1. 1990).

B. Exogenous Factors 1. Plant growth regulators

a. Auxin. Auxin is required for in vitro flower formation on tobacco TCLs (Tran Thanh Van 1973a,b; Cousson and Tran Thanh Van 1981; van den Ende et a1. 1984a; Smulders et a1. 1988a; Mohnen et a1. 1990). Auxin concentrations from 0.1 to 2.2,uM promote floral bud initiation (Table 5.2), whereas higher auxin concentrations (~4.5,uM) have promoted callus formation with reduced organogenesis (Smulders et a1. 1988a).

248

MICHAEL E. COMPTON AND RICHARD E. VEILLEUX

Table 5.2. Response of tobacco thin cell layer explants to media containing different auxins, cytokinins, ethylene, gibberellins and polyamines.

PGR

Tobacco cultivar

Response

Reference

Auxin O.lIJ.M IBA

Samsun

100% flower formation

Cousson & Tran Thanh Van 1981 Mohnen et al. 1990 van den Ende et al. 1984b

O.1-2J!M IBA Samsun 0.1-1J!M NAA Samsun NAA

Samsun

0.45J!M NAA Samsun 2.2J!M NAA 4.51J.M NAA

O.lIJ.M IBA

Xanthi

NAA and CTAUnknown Cytokinin 0.11J.M kinetin Samsun 0.2-2J!M kinetin 1IJ.M BA

Samsun

101J.M BA 0.22-1IJ.M BA

Samsun Sam sun

0.45-11J.M BA

Samsun

Samsun

O.lIJ.M kinetin Xanthi BA Ethylene 0.51J.r 1 1-lOlJ. r1 Gibberellin GAJ Polyamines putrescine spermidine

Unknown

Sam sun

Samsun

Sam sun

Optimal flower formation Optimal flower development Rapid uptake during first 24 h Basal flower formation Uniform bud distribution Promoted callus and reduced flower morphogenesis 100% flower morphogenesis Increased flower morphogenesis

Smulders et al. 1988a,b

Tran Thanh Van & Trinh 1978 Heylen & Vendrig 1988

100% flower morphogenesis Optimal flower formation

Cousson & Tran Thanh Van 1981 Mohnen et al. 1990

100% flower morphogenesis Floral bud abnormalities Greatest flowering response Greatest flowering response 100% flower morphogenesis Increased flower morphogenesis

van den Ende et al. 1984b

Increased flower bud initiation during first seven days Inhibited flower morphogenesis Inhibited flower initiation

Highest during bud formation Wisconsin 38 Promoted flower morphogenesis Samsun

Barendse et al. 1987

van den Ende et al. 1984b Barendse et al. 1987 van der Krieken et al. 1988 Tran Thanh Van & Trinh 1978 Heyden & Vendrig 1988

Smulders et al. 1990

Smulders et al. 1990

Croes et al. 1986a Torrigiani et al. 1987 Kaur-Sawhney et al. 1988

5.

THIN CELL LAYER MORPHOGENESIS

Table 5.2.

249

Continued.

PGR putrescine spermidine

Tobacco cultivar

Response

Wisconsin 38 Promoted vegetative buds Promoted flower morphogenesis [3 H]spermidine Wisconsin 38 Promoted flower morphogenesis

Reference Tiburcio et a1. 1988

Apelbaum et a1. 1988

Synthetic auxins, such as naphthaleneacetic acid (NAA) orO'-(3-chloro-otolyl) acetic acid, have increased flower morphogenesis on tobacco TCLs compared to natural auxins, such as indole-3-acetic acid (IAA) (Heylen and Vendrig 1988; Croes et al. 1985); however, 2,4-dichlorophenoxyacetic acid (2,4-D) has been found to inhibit flower morphogenesis (Heylen and Vendrig 1988). IAA has been the preferred auxin for flower formation on explants of Petunia hybrida (Mulin and Tran Thanh Van 1989b), Torenia fournieri (Chlyah 1973) and inflorescence explants of tomato (Lycopersicon esculentum Mill. cv. Red Alert) (Compton and Veilleux 1991a). Auxin uptake by tobacco TCL explants occurs rapidly after culture initiation. The concentration of free NAA in tobacco TCL explants increased 16-fold during the first 24 h of culture (Barendse et al. 1987). However, auxin may only be required during the first four days of culture for flower bud initiation. Smulders et al. (1988a) reported that flower bud formation proceeded normally when tobacco TCL explants were transferred to auxin-free medium 4 days after initiating explants on flower induction medium. They suggested that competent cells have committed to flower initiation by the fourth day and auxin was no longer required for flower morphogenesis. Floral bud development can be improved, however, by maintaining explants in medium supplemented with 111M auxin (van den Ende et al. 1984a). Flower buds generally form at the basal end of tobacco TCL explants incubated on medium containing 0.1 to l}lM NAA (Croes et al. 1985; Smulders et al. 1988a,b). However, flower buds become uniformly distributed along the explant surface at higher (2.2}lM) NAA concentration (Smulders et al. 1988a). Uniform bud distribution also occurred when auxin inhibitors [2,3,5-triiodobenzoic acid (TIBA) or 1naphthylphthalamic acid (NPA)] were added to floral medium. TIBA and NPA act by blocking auxin movement within the explant (Smulders et al. 1988b). This suggests that auxin is transported to target sites within the explant during flower morphogenesis, and uniform bud distribution can be achieved on TCL explants by increasing the N AA concentration in the

250

MICHAEL E. COMPTON AND RICHARD E. VEILLEUX

medium or by inhibiting the transport of auxin by incorporating auxin transport inhibitors into the medium. b. Cytokinin. In plant cells, cytokinin stimulates cell division (McGaw 1987) and is required for floral bud formation on tobacco TCLs (Cousson and Tran Thanh Van 1981; van den Ende et al. 1984b; Barendse et a1. 1987; Heylen and Vendrig 1988; Mohnen et a1. 1990). Optimal cytokinin concentrations for flower morphogenesis have been found to range from 0.1 to lJLM (Table 5.2). The number of floral buds on tobacco TCLs has been shown to increase linearly as the cytokinin concentration increased from 0.1 to 10JLM (van den Ende et a1. 1984a; Barendse et a1. 1987). However, high cytokinin concentrations (10JLM) have been reported to inhibit flower formation (Hillson and LaMonte 1977) or cause grossly abnormal bud development (van den Ende et a1. 1984b; van der Krieken et a1. 1988). Both kinetin (Wardell and Skoog 1969; Tran Thanh Van et a1. 1974) and benzyladenine (BA) have promoted floral bud formation on tobacco TCL explants (Cousson and Tran Thanh Van 1981; van den Rnde et al. 1984b; Barendse et al. 1987; van der Krieken et a1. 1988; Heylen and Vendrig 1988); however, more floral buds are produced with BA (Heylen and Vendrig 1988). Zeatin promoted meristematic activity on Torenia TCL explants (Tanimoto and Harada 1984), but delayed floral bud formation on tobacco TCLs (Meeks-Wagner et a1. 1989). Kinetin has been used to obtain flower buds on Petunia hybrida (Mulin and Tran Thanh Van 1989b) and tomato (Compton and Veilleux 1991a) explants. Cytokinin (BA) uptake by explants during the first 24 h of culture was found to be less rapid than auxin (NAA) uptake with most (79.4%) of the exogenously supplied BA converted to 7-fJ-D-glucopyranosyl-BA (Barendse et a1. 1987). Approximately 7-12% of the exogenously applied BA remained in the free (active) form. The exact role of the BA metabolite(s) is unclear. c. Gibberellin. Flower initiation on 'Wisconsin 38' tobacco stem segments was inhibited by the addition of 0.01JI,M gibberellic acid (GAJ) to floral bud induction medium (Wardell and Skoog 1969), and flower bud formation on tobacco TCLs was reduced when GA3 was applied to emasculated tobacco flowers prior to in vitro culture (Croes et al. 1986a). Similarly, gibberellin synthesized by developing fruit was suggested to decrease flower morphogenesis on TCL peduncle explants (Croes et a1. 1986a). However, the addition of GA3 to floral bud medium promoted flower maturation on tobacco stem segment explants when applied after the appearance of flower buds (Wardell and Skoog 1969). Hence, gibberellins may be beneficial for promoting floral bud maturation, but should not be added prior to floral bud initiation because of their inhibitory influence on the initiation process.

5.

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251

d. Ethylene. Flower morphogenesis on tobacco TCLs may be controlled by an interaction between ethylene and auxin. Low concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene (O.5JLM) promoted rapid flower bud initiation when floral induction medium contained 1-2.2JLM NAA (Table 5.2), but inhibited flower morphogenesis when medium contained less (0. 22JLM) NAA (Smulders et al. 1990). Higher ethylene concentrations (1 and 10jLM) inhibited flower morphogenesis at 0.22-2.2JLM NAA, but were ineffective at 4.5 or 10JLM NAA. Aminoethoxyvinylglycine (AVG), an inhibitor of ACC synthase, and AgN03, a competitive inhibitor of ethylene, reduced the number of flower buds formed 7 days after culture initiation (Smulders et al. 1990). This suggests that low levels of endogenous ethylene are important in flower initiation. Endogenous ethylene production in TCL explants has been reported to be dependent on the exogenous NAA concentration. Peak ethylene sYnthesis occurs 2-3 days after culture initiation at optimal NAA concentrations (0. 22-1JLM) , but the time to peak ethylene production is extended to 6 days at a higher, normally supraoptimal NAA concentration (4.5JLM) (Smulders et al. 1990). Increased ethylene sYnthesis by explants cultured at high NAA concentrations may contribute to decreased flower morphogenesis. 2. Other growth-promoting substances

a. Polyamines. Polyamines occur in free and conjugated forms in explanted tissues. An increase in free and conjugated polyamines was reported during the formation of floral meristem initials on tobacco (N. tabacum L. cv. Samsun) TCLs (Torrigiani et al. 1987). Exogenously applied spermidine appears to promote floral bud initiation, whereas putrescine has been associated with vegetative bud formation (KaurSawhney et al. 1988; Tiburcio et al. 1988) (Table 5.2). Severalobservations support this. First, the addition of cyclohexylamine, a competitive inhibitor of spermidine synthase, to floral initiation medium inhibited floral bud formation, but promoted vegetative shoot morphogenesis (Kaur-Sawhney et al. 1988). Second, 28010 of tobacco TCL explants formed flower buds when 0.5mM spermidine was added to vegetative shoot induction medium, whereas without added spermidine, 100010 of the TCLs normally produced vegetative shoots (Kaur-Sawhney et al. 1988). Third, Apelbaum et al. (1988) observed that binding of [3H}spermidine to a 18kd protein stimulated floral morphogenesis. The production of a similar size protein during morphogenesis was previously reported by Croes et al. (1986b). Changes in specific activity of two important polyamine enzymes, arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), have

252

MICHAEL E. COMPTON AND RICHARD E. VEILLEUX

been correlated with vegetative and floral bud initiation and development. ADC activity peaked during bud initiation, whereas ODC activity declined (Tiburcio et al. 1988). This was supported by the observation that DL-a-difluoromethylarginine (DFMA), a specific inhibitor of ADC, inhibited bud formation, whereas DL-a-difluoromethylornithine (DFMO), an inhibitor of ODC, promoted bud initiation (Tiburcio et al. 1988). This suggests that polyamines derived through ADC promote bud initiation; polyamines derived through ODC regulate organ growth and development (Tiburcio et al. 1988). b. Oligosaccharins. Oligosaccharins are naturally occurring carbohydrates with biological regulatory functions (Albersheim et al. 1983). Oligosaccharins have been shown to trigger plant defense mechanisms against pathogens and stress and to regulate cell growth and differentiation into roots, flowers, and vegetative buds (Darvill et al. 1989). Tran Thanh Van et al. (1985) tested the ability of pectic fragments (oligosaccharins) isolated from sycamore cell walls to promote de novo flowers, roots, or vegetative shoots on tobacco TCLs. Pectic fragments were isolated by treating sycamore cell walls with either endo-a-1,4polygalacturonase of Aspergillus niger (EPG fragments) or by a partial base-catalyzed solubilization and partial degradation of cell wall components [(base-soluble fragments) (Tran Thanh Van et al. 1985)). Pectic fragments (lp,g ml-1 ) inhibited flowering and promoted vegetative shoot formation on 87% of tobacco TeL explants incubated on flower induction medium (Tran Thanh Van et al. 1985). When medium contained 1p,g ml-1 EPG fragments, vegetative buds formed on callus induction medium and flower morphogenesis occurred on medium normally conducive to vegetative bud formation (Tran Thanh Van et al. 1985). Base-soluble fragments (10p,g mr1) induced vegetative buds on floral induction medium, and roots or callus on vegetative bud medium. Other investigators have observed that oligosaccharins influence morphogenesis on tobacco TCL explants but they have been unable to repeat the dramatic results of Tran Thanh Van et al. (1985). Eberhard et al. (1989) observed that 10p,g ml-1 EPG fragments reduced rooting of explants grown on root induction medium and increased the number of flowers per explant incubated on transition medium (e.g., medium on which moderate flower morphogenesis was already observed) (Mohnen et al. 1990). Flower formation was also increased when transition medium was supplemented with oligosaccharins from tobacco· cell suspension cultures. However, they were not able to reverse morphogenic patterns completely by adding EPG fragments as was Tran Thanh Van et al. (1985). Darvill et al. (1989) observed that EPG fragments (1-10 p,g/ml) isolated from sycamore, citrus, and tobacco cells induced flower formation of tobacco TCL explants incubated on root induction

5.

THIN CELL LAYER MORPHOGENESIS

253

medium (1.5pM IBA and 0.9pM kinetin); however, they did not provide data showing the degree of flowering obtained. Ion-exchange and gelpermeation chromatography revealed that pectic fragments consist primarily of rhamnogalacturonan I and II, and a-1, 4-linked oligogalacturonides. Flower formation only occurred when tobacco TCLs were incubated on media with O.lJLM oligogalacturonides with degrees of polymerization of 10 to 14 (Darvill et ai. 1989). Researchers have yet to discover the mode of action of oligosaccharin extracts; however, oligosaccharins may act by modifying auxin activity, availability, or mobility within explanted tissue. 3. Light. Light intensity plays a significant role in TCL flower bud morphogenesis. Cousson and Tran Thanh Van (1983) observed that flower morphogenesis was decreased when explants incubated on flower induction medium received less than 50 W m-2 illumination. In addition, Mohnen et ai. (1990) observed that TCL flower morphogenesis was reduced when explants incubated on LS medium with 0.5 pM IBA and 0.5JLM kinetin received light intensities greater than 55 JLIIlol m-2 s-t, Higher light intensities (95 and 115 JLIIlol m-2 S-l) have been conducive to flower formation when TCLs were incubated on medium with increased (4P.M) IBA levels (Mohnen et al. 1990); however, further investigation revealed that flower formation on TCLs incubated at high light intensities on medium with increased IBA concentrations was promoted by the photodegradation of IBA in the medium. Light quality (source) is not critical in the obtention of flower buds on tobacco TCLs. Equivalent numbers of flowers have been obtained among explants illuminated with cool- or warm-white fluorescent lamps, Growlux, or natural fluorescent lights (Mohnen et al. 1990). Hence, a majority of the researchers chose to illuminate TCL explants with coolwhite fluorescent lamps. The timing of light application is extremely important to obtain flower morphogenesis on tobacco TCLs. Cousson and Tran Thanh Van (1983) observed that TCL explants receiving 50 W m-2 illumination only on days 2,4, or 6 failed to produce more flowers than explants incubated in continuous darkness. However, the flowering response was increased to 80% when explants were illuminated only on days 6-11. Although providing illumination between days 6 and 11 increased flower morphogenesis over dark controls, the best results may be obtained by providing TCL explants illumination throughout the course of experiments. It is common practice to incubate TCLs under a 16-h photoperiod. Under these conditions, 25 of 30 Nicotiana lines produced flowers. Most competent Nicotiana genotypes are day neutral (DN) (Le., flowering is

254

MICHAEL E. COMPTON AND RICHARD E. VEILLEUX

independent of day length), whereas nonresponsive Nicotiana are either long day (LD) requiring short nights to promote flowering, or short day (SD) requiring long nights (Trinh and Tran Thanh Van 1981; Kamate et al. 1981; Tran Thanh Van and Cousson 1982; Bridgen 1984; Bridgen and Veilleux 1988). Flowering has been· obtained when TCLs taken from pedicels of SD cultivars were given an 8-h photoperiod. All (100%) of the 'Maryland Mammoth' (Rajeevan and Lang 1987) and 89% of the 'White Burley' (Altamura et a1. 1989) TCL explants formed flowers when given the appropriate SD photoperiod. Rajeevan and Lang (1987) stated that flower morphogenesis on 'Maryland Mammoth' TCLs was independent of photoperiod; however, Altamura et a1. (1989) demonstrated that the number of 'White Burley' TCLs that produced flowers and the number of flowers per TCL were increased by providing an 8-h photoperiod. Others (Kamate et a1. 1981; Bridgen and Veilleux 1985) have attempted to induce flowering on TCLs of SD cultivars by providing inductive photoperiods; but they we~e unsuccessful, possibly because they used less responsive (peduncle) explants. 4. Carbon source. Sugars are necessary sources of carbon in many plant tissue culture media. One hundred percent flower formation has been achieved on tobacco TCLs when Murashige and Skoog (MS) or Linsmaier and Skoog (LS) medium was supplemented with 167 mM glucose (Cousson and Tran Thanh Van 1981,1983; van den Ende et al. 1984a, b; Croes et al. 1985, 1986a,b; Barendse et a1. 1987; Bridgen and Veilleux 1988), sucrose (Tran Thanh Van 1973; Tran Thanh Van et a1. 1974; Tran Thanh Van and Trinh 1978), or equimolar (83 mM) concentrations of glucose and sucrose (Cousson and Tran Thanh Van 1983). However, a majority of the TCL researchers chose to supplement their media with glucose (about 30 g 1-1 ). 5. Agar-solidified vs. liquid medium. Flower formation has been accomplished by incubating tobacco TCLs on agar solidified (0.6-1%) medium (Tran Thanh Van 1977; Trinh and Tran Thanh Van 1981; van den Ende et a1. 1984a,b; Barendse et a1. 1987; van der Krieken et a1. 1988; Smulders et al. 1988), on liquid medium with nylon mesh (Smulders et al. 1990), on filter paper (Than Thanh Van and Trinh 1978) or glass beads (Cousson and Tran Thanh Van 1981), or floated on liquid medium without physical support (Than Thanh Van 1985; Eberhard et al. 1989; MeeksWagner et al. 1989; Mohnen et al. 1990; Neale et al. 1990). Much controversy exists in the literature as to the benefit of agar-solidified or liquid medium for flower morphogenesis. Studies show that reducing the agar concentration in the medium from 1 to 0.5% resulted in a 15% reduction in flower morphogenesis (Than Thanh Van and Trinh 1978). In contrast,

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Mohnen et a1. (1990) suggested that PGRs and other morphogenic substances may become trapped in the agar matrix and the use of agarsolidified medium should be avoided. In our laboratory we incubated tomato pedicel explants on agar-solidified medium, on liquid medium with nylon mesh or nutrient filter membranes as support, or floated directly on liquid medium. In these experiments, flowering shoots were only obtained on explants incubated on agar-solidified medium (unpublished results). This suggests a differential response among plant species to medium solidification.

c. Genetic Transformation of Flowering Shoots The production of transformed flowering shoots has been obtained by co-cultivating TCLs of diploid and haploid N. plurnbaginifolia with a disarmed strain of Agrobacteriurn tumefaciens (strain GV3111SE with the disarmed plasmid pTiB6S3SE). The co-integrate pTiB6S3SE: :pMONC9 contains the chimeric genes NOS-nptII (nopaline synthase-neomycin phosphotransferase) and rbcS-8B-CAT. The latter is composed of the 5' upstream region of the rbcS-8B gene of N. plumbaginifolia and the coding sequence of the bacterial chloramphenicol acetyl transferase (CAT) gene. TCLs were obtained from flowering branches and floated on liquid medium containing A. tumefaciens (1(f cells/ml) for 72 h. Putatively transformed shoots were selected on flower bud induction medium (111M each of kinetin and IAA) supplemented with 500/ig mr1 carbenicillin and 100 /ig mr 1 kanamycin. TCLs with 4-6 flowering shoots were observed 2-3 weeks after transfer to selective media and 40% of these shoots developed roots within 2 weeks of transfer to medium supplemented with 100 /ig ml-1 kanamycin. Seeds were obtained from fertile flowers of both diploid and dihaploid (haploid that spontaneously doubled) TCLs and germinated on MS medium with 100 /ig mr 1 kanamycin to select seedlings that carried the NOS-npt II gene. Approximately 75% (1751/2335) of the diploid and all (3705) of the dihaploid seedlings germinated on medium supplemented with kanamycin, suggesting that the NOS-npt II gene was dominantly inherited. All transformed plants that contained the NOS-npt II gene also expressed CAT activity and, thus, carried the rbcS-8B-CAT chimeric gene. Because the nuclear gene rbcS-8B is expressed in an organ-specific manner, Trinh et a1. (1987) tested for CAT activity in the leaves, flowers, and roots of transgenic plants. They observed that the highest CAT activity occurred in the leaves, confirming the regulated expression of rbcS-8B-CAT in transgenic flowering shoots from TCL explants. The ability to obtain transformed seed from flowering shoots may increase

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the transformation efficiency by reducing the time required to obtain transformed plants.

III. VEGETATIVE SHOOT MORPHOGENESIS A. Growth Regulator Requirements ofVarious Species A wide range of species has demonstrated competence for de novo vegetative shoot proliferation on TCLs. These include Begonia rex Putz. (Chlyah and Tran Thanh Van 1975), Beta vulgaris L. (Detrez et a1. 1988), Brassica napus L. (Klimaszewska and Keller 1985), Bryophyllum daigremontianum (Bigot 1976), Lycopersicon esculentum Mill. (Compton and Veilleux 1991a), Nicotiana tabacum (Tran Thanh Van 1973b), Petunia hybrida Hort. (Mulin and Tran Thanh Van 1989b.), Psophocarpus tetragonolobus (L.) DC. (Trinh et a1. 1981), and Torenia fournieri (Chlyah 1973). Generally, a high cytokinin-to-auxin ratio (10-100:1) leads to vegetative shoot morphogenesis. Vegetative bud formation occurred on tobacco TCLs incubated on medium supplemented with 10JLM BA and 1P.M IAA (Tran Thanh Van et a1. 1974) (Fig. 5.4a). Higher PGR concentrations were required for shoot morphogenesis on TCLs of Brassica napus (44-66JLM BA and 2.2p.M NAA) and Beta vulgarus (22p.M BA and 2.7p.M NAA) (Klimaszewska and Keller 1985 and Detrez et a1. 1988, respectively). Petunia hybrida TCLs formed shoots on medium supplemented with as little as lJLM each of kinetin and IAA (Mulin and Tran Thanh Van 1989b). In tomato, vegetative shoots formed when explants were incubated on medium supplemented with 10JLM zeatin, or 10JLM BA, and O.OOlJLM IAA, Le., a 10,000:1 cytokinin:auxin ratio (Compton and Veilleux 1991a) (Fig. 5.4b). Vegetative shoot morphogenesis was reduced when medium was prepared with other cytokinins (kinetin, 2iP) or auxins (NAA,IBA). By microscopic analysis of TCLs after various days of incubation, it has been found that shoots originate from subepidermal parenchyma cells (Klimaszewska and Keller 1985; Detrez et a1. 1988; Compton and Veilleux, 1991a). The first cell divisions have been detected after three days of culture for Brassica napus (Klimaszewska and Keller 1985), 7 days for 'Ohio 7814' tomato (Compton and Veilleux 1991a), and 10 days for sugar beet (Detrez et a1. 1988). In tomato, areas of active cell division have been demonstrated to occur randomly throughout the subepidermal layer 14 days after culture initiation. These meristematic regions developed 1-2-cm shoots within 8 weeks (Compton and Veilleux 1991a). In a comparison of shoot morphogenic potential of tomato tissue

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Figure 5.4. Vegetative shoot production on a tobacco TCL (60X) (a) and tomato pedicel explant (X20) (b) (from Compton and Veilleux 1991a).

culture systems, Compton and Veilleux (1991b) observed that more shoots were produced on pedicel explants than on cotyledon calli. This trend occurred for 9 of the 12 genetic lines (Fig. 5.5). Although the frequency of responding explants was less for pedicel explants than for cotyledon calli, the mean number of shoots per TCL explant was greater and compensated for the decreased explant response (Compton 1990).

B. Genetic Transformation of Vegetative Shoots TCLs can be used as an explant source for the obtention of transgenic vegetative shoots. High (40-50%) transformation frequencies have been reported among shoots regenerated from TCLs of Brassica napus ssp. oleifera cv. Westar (Charest et a1. 1988) and N. plumbaginifolia (Trinh et a1. 1987). The highest transformation rates occurred when B. napus TeL explants were given a 4-day incubation period on a feeder layer of

258

MICHAEL E. COMPTON AND RICHARD E. VEILLEUX N

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Genotype Figure 5.5. Number of shoots per responding explant on cotyledon calli and pedicel explants of 'Large Red Cherry' (A), 'Red Alert' (B), 'LA 1430' (C), 'LA 1526' (D), and reciprocal hybrids. (from Compton and Veilleux 1991b).

B. napus cells derived from suspension culture prior to a 30s inoculation with Agrobacterium tumefaciens (10 9 cells ml-1 ). TCL explants were cocultivated with A. tumefaciens for 2 days before the addition of antibiotics. Longer inoculation and co-cultivation periods resulted in explant necrosis and uncontrollable bacterial growth. Transformed shoots were selected for kanamycin resistance by transferring unrooted shoots to B5 medium containing 15J.Lg ml-1 kanamycin. The surviving shoots were transferred to higher levels (80J.Lg ml-1 ) of kanamycin for further selection. Kanamycin resistance and nopaline synthase activity were transferred to the selfed progeny of the transgenic plants in ratios of 9:6, 8:5, 7:4, 12:4, or 7:8 depending on the Agrobacterium strain used. Putatively transformed shoots were obtained on N. plumbaginifolia TCLs two weeks after co-cultivation with A. tumefaciens (Trinh et a1. 1987). About half of the shoots developed roots on rooting medium that contained 100J.Lg ml-1 kanamycin. Fifty of the kanamycin-resistant plants were chosen at random and analyzed for CAT activity. All plants that were kanamycin resistant also displayed CAT activity and, therefore, contained the rbcS-8B-CAT gene.

IV. SOMATIC EMBRYOGENESIS Somatic embryogenesis has been reported on TCL explants obtained from sunflower (Helianthus annuus L.) hypocotyls (Pelissier et a1. 1990).

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TCL explants were initially incubated in the dark in liquid MS medium plus 3% sucrose, 0.2% casein hydrolysate, 2% coconut water, 1 mg-1 NAA, and 1 mg-1 BA for 5 days. Primary embryos formed 8 days after transfer to liquid B5 medium supplemented with 9% sucrose. Secondary embryos formed when the explants were transferred to solidified MS medium with 12% sucrose and 0.2 mg-1 BA. The embryos were then transferred to solidified B5 medium plus 6% sucrose and 0.05 mg-1 NAA for 2 weeks to encourage further development. Somatic embryos were germinated in the light on solidified B5 medium with 3% sucrose and 0.1 mg-1 lAA. A range of 150-200 embryos was harvested from each flask (4 TCL explants/flask). Fertile plants were obtained after a total of 12 weeks. Somatic embryogenesis occurred on sunflower TCLs in the same manner as flower and shoot morphogenesis on tobacco TCLs. The first cell divisions were observed in the epidermal and cortical layers during the first 5 days of culture. Embryos were then observed on the surface of the epidermis after an additional 8 days. Other explants, such as the epidermis alone, hypocotyl without the epidermis, or cortical tissue alone, did not form embryos.

v. CONCLUSIONS The TCL tissue culture system is unique in that flowers, shoots, and somatic embryos can be obtained directly from differentiated parenchyma cells without callus formation. This type of morphogenic scheme allows one to observe the direct effects of the imposed treatments without interference from metabolic sources and sinks, such as meristems, roots, buds and callus. Direct morphogenesis may also avoid genetic changes that have been frequently reported after organogenesis from callus. De novo flowers of tobacco may be obtained in vitro by selecting explants from the apical region of flowering plants and incubating them in medium containing 0.1 to 2.2JlM auxin and lJlM cytokinin. Low ethylene concentrations may stimulate flower morphogenesis by altering explant sensitivity to auxin. Other growth-regulating substances, such as polyamines and oligosaccharins, have been implicated in flower morphogenesis, but their exact role has yet to be determined. Competence for de novo flower formation has been transferred to F1 and F2 hybrids and partial somatic hybrids between responsive and nonresponsive genotypes. De novo flower morphogenesis can also be improved by reducing plants to the haploid state. An important breakthrough in flower morphogenesis occurred when

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six gene families (FB7) associated with flower initiation were isolated from tobacco TCL explants. Three of these genes were identified as single or low copy number sequences and were expressed in TCL explants and plants grown from seed. It was later found that the gene sequences encoded for pathogen-related proteins (chitinase, f3-1, 3-glucanase, osmotin). The association of pathogen-related proteins with flower formation is intriguing and warrants further investigation. De novo vegetative shoot morphogenesis occurs when TCL explants are derived from vegetative plants, or from the base of flowering plants, and incubated in medium supplemented with increased cytokinin (1D100,uM). For tomato, shoot morphogenesis can be promoted by incubating pedicel explants in medium containing 10JlM zeatin. Somatic embryogenesis has been reported on TCL explants incubated in the dark in liquid B5 medium with 9% sucrose. Although this phenomenon has only been reported in sunflower, it is a recent breakthrough that may soon be extended to other species. Morphogenesis of flowers, vegetative shoots, and somatic embryos on TCLs can be employed to facilitate genetic transformation. TCL explants have been used efficiently to transform vegetative shoots of Brassica napus and vegetative and flowering shoots of Nicotiana plumbaginifolia. Shoot morphogenesis after genetic transformation of TCLs results in the production of genetically transformed shoots that can be acclimatized to greenhouse and field conditions. De novo flower morphogenesis after genetic transformation of TCLs results in the rapid production of genetically transformed seed that can be planted directly in the field, thus bypassing the sometimes cumbersome acclimatization steps associated with establishing in vitro plantlets. In addition, the development of a transformation method for TCL somatic embryos may further increase this system's desirability.

UTERATURE CITED Aghion-Prat, D. 1965. Floral meristem-organizing gradient in tobacco stems. Nature 207:1211.

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___ . 1986. Fundamental and applied aspects of differentiation in vitro and in vivo. p. 316-335. In: D. A. Evans, W. R. Sharp and P. V. Ammirato (eds.), Handbook of plant cell culture. Vol. 4. Techniques and applications. Macmillan Publishing Company, New York. Tran Thanh Van, K. M., N. T. Dien, and A. Chlyah. 1974. Regulation of organogenesis in small explants of superficial tissue of Nicotiana tabacum L. Planta 119:149-159. Tran Thanh Van, K., P. Toubart, A. Cousson, A. G. Darvill, D. J. Gollin, P. Chelf, and P. Albersheim. 1985. Manipulation of the morphogenetic pathways of tobacco explants by oligosaccharins. Nature 314:615-617. Trinh, T. H., and K. Tran Thanh Van. 1981. Formation in vitro de fleurs Ii partir de couches cellulaires minces epidermiques et sous-epidermiques diploids et haploides chez Ie Nicotiana tabacum L. et chez Ie Nicotiana plumbaginifolia Vivo Z. Pflanzenphysiol. 101:1-8. ___ . 1983. Influence de l'interaction genome-cytoplasme sur la formation de fleurs in vivo et in vitro chez les hybrides entre Nicotiana plumbaginifolia et Nicotiana tabacum. Can. J. Bot. 61:3514-3522. Trinh, T. H., H. Lie-Schricke, and K. Tran Thanh Van. 1981. Formation directe de bourgeon Ii partir des fragments et des couches cellulaires minces de differents organes chez Ie Psophocarpus tetragonolobus (L.) DC. Z. Pflanzenphysiol. 102:127-139. Trinh, T. H., S. Mante, E.-C. Pua and N.-H. Chua. 1987. Rapid production of transgenic flowering shoots and F1 progeny from Nicotiana plumbaginifolia epidermal peels. BiolTechnology 5:1081-1084. van den Ende, G., A. F. Croes, A. Kemp, and G. W. M. Barendse. 1984a. Development of flower buds in thin-layer cultures of floral stalk tissue from tobacco: role of hormones in different stages. Physiol. Plant. 61:114-118. van den Ende, G., A. F. Croes, A. Kemp, G. W. M. Barendse, and M. Kroh. 1984b. Floral morphogenesis in thin-layer tissue cultures of Nicotiana tabacum. Physiol. Plant. 62:8388. van der Krieken, W. M., A. F. Croes, G. W. M. Barendse, and G. J. Wullems. 1988. Uptake and metabolism of benzyladenine in the early stage of flower bud development in vitro in tobacco. Physiol. Plant. 74:113-118. Wardell, W. L., and F. Skoog. 1969. Flower formation in excised tobacco stem segments; I. Methodology and effects of plant hormones. Plant Physiol. 44:1402-1406. ___ .1973. Flower formation in excised tobacco stem segments. III. Deoxyribonucleic acid content in stem tissue of vegetative and flowering tobacco plants. Plant Physiol. 52:215-220.

6 Tissue and Cell Cultures of Woody Legumes*, ** R. N. Trigiano Department of Ornamental Horticulture and Landscape Design Institute of Agriculture University of Tennessee Knoxville, TN 37901-1071 R.L. Geneve Department of Horticulture and Landscape Architecture University of Kentucky Lexington, KY 40546

S. A. Merkle School of Forest Resources University of Georgia Athens, GA 30602 ]. E. Preece Department of Plant and Soil Science Southern Illinois University Carbondale, IL 62901-4415

I. II.

Introduction In Vitro Propagation A. Axillary Bud Proliferation

"'Literature search concluded June 1991. "Abbreviations used in this review are as follows. Basal media: B5 (Gamborg et al. 1968]. BL (Blaydes 1966]. BNM (Bonner 1943], GD (Gresshoff and Doy 1972], MS (Murashige and Skoog 1962], SH(SchenkandHildebrandt 1972], WPM (Lloyd and McCown 1980], and WM (White 1963). Growth regulators and other organic compounds: BA (N-(phenylmethyl)-lH-purine-6amine), CW (Coconut water], 2,4-D ((2.4-dichiorophenoxy]acetic acid), GAJ (gibberellic acid) [(10'. 2f3,4aO', 4bf3. 1013]-2 ,4a. 7-trihydroxy-l-methyl-8-methylenegibb-3-ene-l,10-dicarboxylic acid 1.40'-IactoneJ, IAA (lH-indoIe-3-acetic acid), IBA (lH-indole-3-butyric acid), 2iP [N-(3-methyl-2-butenyl)lH-purine-6-amine], KIN (kinetin) [N-(2-furanylmethyI)-lHpurine-6-amine}, NAA (l-naphthaleneacetic acid), PIC (picloran] (4-amino-3.5,6-trichloro2-pyridinecarboxylic acid), ZEA (zeatin] [2-methyI-4-(lH-purine-6-yiamino]-2-buten-l-oI]. 265

266

III.

IV.

V. VI.

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

J.

E. PREECE

B. Organogenesis C. Somatic Embryogenesis Crop Improvement A. Protoplast Culture B. Androgenesis C. Genetic Transformation Secondary Metabolite Production A. Alkaloids B. Anthraquinone Compounds C. Rotenoids D. Steroids E. Antimicrobial Activity F. Other Secondary Products In Vitro Studies of Nitrogen Fixation Concluding Remarks Literature Cited

I. INTRODUCTION The Leguminosae is one of the largest and most diverse plant families containing between 650-750 genera and over 18,000 species. Only the grass (Poaceae) and orchid (Orchidaceae) families contain more species. The Leguminosae is usually separated into three subfamilies, which include the Papilionoideae, Mimosoideae, and Caesalpiniodeae. However, some scientists elevate these subfamilies to family status. This can lead to confusion because the name Fabaceae has been used to replace Papilionoideae, when the subfamily is considered a distinct family. Also, Fabaceae has been used to replace the entire family name Leguminosae by taxonomists to conform totheaceae-ending common for other family names. Additional confusion can occur in taxonomy at the tribe, genus, or species level within the Leguminosae. Initial taxonomic classifications based on floral or seed morphology have given way to more sophisticated systematics utilizing the cryptic features of pollen morphology, chromosome number, Rhizobium nodulation, chemotaxonomy, and the fossil record to evaluate the relationships between genera. Reclassifications and revisions can be expected in a large family with such a diverse population. The woody species within the Leguminosae are equally as diverse as other members of the family. Woody species include trees, shrubs, and vines that are widely adapted to varied soil types, climates, and habitats. These species are found as major components of tropical rainforests, stubborn features of arid deserts, and indigenous plants of the arctic circle. Woody members of the Ceasalpinioid subfamily are the phylogenetically most ancient members of the Leguminosae. The

6.

TISSUE AND CELL CULTURES OF WOODY LEGUMES

267

Ceasalpinioideae species have been proposed as the evolutionary progenitors of both the Mimosoideae and Papilionoideae (Polhill et al. 1981). Additionally, nearly two-thirds of the species in the Mimosoideae occur in the predominantly woody genera of Acacia, Inga, and Mimosa. As well as being central to the evolution of the Leguminosae, woody legume species are also important ecologically and economically (Table 6.1). Woody legumes can be the predominant species in certain ecosystems, especially in the tropics. The impact of these species in tropical rainforests is enormous and includes a substantial amount of nitrogen fixation through the symbiotic association between legumes and Rhizobium. Approximately 90% of the Papilionoids and Mimosoids fix nitrogen, whereas only 34% of the Caesalpinioids have been shown to have a Rhizobium association (Brewbaker 1987). However, species that do not fix nitrogen apparently exploit mycorrhizal symbiosis to improve their nutritional status. Woody legumes have had a rich association with human history and development. Some of the earliest written records contain references to their importance as supplements to the human diet, use as animal fodder, standard weight measurements for commerce, and their involvement in folklore and medicine (Allen and Allen 1981). Early records are testaments to the important use of gum arabic (Acacia senegal), carob [Ceratonia siliqua), senna (Cassia spp.), and others. There is considerable interest in the role woody legumes can play in sustainable agriculture, especially in arid and tropical regions. Solutions to food problems of many agriculturally underdeveloped tropical countries do not lie in increased food production through the emulation or reliance on traditional monocultures typical to the affluent countries in temperate regions (Dover and Talbot 1987). Instead, reliable food productionmust be increased in the regions that experience limited food supplies. To reach this goal, sustainable agricultural systems that utilize limited resources and reduce the impact of production on fragile arid and tropical ecosystems need to be employed. To this end, woody legumes have figured prominently in the development of agroforestry systems (Brewbaker 1987). Agroforestry is the integration of woody tree or shrub species into traditional crop or livestock production (Gholz 1987). Woody legumes offer unique qualities desirable in an agroforestry system (Brewbaker 1987). Woody legume species are widely adaptable, often producing a sparse crown that allows light penetration to cocultivated crops and providing additional soil nutrition through leaf litter or nitrogen fixation. The adoption of agroforestry systems also offers potential relief from the substantial deforestation in the tropics. The potential uses of woody legumes in agroforestry are listed in Table 6.2.

~

Table 6.1.

Woody legume genera with potential ecological or economical value. z

C\:)

Secondary products Subfamily Genera Caesalpinioideae Acrocarpus AfzeIia

Fodder Forest or Products Forage

x

Agroforestry Nurse Trees

Orna- Pharmemental ceutical Soap

Gum

Fish Stupe- InsectiTannin fier cide Comments

x

x x

Amherstia

Baikiaea

x

x

Bauhinia

x

x

BerIinia

x

Brachystegia

x

x

x

x

x

x

ZTable adapted from Allen and Allen (1981), Brewbaker (1987), Duke (1981), and NAS (1979).

One of the largest trees (60 m) in the Indian rainforest. Seed produces a yellow dye. Ornamental seeds used for jewelry. Considered one of the most attractive flowering trees in the world. The attractive flower is the largest in the legume family. The wood is marketed as "Rhodesian Teak." Large trees planted for ornamental flowers resembling orchid blooms. Seeds produce an oil used for cosmetics. Pods are edible. Wood sold as "Rose Zebrams,"

Table 6.1.

Continued. Secondary products

Subfamily Genera

Fodder Forest or Products Forage

Brownea

Burkea Caesalpinia

Ceratonia

t-.:l

Agroforestry Nurse Trees

Orna- Pharmemental ceutical Soap

Gum

Fish Stupe- InsectiTannin fier cide Comments One of the most attractive flowering trees of South America.

X

x

X

X

X

X

X

X

X

X

X

X

X

X

X

A large genus containing attractive flowering trees. Pods of some species contain up to 50% tannins and are a source for gallic esters. Some species produced important dyes. Carob has been cultivated since antiquity for its leaves used as fodder and its edible pods. Carob seed gum is high quality and extensively used. Carob pod powder is a substitute for chocolate. Carob seed was used as a jeweler's weight and the term "carat" is derived from the arabic word for carob.

0)

co

continued

N 'J 0

Table 6.1.

Continued. ---------

Secondary products Subfamily Genera

Fodder Forest or Products Forage

Gymnocladus

Agroforestry Nurse Trees

Orna- Pharmemental ceutical Soap X

Haematoxylon

X

Hardwickia

X

Heterostemon

Gum

X

X

X

Hymenaea

X

Intsia

X

Kingiodendron Lysidice Maniltoa

X X

X

Fish Stupe- InsectiTannin fier cide Comments Historically seeds were roasted as a substitute for coffee, however pulp in pods is poisonous. Two biological stains of importance are produced in "Logwood"- hematoxylin and brazelein. Bark used to produce paper and rope. Resin used to produce a wood perservative. Orchid-like flowers and attractive foliage. Stripped bark used to make native canoes. Gum used for varnishes or polishes. Timber has commercial value for furniture. Bark yields a dye used to stain textiles.

X X X

Ornamental in China.

Table 6.1.

Continued. Secondary products

Subfamily Genera

Fodder Forest or Products Forage

Mora

X

Pahudia

X

Agroforestry Nurse Trees

X X

Piliostigma

N

"-l ....

X

X

X

X

Cercidium

X

Cercis

X

Colophospermum

Gum

Fish Stupe- InsectiTannin fier cide Comments

X

Parkinsonia Peltophorum

Orna- Pharmemental ceutical Soap

X

X

X

X

X

Produces the largest dicot seed weighing over 100 grams. Valued wood product from the Pacific Islands. Drought tolerant ornamental. Ornamental trees bloom twice a year. Ecologically important as a pioneer species in tropical Africa. The "Paloverde" of southwest North America. Attractive ornamental with flowers appearing before the leaves. Flowering branches have been used as cutflowers. Acrid tasing flowers used as a garnish. "Mopane" is an important shrub or small tree in the savannas of Africa browsed by wild game. continued

~

Table 6.1.

Continued.

N

Secondary products Subfamily Genera

Fodder Forest or Products Forage

Copaifera

x

Cynometra

X

Daniellia

X

Delonix

Agroforestry Nurse Trees

Orna- Pharmemental ceutical Soap

X

X

Dialium

X

Distemonanthus

X

Eperua

X

X

Gum

Fish Stupe- InsectiTannin fier cide Comments Trees produce a resin used as "Copal Resin." Used in paints, varnishes and lacquers. Unripe fruits of C. cauIiflora have an apple flavor and are sold as "Nam-nam" in India. Balsam produced from bark. Timber sold as "Ogea." Ornamental known as the "Royal Poinciana" or "Peacock Flower." Pods eaten as a fruit in local markets. Bark used in local medicines. Wood sold as "Ayan" or "Satinwood." A handsome flowering tree. Wood known as "Wallaba."

Table 6.1.

Continued. Secondary products

Subfamily Genera Erythrophleum

Fodder Forest or Products Forage X

Gleditsia

X

Guibourtia

X

Prioria

X

Schizolobium N

"'"

X

Gossweilerodendron

Saraca

Agroforestry Nurse Trees

Orna- Pharmemental ceutical Soap X

X

X

X

Gum X

X X

X X

Fish Stupe- InsectiTannin fier cide Comments X

"Sasswood" is one of the largest trees in Africa. Alkaloids of this genus of intense interest as one of the few Caesalpinoids producing significant alkaloids. Seed pods used as fodder with orchards sometimes planted for cattle browse. One of the largest trees of the African rainforest. Sold as "Nigerian Cedar" or "Agba Wood." Timber known as "Rhodesian Mahogany." The resin has some commercial value. Wood similar to mahogany. Seeds are edible. Often planted around temples as a sacred tree. Valued for ornamental flowers and fast growth.

~

continued

~

~

Table 6.1.

Continued. Secondary products

Subfamily Genera

Fodder Forest or Products Forage

Sindora

x

Swartzia Tamarindus

x

Agroforestry Nurse Trees

x

Orna- Pharmemental ceutical Soap

x

x

X

Trachylobium

Gum

Fish Stupe- InsectiTannin fier cide Comments

X

X X

X

X

Vouacapoua Zollernia Papilionoideae Abrus

Large trees. "Supa Oil" is extracted from the sap which drips from cuts in the bark. Pods used for cattle feed. Pulp in the seed pod used as a confection, beverage and an alcoholic drink. Oil from seed used as a varnish. Seeds are edible or ground into a starch. High quality resin used for varnishes. Timber known as "Partridgewood" from Brazil.

X

X

Jequirity seeds were used by goldsmiths in Asia as an official, uniform measuring weight. Each seed is approximately 0.133 g. Seeds contain abrin and are extremely toxic.

Table 8.1.

Continued. Secondary products

Subfamily Genera

Fodder Forest or Products Forage

Agroforestry Nurse Trees

Orna- Pharmemental ceutical Soap

Gum

Fish Stupe- InsectiTannin fier cide Comments

x

Alhagi

Planted for soil erosion. A. camelorum has become an

Amburana

X

Ammothmnus

X

Amphora Anagyris'

Andira

Antheoporum

X

X X

X

X

X

X

X

introduced weed pest in the southwest USA. Sweet gum exudation eaten by native Arabs. Oil extracted from the seeds are used as a fragrance to scent soaps and tobacco products. Source for quinolizidine alkaloids. Produces a red dye used for coloring silk. Pods contain an extract with insecticidal properties. Leaves used to produce "Herba Anagyris," a purgative used in Greece. Bark and seeds contain the alkaloids berberine and andirine. Seed contains small amounts of rotenone.

I:\)

'I

en

continued

_

...

-

_---_. __

~

..

~

..

~

..

_

_

_

....

-

_._----

~

_~_.-

-

..

~

_

-

-

~

_

....

.

~

~

Continued. _

Table 6.1.

.

-..:J O'l

~

N

_

276

..

_---

Secondary products Subfamily Genera

Fodder Forest or Products Forage

Astragalus

Baphia

X

Barklya Bolusanthus

X

Agroforestry Nurse Trees

X

X

X

X

Gum X

X X

Bossiaea

X

Brya

X

Butea

X

Calpurnia

Orna- Pharmemental ceutical Soap

X

X

X

X

X

X

X

Fish Stupe- InsectiTannin fier cide Comments The largest genus in the legume family. Gum historically important as "Tragacanth Gum." Wood marketed as "African Sandalwood:' B. nitada was a significant source of red dye. Attractive flowering habit earned the name of "Tree Wisteria." Papilionaceous flowers produced on leafless branches. Wood sold as "Jamaican Ebony:' Also called "Torchwood" because of inflamable resins in wood. Bark is a source of fiiber for rope. Flowers make a yellow dye.

Table 6.1.

Continued. Secondary products

Subfamily Genera

Fodder Forest or Products Forage

Caragana Castanospermurn

Agroforestry Nurse Trees

Orna- Pharmemental ceutical Soap

Gum

Fish Stupe- InsectiTannin fier cide Comments Valuable for its tolerance to cold, dry environments. Seeds are edible after roasting. Source of honey. Ornamental species called "Flame Pea." Wood and roots yield a yellow dye. Often called "Bladder Senna," the leaves of some species have the same properties as Cassia. Leaves used as a substitute for tea. Cultivated as "Brooms." Source of quinodizidine alkaloids.

X X

Chamaecytisus Chorizema

X X

X

X

X

Cladrastis

X

Colutea

X

X X

X

X

X

X

X

X

Cyclopia Cytisus

Dahlstedtia Dalbergia

N '1 '1

Dalea

X X

X

X

Important timber crop commonly called "Rosewood" because of the fragrance.

X

continued

Table 6.1.

Continued. Secondary products

Subfamily Genera

Fodder Forest or Products Forage

Agroforestry Nurse Trees

Orua- Pharmemental ceutical Soap

Geoffroea

Gliricidia

X

X

Gourliea

X

X

Hovea

~

~

X

X

X

X

Jacksonia

to

X

X

Inocarpus

Laburnum Lonchocarpus

Gum

Fish Stupe- InsectiTannin fier cide Comments

X

X X

X X

Roasted seeds of G. superba are edible. Bark extracts have been used medicinally. Commonly used to shade coffee or support vanilla vines. Pods eaten by cattle and also fermented to make an alcoholic beverage. Attractive Australian ornamental. Edible seed used as a food stuff by natives. Called "Polynesian or Tahitian Chestnut." Australian "Golden Rain Tree." Seeds are extremely toxic. One of the "Rain Trees" of Africa where an insect exudes water while feeding on the tree's sap producing "Rain." Roots contain large amounts of Rotenone. continued

t\J

(Xl

Table 6.1.

Continued,

0

Secondary products Subfamily Genera

Fodder Forest or Products Forage

Lotononis Maackia

Agroforestry Nurse Trees

Orua- Pharmemental ceutical Soap

X X

X

Millettia

X

X X

Mucuna

X

X

X

X

Myroxylon

X

Olneya

Piscidia

X

X

Mundulea

Ougeinia Pericopsis

Gum

Fish Stupe- InsectiTannin fier cide Comments

X X X

X

X

X

X

X

Has potential as a pasture legume. An Asian ornamental similar to the North American Cladrastis. Drooping flower panicles resemble Wisteria. Primarily used as a cover crop. Cooked, unripe seeds are edible. A source for LDopa. Alkaloids originally thought to be rotenone, but alkaloids have a high human toxicity. Source of "Peru Balsam" oil used as a perfume. Gum used as a flavoring. Southwestern U.S. "Sonoran Desertwood,"

X

X

X

Asian species produce a fine product for woodworking. Called "Fish-Fuddle Tree."

Table 6.1.

Continued. Secondary products

Subfamily Genera

Fodder Forest or Products Forage

Agroforestry Nurse Trees

Orna- Pharmemental ceutical Soap

Platymiscium Pongamia

X X

X

X

Pterocarpus

X

X

X

Robinia

X

X

X

Sarothamnus

X

Sesbania

X

Sophora

Gum

Fish Stupe- InsectiTannin fier cide Comments

X

X

X

X

X

X X

X

X

X

X

Large trees native to Brazil. Seed extracts have potential medicinal value. Large trees indigenous to the tropics. Gum is red and used to produce dyes. Important timber crop in India. Planted to reclaim land strip mined for coal in the U. S. Flowers attractive for honey production. Bark yields a yellow dye. Stems used to make brooms. Flowers, buds and pods are edible. Valued for soil enrichment, erosion control and forage for the tropics. Scholar tree of the Orient. Alkoloids present in Sophora are of commercial interest. "Mescalbean" has hallucinogenic properties.

l\,)

CXl ....

continued

~

Table 6.1.

Continued.

~

Subfamily Genera

Fodder or Forest Products Forage

Agroforestry Nurse Trees

Sphenostylis Taralea

Ulex

Orna- Pharmemental ceutieal Soap

Gum

Fish Stupe- InsectiTannin fier cide Comments

X X

Tephrosia

Tipuana

Secondary products

X

X

X X

X

X

X

X

X

Virgilia

X

Wisteria

X

X

X

Flowers, seeds and storage roots are edible. An industrial quality oil is extracted from the seeds. An alternative to Derris for rotenone production. Also contains tephrosin, an alkaloid with similar properties to rotenone. Ornamental tree called the "Pride of Bolivia." Often grown for soil erosion control or as a "Living Fence." Limited use as of fodder because of presence of alkaloids. The common Africana name means "Choice Tree" reflected in the showy fragrant flowers. An attractive ornamental vine with flowering panicles reaching up to two feet.

Table 6.1.

Continued. Secondary products

Subfamily Genera Mimo8oideae Acacia

Fodder or Forest Products Forage

X

Adenanthera

X

Albizia Anadenanthera

X X

Agroforestry Nurse Trees

Oma- Pharmemental ceutical Soap

X

X

X

X

X

X

X

X

Gum

Fish Stupe- InsectiTannin fier cide Comments

X

X

X X

X

Largest genus in mimosoid subfamily. Acacia contains many agronomically important species which have been used for centuries. Yields high quality and commercially important "Gum Arabic." Circassian seed has been used as a standard measurement (0.26-0.32 g) for evaluating jewel weight. Ornamental seeds used for jewelry.

X

X

X

X Seeds are used to produce an hallucinogenic snuff. Its use was first described in 1496 after Columbus' second voyage to the new world.

N

O:l

w

continued

N

CD

"'"

Table 6.1.

Continued. Secondary products

Subfamily Genera Calliandra

Fodder Forest or Products Forage X

X

X

Cassia

Dichrostachys

X

Entada

X

Agroforestry Nurse Trees

Orna- Pharmemental ceutical Soap

X

X

Gum

X

Fish Stupe- InsectiTannin fier cide Comments

X

X

X

X

X

X

X

A large genus containing ornamental trees known as "Powder Puffs." Extracts from the bark of C. houstonH have been used as a substitute for quinine. A large genus comprising trees, shrubs and herbs. The woody species have generally been used as handsome ornamentals and shade for tea and coffee plantations. Leaves of some species are the source of the laxative senna. Species used to reforest cleared areas. Pods eaten by wildlife species. Bark decoctions used as native medicines. Noted for large size of pods which have been used to make jewelry boxes.

Table 6.1.

Continued. Secondary products

Subfamily Genera

~

00

Fodder Forest or Products Forage

Enterolobium

X

lnga

X

Leucaena

X

Lysiloma

X

Mimosa

X

Agroforestry Nurse Trees

Oma- Pharmemental ceutical Soap X

X

Gum

Fish Stupe- lnsectiTannin fier cide Comments X

X

X

X

X

X

X

X

X

X

X

X

"Elephant Tree" refers to shape and size of pod. Gum used in native medicines. Historic association as a companion crop for shading coffee and cacao. Edible pods and seeds calleld "Ice Cream Beans" are used to flavor desserts. Nutritious fodder crop. However. its use is limited to ruminants because of a toxin "Mimosine." Initial symptoms include hair loss. Important timber tree in Central America but is in small supply. Genus contains the novel sensitive plants. The leaves respond to touch or temperature by folding leaflets. The common name mimosa also applies to Acacia and Albizia.

C11

continued

IS)

CD CD

Table 6.1.

Continued. Secondary products

Subfamily Genera

Fodder Forest or Products Forage X

Agroforestry Nurse Trees

Parkia

X

Pentaclethra

X

Piptadenia

X

Pithecellobium

X

X

X

Prosopis

X

X

X

X

Orna- Pharmemental ceutical Soap X

X

Gum

X

Fish Stupe- InsectiTannin fier cide Comments X

X

X

X

X

X

X

Flowers pollinated by bats. Pulp from pods used as a native food stuff. Seeds roasted and eaten. Seed contain a high percentage of oil extracted for soap or lubricants. Seeds are edible. Oil called "Owala Butter." Seed extracted to form an hallucinogenic substance used by natives of South America. A large genus containing species used for ornamentals. dyes. coffee substitutes and edible seeds. Useful plants for arid areas with both ecological and economic value. Common called "Mesquite." Pods are edible to cattle and humans. Used to make alcoholic beverages (mescal). Source for a honey crop in Hawaii.

Table 8.1.

Continued. Secondary products

Subfamily Genera

~

Q)

-..:J

Fodder Forest or Products Forage

Samanea

X

Tetrapleura

X

X

Agroforestry Nurse Trees X

Orna- Pharmemental ceutical Soap

Gum

Fish Stupe- InsectiTannin fier cide Comments Species for S. saman grow to tremendous size and spread. Leaflets fold at night. Pods eaten by livestock.

X

X

X

288 Table 6.2.

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

J. E. PREECE

Potential economic and ecological importance of woody legume species.

Soil Improvement • nitrogen fixation • erosion control, wind breaks • reclamation of mining sites • green manure

Human Consumption or Utilization • pods or seeds are high sources of protein • • •

gums, oils secondary products, pesticides ornamental value

Timber • lumber, carpentry • pulp for paper production

Livestock Interactions • fodder or forage • "living fences" to limit animal movement

•

•

fuel wood or charcoal

shade to reduce temperatures

Nurse Crops • shade for specialty crops (coffee, tea, cacao) • support for specialty vine crops (vanilla, black pepper)

There has been relatively little effort expended toward crop improvement either through conventional or unconventional breeding efforts considering the potential usefulness demonstrated by woody legumes. Tissue culture is becoming an important component of an integrated approach toward crop improvement (ie. in Lueceana spp. and Prosopis spp.). Tissue culture offers great potential for the rapid multiplication of superior individuals. This is important for many woody legume species that have long maturation periods or are difficult to multiply through conventional vegetative propagation. Several important species cannot easily be improved through selection of "elite" trees for seed production owing to self-incompatibility barriers or low seed viability. Tissue culture may eventually provide the primary means for the clonal propagation of superior individuals or can serve to enhance conventional breeding efforts by increasing individuals from intra- or interspecific hybrids. Tissue culture also provides an alternative for production of alkaloids and other secondary products. Woody legumes continue to be an important source for useful secondary products. This review's objective is to highlight the ecological and economic importance of woody legumes and to provide an overview of tissue culture protocols used for the multiplication or improvement of selected species. Additionally, the use of tissue culture techniques for the production of secondary products and basic studies of nitrogen fixation is reviewed.

6.

TISSUE AND CELL CULTURES OF WOODY LEGUMES

289

II. IN VITRO PROPAGATION A. Axillary Bud Proliferation

The successful propagation of woody perennial species through tissue culture has been a relatively recent accomplishment (Winton 1978; Dunstan and Thorpe 1986). Many woody angiosperms are amenable to standard micropropagation systems that employ axillary bud proliferation for shoot multiplication in vitro (Dunstan and Thorpe 1986; Zimmerman 1986). The basic micropropagation protocol has been detailed in previous reviews (Murashige 1974; Hu and Wang 1983). Micropropagation usually involves four stages including initiation of cultures, multiplication of shoots, rooting of shoots, and acclimatization of plants. Many woody species respond on various modifications of an MS medium, but some benefit from lower salts media based on B5 or WPM. The multiplication stage usually requires a medium containing either a cytokinin or a combination of cytokinin and auxin for multiple shoot development. Resulting shoots can be used for remultiplication or rooted either in vitro or ex vitro. Root formation may require an auxin treatment (Dunstan and Turner 1984). Micropropagation through axillary bud proliferation is often preferred by commercial tissue culture laboratories because there is less clonal variation when compared to systems where shoots arise through adventitious means (Dunstan and Thorpe 1984; Geneve 1990). The woody species in the Leguminosae that have been cultured successfully in vitro from apical or axillary meristems are listed in Table 6.3 along with a summarized protocol. The list is relatively large considering the generally recalcitrant nature of woody species and the difficulty many researchers have encountered with some of the commercially important herbaceous members of the legume family (Hammatt et al. 1986). Although this list contains only a handful of the woody legume species with potential for economic exploitation (see Table 6.1), woody legumes have received attention comparable to that given other woody angiosperm families (Rosaceae, Ericaceae, Salicaceae) for in vitro culture. Commercial tissue culture laboratories have micropropagated several woody legumes, such as Gymocladus (McCown and Barker 1989), but detailed information concerning this work is usually unavailable in the literature. Many of the difficulties encountered during the micropropagation of woody legumes are common to woody plant micropropagation in general and are directly attributable to the woody nature of the explants and their maturation status. Most notably, difficulties arise because of excessive phenolic-like (dark colored) exudation associated with culture initiation and the recalcitrant nature of shoot or root formation related to the ontogenetic stage of maturity inherent in a perennial woody crop.

~

co

0

Table 6.3.

Axillary bud prolifera tion of woody legume species. Explant Source

Species

Seedling

Acacia alb ida DeIile

Nodal stem segment Nodal stem segment In vitro derived shoots

A. auricuIif ormis A. Cunn, exBenth . A. koa A. Gray

A. ligulata A. Cunn, exBenth .

Nodal stem segment Nodal stem segment

A. melanox ylon R. Br. Albizia odoratissima (WiIld.) Benth. A. procera (Rom.) Benth. Ceratoni a oreothau ma Hill, Lewis and Verd,

Mature

Shoot tip Nodal stem segment Nodal stem segment

Shoot Formati on Medium

PGR (IlM)

Modifie d MS

Root Formati on Medium

PGR (IlM)

Acclima tized

Referenc e

26.9 NAA + Modifie d 13.3 BA MS

None

Yes

Duhoux and Davies 1985

B5

1 BA

B5

1NAAo r1 lAA

Not reported

Mittal et aI. 1989

Modifie d MS

4.4 BA

Modifie d 1.5IBA

Yes

Skolmen and Mapes 1978

Modifie d MS

0.5 KIN 0.5 BA

Modifie d MS

None

Yes

William s et al. 1985.

MS

1-5 lAA + 1 BA

MS

10 IBA

Not reported

Meyers and van Staden 1987

MS

4.4 BA

1/2

Yes

MS

8.9BA

1j2 MS (no agar)

4.9 IBA or 5.71AA 4.9 IBA + 2.2 BA

Yes

Phukan and Mitra 1983 Roy and Datta 1985

MS

4.4-8.9 BA

1/2

Yes

Woods 1985

+

MS

MS

2.9-5.7 lAA

Table 6.3.

Continu ed. Shoot Formatio n

Explant Source

Root Formati on Acclima tized

Referenc e

Yes

Sebastia n and McComb 1986

4.9 IBA

Not reported

Thomas and Mehta 1983

Ex vitro

2460 IBA 30 min basal dip

Yes

Bennett 1987

10 BA

1/2 WPM or ex vitro

300 IBA for 15 days

Yes

Yusnita et aI. 1990; Geneve et al. 1990b

MS

10 BA

112 MS

10 NAA

Yes

Grubisic and Culafic 1986

-

8.9 BA

Liquid

1.5IBA

Yes

Bignami 1984

PGR (,!M)

Species

Seedling

Mature

Medium

PGR (J!M)

Medium

C. siliqua L.

Shoot tip

Nodal stem segment Shoot-ti p or nodal stem segment

MS

5 ZEA

10 IBA 1/2 MS 1 wk in dark

Modifie d MS

Initiatio n 5.4 NAA+ 8.9 BA multiplication 0.5 IBA + 2 BA+ 5.8 GA

Modifie d MS

WPM

22.2 BA

WPM

C. siliqua

Cercis canaden sis var. mexican a L.

Nodal stem segment

C. canaden sis var. alba L.

Nodal stem segment Shoot-ti p or nodal stem segment

C. siliquast rum L.

C. siliquast rum

Nodal stem segment

Nodal stem segment

continue d N

c.o

....

N

co

N

Table 6.3.

Continued. Explant Source

Shoot Formation

-Species

Cladrastis lutea (Michx.) K. Koch Dalbergia latifoIia Roxb.

D. sisson Roxb. ex DC

Seedling

D. sissoo

Gleditsia triacanthos L.

Mature

Medium

PGR(p.M)

Medium

Axillary bud

MS

2.2-4.5 BA

1/2

Modified MS

4.4-13.3 BA + 2.3 KIN

1/2

MS

Modified MS Modified MS

Shoot-tip or nodal stem segment from in vitro derived shoots Nodal stem segment from in vitro derived shoots

D. sissoo Shoot-tip

Root Formation

Axillary bud Nodal stem segment

Nodal stern WM segment

PGR(p.M)

Acclim atized

Reference

MS

4.9 IBA

No

MS

5.4 NAA+ 5.7 IAA + 4.9 IBA for 72 h

Yes

Weaver and Trigiano 1989 Ravishankar Rai and Chandra 1989

1.1BA

Ex vitro

O.Ol%IAA 5 minute basal dip

Yes

Suwal et al. 1988

2.7NAA+ 4.4 BA 2.9 IAA + 4.6 KIN

Modified MS Modified MS (reduced agar cone.) -

2.7NAA+ 4.4 BA 2.9 IAA + 4.6 KIN

Yes

Dawra et al. 1984.

Not reported

Datta et al. 1983

5.4 NAA+ 0.4 BA

-

Rogozinska 1968

Table 6.3.

Continued. Explant Source Seedling

Mature

Medium

PGR (}.tM)

Medium

PGR(JA.M)

Acclimatized

Reference

GymnocIadus dioicus L.

Nodal stem segment

Nodal stem segment

Modified WPM

10 BA

573 NM for 2 minutes basal dip

Yes

Geneve et a1. 1990a

Leuceana Ieucocephala (Lorn.) de Wit

Nodal stem segment (seedling to 2 yrs old)

Modified MS

3 BA

Seedling derived microshoots rooted ex vitro l/2MS

5IM

Yes

Dhawan 1988; Dhawan and Bhojwani 1987a; c; 1985, 1984

MS

5.7IM

MS

5.7IM

Not reported

Datta and Datta 1985

Initiation MS multiplication l/2MS

Initiation 8.9-13.3 BA multiplication none

1/2

9.8 IBA+ 0.2 KIN

Yes

Goyal et a1. 1985

Modified MS

13.3 BA + 13.9 KIN

-

-

Not reported

Dhawan 1988

L. leucocephala

Ougeinia dalbergioides Benth. N

W

Root Formation

Species

L. leucocephala

to

Shoot Formation

Nodal stem segment Nodal stem segment (14-mo.old plant) Nodal stem segment

MS

continued

N

co

N::>

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.

TISSUE AND CELL CULTURES OF WOODY LEGUMES

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.

TISSUE AND CELL CULTURES OF WOODY LEGUMES

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.

TISSUE AND CELL CULTURES OF WOODY LEGUMES

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

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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

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

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.

TISSUE AND CELL CULTURES OF WOODY LEGUMES

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

Medium and Regulators

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

Medium and Regulators

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

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

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

6.

TISSUE AND CELL CULTURES OF WOODY LEGUMES

321

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

322

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

J. E.

PREECE

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.

6.

TISSUE AND CELL CULTURES OF WOODY LEGUMES

323

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.

324

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PREECE

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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50:199--204. Sebastian, K. T.. and J. A. McComb. 1986. A micropropagation method for carob (Ceratonia siliqua L.). Sci. Hort. 28:127-131. Sha, L., B. H. McCown. and L. A. Peterson. 1985. Occurrence and cause of shoot-tip necrosis in shoot cultures. J. Am. Soc. Hort. Sci. 110:631-634. Sharma, S., and N. Chandra. 1988. Organogenesis and plantlet formation in Dalbergia sissoo Roxb. J. Plant Physiol. 132:145-147. Sharma, R., and P. Khanna. 1975. Production of rotenoids from Tephrosia spp. in vivo & in vitro tissue cultures. Indian J. Expt. BioI. 13:84-85. Shekhawat. N. S. andA. Kackar.1987. ProtoplastsofProsopsiscineraria (L.)Duce: isolation and culture. Nitrogen Fixing Tree Res. Rpt. 5:51-53. Sinha, R. K., and R. Mallick. 1989. In vitro propagation of Sesbania grandiflora [L.) Poir through callus culture. Cell and Chromosome Res. 12:1-8. Skolmen, R. G. 1986. Acacia (Acacia KoaGray). p. 375-384. In: Y. P. S. Bajaj(ed.). Biotechnology In Agriculture and Forestry. Vol. 1. Trees I. Springer-Verlag, Berlin. Skolmen. R. G., and M. O. Mapes. 1978. After care procedures required for field survival of tissue culture propagated Acacia koa. Proc. Int. Plant Prop. Soc. 28:156-164. ___ . 1976. Acacia koa Gray plantlets from somatic callus tissue. J. Hered. 67:114-115. Smith. M. A. L., and A. A. Obeidy. 1991. In vitro rescue of a mature male Kentucky coffeetree (Gymnoc1adus dioicus L.) genotype. HortScience 26:749. Stuart. D. A.. J. Nelson, S. G. Strickland, and J. W. Nichol. 1985. Factors affecting developmental processes in alfalfa cell cultures. p. 59-73. In: R. R. Henke. K. W. Hughes, M. P. Constantin. and A. HOllaender [eds.). Tissue Culture In Forestry and Agriculture. Plenum Press, New York. Sudhadevi. A. M.. and K. Nataraja. 1987a. Establishment of planUets in hypocotyl cultures of Dalbergia latifolia Roxb. Indian J. For. 10:1-6. ___ . 1987b. In vitro regeneration and establishment of plantlets in stem cultures of Dalbergia latifolia Roxb. Indian Forester 113:501-506. Suwa!' B.. A. Karki, and S. B. Rajbhandary. 1988. The in vitro proliferation of forest trees. 1. Dalbergia sissoo Roxb. ex Dc. Silvae. Genet. 37:26-28. Tabone, T. J., P. Felker. R. L. Bingham, I. Reyes. and S. Loughrey. 1986. Techniques in the shoot multiplication of the leguminous tree Prosopis alba clone Hz Vso • For. Eco!. Mgt. 16:191-200. Thomas. E. 1981. Plant regeneration from shoot culture derived protoplasts of tetraploid potato (Solanum tuberosum.) Plant Sci. Lett. 23:81-88. Thomas. V.. and A. R. Mehta. 1983. Effect of phloroglucinol on shoot growth and initiation of roots in carob tree cultures grown in vitro. p. 451-457. In: S. K. Sen and K. L. Giles (eds.). Plant Cell Culture In Crop Improvement. Plenum Press, New York. Tomar, U. K.. and S. C. Gupta. 1990. Some factors affecting somatic embryogenesis in callus cultures of a leguminous tree-Albizia richardiana King. VII IntI. Congr. Plant Cell and Tissue Cult. Abstr., Amsterdam. The Netherlands, p. 253. ___ . 1988a. In vitro plant regeneration of leguminous trees (Albizia spp). Plant Cell Rpt. 7:385-388. ___ . 1988b. Somatic embryogenesis and organogenesis in callus cultures of a tree legume-Albizia richardiana King., Plant Cell Rpt. 7:70-73. ___ .1986. Organogenesis and somatic embryogenesis in leguminous trees (Albizia spp.) VI IntI. Congr. of Plant Tissue and Cell Cult. Abstr.. Minneapolis. MN. p. 27. Trigiano, R. N., and R. M. Beaty. 1989. Direct and indirect somatic embryogenesis in cultures of Cercis canadensis. HortScience 24:129. Trigiano, R. N., R. M. Beaty. and E. T. Graham. 1988. Somatic embryogenesis from immature embryos of redbud [Cercis canadensis.) Plant Cell Rpt. 7:148-150.

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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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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

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MIKLOS FAUST AND SHIOW Y. WANG

I--?

~

ARGININE

~O~ ~0 a

ASPARTATE

~ULLINE ORNITHINE

AGMATINE

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NHa~

PUTRESCINE ~ SPERMIDINE ~ SPERMINE

5 -METHYLTHlOAOENOSINE

IMTA)

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S-ADENOSYLMETHIONINE IAdoMetl

® ADENINE

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Figure 7.1. Schematic ill ustration of polyamine biosynthetic pathways. Note: Not all intermediates or endproducts are shown: (1) Ornithine decarboxylase (ODC); (2) Arginine Decarboxylase (ADC); (3) Spermidine synthase; (4) Spermine synthase; (5) MTA nucleosidase; (6) ACC synthase; (7) AdoMet decarboxylase.

activity. When elongation and nonmitotic processes are affected ADC activity is involved (Galston and Kaur-Sawhney, 19B7a). In most cases where putrescine accumulation was due to stress, ADC was activated by de novo synthesis and ODC inhibitors were ineffective to block putrescine biosynthesis. This prompted Galston and Kaur-Sawhney (1990) to label ADC as a general stress enzyme. Spermidine and spermine are synthesized from putrescine by addition of aminopropyl groups. One or two aminopropyl group(s) is/are transferred from decarboxylated S-adenosylmethionine (AdoMet), which is an intermediate of ethylene biosynthesis. This interlinks polyamine and ethylene biosynthesis. Alternatively, AdoMet can be metabolized successively to 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene. Because of this interlink between polyamine and ethylene biosynthesis, the question has been raised as to whether competition exists for AdoMet between the ACC-ethylene pathway and spermidine and spermine biosynthesis (Even-Chen et al. 1982). While in carnation flowers the two uses of AdoMet appear competitive (Roberts et al. 1984),

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in avocado during fruit development and ripening (Kushad et al. 1988) and in water-stressed apple leaves (Wang and Steffens 1985) no competition was observed. In general, the enzymes of amino acid metabolism are compartmentalized in plant cells (Miflin and Lea 1977); this appears to be the case for polyamine metabolism as well (Slocum et al. 1984). ODC is mostly associated with nuclear chromatin, although cytoplasmic ODC activity has been detected in most extracts (Slocum et al. 1984). ADC is located in the cytosol, whereas spermine synthase, once reported to occur in the chloroplast (Cohen et al. 1981), now is recognized not to be compartmented into specialized organelles (Yamanoha and Cohen 1985).

III. POLYAMINES AND PLANT DEVELOPMENT

A. Embryogenesis Changes in polyamine levels are associated with morphogenetic changes and with developmental processes of plants. Such associations have been detected during both in vivo differentiation and in vitro morphogenesis. Considering the effects of polyamines on morphogenesis, we have to distinguish morphogenetic effects (1) that are associated with rapid growth and/or macromolecular synthesis, such as fruit set, and (2) those where cell division need to be temporarily arrested for morphogenesis to follow, such as in case of flower bud initiation. It is not clear in which category somatic embryogenesis or root initiation should be classified. In both cases initiation, a qualitative change that may require arresting of cell division temporarily, is followed by a macromolecular synthesis and visible growth. Because initiation of both embryogenesis and root formation cannot be readily determined, these processes can only be measured by the appearance of embryos or roots, which also include the growth phase. It is probable that the growth phase of embryogenesis or root growth require polyamines, but whether exclusion of polyamines for initiation of these processes is necessary or not is unknown. The evidence points toward a requirement for polyamines during the overall differentiation process. Carrot cells increase putrescine content when shifted from proliferation medium to 2,4-D free embryogenesis medium (Montague et al. 1978). These increased levels are correlated with increased ADC activity (Montague et al. 1979). The addition of DFMA (an ADC inhibitor) to carrot cultures blocked the transition to embryogenesis, which could be relieved by addition of putrescine (Feirer

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MIKLOS FAUST AND SHIOW Y. WANG

et a1. 1984). The spermidine synthesis inhibitors, di-cyclohexylamine (DCHA) and methylglyoxal-bis(guanylhydrazone), (MGBG) reduced growth, embryogenesis and spermidine levels in carrot cultures (Feirer et a1. 1985). In carrot suspension cultures DFMO had no significant effect on the growth of carrot cells (Fallon and Phillips 1988), but embryos treated with DFMO developed into normal plants at a higher rate than the untreated embryos (Mengoli et a1. 1989). Embryogenesis usually requires 2,4-D free medium, whereas the presence of 2,4-D promotes callus formation. When differentiating embryos were transferred from minus 2,4-D medium to plus 2,4-D medium containing DFMO embryogenesis continued, but when DFMO was withdrawn, all cells reverted to unorganized growth within a few days (Minocha 1988). By using carrot cells and the inhibitors DFMA and DFMO and auxinfree or auxin-enriched media, Robie and Minocha (1989) came to the conclusion that DFMO promotes polyamine biosynthesis by decreasing the levels of S-adenosylmethionine and ethylene biosynthesis. This allows embryogenesis to occur in the presence of auxin because ethylene inhibits embryogenesis and auxin is known to promote ethylene synthesis (Minocha et a1. 1990). This theory is underlined by the use of a mutant line of carrot (WOOlC) that contains high internal levels of auxin and would not go through embryogenesis even when p laced on auxin-free medium. This mutant also fails to increase ADC and AdoMet decarboxylase levels (Fienberg et a1. 1984). A lag period exists after the cultures are exposed to embryogenic conditions and polyamine synthesis. This is very similar to that observed in flower bud development (discussed later) and raises the question whether the induction process and the later observed increase in polyamines are one or two processes. The dramatic increase in polyamine levels occurs from 10 to 24 days after cultures are transferred to embryogenic medium in Passiflora (Desai and Mehta 1985), and in carrot 24 h after cells are transferred to embryogenic culture (Montague et a1. 1978). This is a long lag period, especially in case of Passiflora, and perhaps induction has taken place before the increase in polyamines and, in turn, their interaction with other factors to promote embryogenesis. Desaie and Metha (1985) concluded based on histological evidence that the rise in putrescine occurred before the appearance of root and shoot primordia. However, the histological evidence of morphogenesis is a relatively late event. The event of induction, the first step in morphogenesis, and its dependence on polyamines is not known at this time. Induction versus morphogenesis will be discussed in more details in connection with flowering and root formation. Exogenously applied putrescine was successful in promoting

7.

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regeneration of plants. In apple leaf tissues, treatments that stimulated regeneration were more effective in the presence of putrescine Games et a1. 1988). Naphthaleneacetic acid (NAA) interacted with spermidine in regeneration of Ipomoea, whereas the interaction with putrescine was not clear (Eilers et a1. 1988). Differentiation of protoplast cultures of Petunia were inhibited by DFMO and DFMA and this effect was only partially reversed by putresine or spermidine (Kaur-Sawhney et a1. 1989). B. Rapid Growth As mentioned before, polyamines usually increase when rapid cell division occurs. Polyamine titers parallelled changes in macromolecular synthesis in Phaseolus cotyledons and shoots (Bagni 1970). Similar association has been reported between polyamine levels and growth of the embryonic axis of Lathyrus seedlings (Ramakrishna and Adiga 1975). Maturation of tomato ovaries following fertilization (Cohen et a1. 1982), or auxin-induced parthenocarpic development (Mizrahi and Heimer 1982) also appear to be regulated by polyamine availability. In apple, during the period after bloom, polyamine content was high when fruit set occurred and protein synthesis was rapid (Biasi et a1. 1988). Putrescine, when applied exogenously during the beginning of bloom, increased fruit set of apple (Costa et a1. 1986), olive (Rugini et al. 1986) and pears (Crisosto et a1. 1986). Polyamine synthesis is also associated with intense growth processes during in vivo tuber formation of Jerusalem arthichoke (Bagni et a1. 1983). C. Flower Initiation Apparently polyamine synthesis must be inhibited for initiation of the differentiation process. In Iris hollandica, spermidine is a marker for floral induction (Fiala et a1. 1988). In Brassica callus cultures cell differentiation does not occur (Sethi et a1. 1988) until the cells move through the G1-S-G2 phases of cell cycle and culminate in mitosis. However, when the cellcycle is arrested in the G1 or G2 phases differentiation starts (Sethi et a1. 1988). Addition of polyamine to callus cultures increased growth, and further dramatic increase in growth was obtained when both polyamine and IAA were combined in the media (Sethi et a1. 1988). MGBG retarded polyamine synthesis and cell division, but increased cell differentiation (Sethi et a1. 1988). Polyamine levels of potato also change when differentiation occurs. Free polyamine levels are low in the ovules as cellular endosperm develop, but putrescine and spermidine levels increase before the latter stages of embryogenesis (Olson and Novak 1988). From these data it appears that polyamines are low at the time of

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MIKLOS FAUST AND SHIOW Y. WANG

flower or embryo initiation and they increase only when embryogenesis is at its later stages of development. There are a number of circumstances that indicate a lag period between the initiation and evocation of flower development. Bernier et a1. (1981) describes the need for cell synchronization to occur before evocation of floral development in several test plants. Apparently, during the evocation, cells wait (arrested) in the G2 stage until all the cells in the apical dome reach this stage and become synchronized. Synchronization is followed by rapid cell division when the differentiation-associated growth begins. This lag period corresponds well with the data of Sethi et a1. (1988) and may be also related to the requirement of low polyamine levels. In apples, the time of flower bud initiation is separated from the time of morphogenetic changes observable in the bud by about 4 to 6 weeks. The morphogenetic changes are associated with macromolecular development (Buban and Faust 1982). It is well known that N supplied in the form of ammonium to apples trees for as little as 24 h greatly promotes flower bud formation (Edwards 1986). Arginine concentrations rise in the stems subtending axilliary buds following exposure to ammonium (Edwards 1986). Arginine is a precursor of polyamines. Infusion of polyamines induced the same response in flowering as ammonium ions (Edwards 1986). It must be noted that the buds, in all probability, passed the flower initiation stage when Edwards applied ammonia and they have been in the stage when macromolecular synthesis occurs. Thus it is understandable that either ammonium or polyamines had a positive effect on flower morphogenesis but not on initiation. In citrus, flower formation is promoted by drought or low temperature. Lovatt (1990) monitored leaf N content during drought or low temperature exposure. In oranges exposed to low temperature stress, the total leaf N did not change but the ammonia fraction accumulated in proportion to the stress and the trees produced more flowers. The accumulation of ammonia also induced a de novo arginine biosynthetic pathway. Similar results were obtained in lemons, either by subjecting the trees to water deficit or by foliar applications of urea. Both increased the leaf concentration of ammonia. It is not clear when the initiation of flowers occurs in relation to the increase of ammonia levels in the citrus leaves and whether the response is mediated through polyamines or not. Initiation of flower buds of temperate zone fruit trees is followed by differentiation, a process that involves limited growth and proceeds through dormancy. Buds resume growth after dormancy. In cherry flower buds, polyamines (putrescine, spermidine, and spermine) are present in all stages of bud development after leaf fall (Wang et a1. 1985). Polymine

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content in the buds of Prunus avium and P. serrulata is low during dormancy and increases rapidly with the beginning of active metabolism and resumption of growth (Wang et ai. 1985). The level of polyamine per unit of DNA in the activly growing buds is much higher than in the dormant buds. It appears that if differentiation requires polyamines, the requirement is not as high as required for active growth to occur. The ratio of polyamines (nmol) to DNA (p.g) increased from 2.78 in differentiating dormant buds to 5.56 in actively growing buds of P. avium and from 3.86-5.56 in P. serrulata (Wang et aI. 1985). In this case, the increase of polyamines did not occur during differentiation, but only when active growth was resumed.

D. Pollen Formation Information on the effect of polyamine on pollen tube growth is limited. Increases in polyamine concentration precedes the emergence of pollen tubes in apple (Bagni et aI. 1981). Spermidine-stimulated pollen tube growth in Catharanthus roseus at 10-5 M and the inhibitory effect of MGBG was reversed by 5 X 10-4 M spermidine (Prakash et ai. 1988). Germination and pollen tube growth of lily pollen was inhibited by DFMO and DFMA and putrescine or spermidine totally reversed this effect. Incubation of pollen in polyamines increased pollen tube growth (Rajam 1989). In apple pollen, polyamines decrease RNAse activity (Speranza et al 1984). In vitro studies indicate that the inhibition of nucleases by polyamines may depend on the interaction of polyamines with the substrate and not with the enzyme. Nevertheless, this may indicate the mechanism that may operate also in vivo (Smith 1985). Polyamines may prevent pollen formation. The floral organs of male sterile, stamenless mutant of tomato contain significantly higher level of polyamines than those of the normal plants (Rastogi and Sawhney 1990a). All 3 polyamines were higher in the sepals, petals, stamens, and gynoecium of the mutant plant compared to the normal plants. Low temperature restored normal stamen development in the mutant and this was associated with lower polyamine concentrations and ODC activity (Sawhney 1983). When excised normal flowers were cultured in vitro on putrescine-or spermidine-supplemented media, putrescine at 10-4 M inhibited stamen formation; and at 10-3 M concentration reduced the growth of all parts of the flower. Spermidine inhibited the growth of stamens at a much lower concentration (10~ M) without affecting the growth of the other floral organs. However, at 10-4 M spermidine the other floral organs were also affected (Rastogi and Sawhney 1990b). Similar

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MIKLOS FAUST AND SHIOW Y. WANG

floral abnormalities were reported in Petunia mutants, which were also associated with elevated polyamine levels (Gerats et a1. 1988).

E. Fruit Development There is information that indicates a correlation between polyamine concentrations and fruit development. In apple, polyamine levels, especially bound spermine, are high during the early periods of fruit growth (Biasi et a1. 1988). In orange flowers 80% of total polyamine content is localized in the reproductive organs and they markedly increased during the early stages of flower development (Kushad et a1. 1990). In mandarin orange, polyamine levels (putrescine, spermidine) and ADC and ODC activities were also high during the early stages of fruit development (Nathan et a1. 1984). In avocado, similar correlations have been reported between the cell division and putrescine and spermine levels (Apelbaum 1986; Winer and Apelbaum 1986). Application of putrescine at early stages of fruit development increased tomato fruit growth (Cohen et a1. 1982), fruit set, and yield of apples (Costa et a1. 1984; Costa et a1. 1986) and olives (Rugini et a1. 1986). The increase was especially important in self-incompatible olive cultivars (Rugini and Mencuccini 1985). The results of exogenous application of polyamines is not uniform. Volz and Knight (1986) could not increase fruit set in apples with polyamine treatments. In most fruits, fruit set involves rapid cell division for about 3 weeks after anthesis (Faust 1989). Therefore, correlations between high polyamine levels and fruit set or application of polyamines and positive results in fruit set should be interpreted as an association between polyamine levels and rapid growth regardless of whether this growth occurs in the fruit or in any other organs.

F. Root Formation The possible correlation between root formation and polyamine content of rootlets also have been investigated. In Phaseolus (Jarvis et a1. 1985; Kakkarand Rai 1987) and in mung beans (Friedman et a1. 1982] correlations were observed between polyamine accumulation and initial stages of adventitious root formation. In case of Phaseolus, application of AdoMet decarboxylase inhibitor, MGBG, reduced endogenous levels of spermine and spermidine and inhibited root induction in the absence or presence of indolebutyric acid (Jarvis et a1. 1985). In Kakkar and Rai's studies (1987) spermine was also associated with enhanced rooting. In mung beans, polyamine titers and estimates of RNA, DNA, and protein content were similar and all followed root formation. Shyr and Kao (1985)

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found similarly enhanced rooting with ornithine, as well as spermine and spermidine, although MGBG inhibited rooting. Apparently, in mung beans both pathways ODC and ADC contribute equally to IBAstimulated increase in polyamines (Friedman et a1. 1985). In English ivy (Geneve and Hackett 1990), adventitious root formation proceeds through three stages. The root induction stage (0-6 days) precedes the first cell division; the meristem organization stage (6-9 days) results in root primordia; and the root development and elongation stage (9-18 days) leads to visible root protrusion through the epidermis. The increase in putrescine was associated with the third stage. Ethylene treatment prevented the elongation of root primordia but did not influence the previous steps. There is a differential rooting response between the juvenile and the mature phase of English ivy. Neither polyamine nor ethylene appear to be directly involved in this differential rooting response (Geneve and Kester 1991). Both ethylene and polyamines exert their influence after the induction stage of the adventitious root formation (Geneve and Hackett 1990). Rooting in apple, as measured by the appearance of roots, was associated with an increase in polyamine levels, but in contrast to the reports on Phaseolus, DMFO was more effective than DFMA in reducing rooting (Wang and Faust 1986). Similarly, in cherry, inhibition of polyamine synthesis by DFMA prevented root formation, which was reversed by exogenous application of putrescine (Biondi et a1. 1990). Rooting was not reduced by MGBG. AVG (an ethyene inhibitor) reduced ethylene formation sevenfold, slightly stimulated labeled methionine incorporation into spermine, but did not affect rooting (Biondi et a1. 1990). Bagni et a1. (1983) reported that ODC activity was the greatest at the root tip in Helianthus tuberosum, where cell division is the most active. Therefore it is not surprising that, in general, the ODC pathway is more associated with root formation than the ADC pathway and the polyamine titer in case of root formation closely follows the titer of nucleic acids.

G. Internode Elongation Short internode is usually considered a manifestation of low gibberellin levels in the stem tissue. Dwarf plants of peas, rice, wheat, maize, beans, Cucurbita maxima, and apples were examined for GA content and possible metabolic blocks in the biosynthetic pathway of particular GAs. Daietal. (1982) found that indwarfpeas treated withGA, activity of ADC and the levels of putrescine and spermidine rose in parallel with the promotion of internode elongation by GA. The content of polyamines per gram of tissue remained constant, but, naturally, the

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content per stem section was higher in the longer internode of GA-treated plants. The involvement of polyamine in stem elongation was further implicated by Kaur-Sawhney et a1. (1985) who treated 9-day-old pea seedlings with GA and found an increase ADC 9 h after treatment, whereas stem elongation occured 12 h after the treatment. DFMA added to the GA treatment inhibited 70% of stem elongation. Smith et aL (1985) used genetically different peas that grew to different height and their internode length differed (slender, tall, dwarf and nana). The quantity of polyamines correlated with internode elongation and when DFMO and DFMA were applied they inhibited elongation of internodes. In a subsequent study, Smith (1990) showed that polyamines, indeed, are involved in GA-promotion of internodal growth. She concluded that polyamines are needed for normal growth but not as mediators of GA action. It appeared that polyamines are needed for the cell division phase of internodal growth. IV. STRESS-INDUCED CHANGES IN POLYAMINE CONTENT As indicated at the beginning, there is a strong association between stress and polyamine synthesis. Regardless of the type of stress (nutrient deficiency, cold, drought, or salt induced), polyamines increase. The increase is usually in putrescine and involves the ADC system of biosynthesis. The reason for this accumulation is unknown. It is not clear if polyamines are involved in stress tolerance or if they are simply symptoms of stress. There are several examples of increased resistance to stress being correIated with polyamine levels (Ormrod and Beckerson 1986; Bors et a1. 1989; Kramerand Wang 1989; Krameret a1. 1991) and, as later evidence indicates, they may accumulate as a result of stress but they also may protect cells in case of certain stresses.

A. Nutrient Stress Amino acids usually accumulate as a result of stress (Stewart and Larher 1980). It is well documented that arginine, citrulline, and ornithine accumulate during P deficiency in bananas (Freiberg and Steward 1960), in citrus rootstock (Nemec and Meredith 1981), and citrus and poncirus species (Rabe and Lovatt 1984); this is likely to increase polyamine levels. Putrescine accumulates in potassium deficient peas (Klein et a1. 1979) and black currents (Murti et al. 1971). Murti et al. (1971) suggested that putrescine can compensate for about 30% of the K+ in black current leaves and, thus, it may function in maintaining ionic balance in the tissues.

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Conditions caused by K deficiency can be mimicked by externally applied excess hydrogen ions. When cotyledons of Cucumis sativus were exposed to 5-10 mM HCL (Suresh et a1. 1978), putrescine concentration and ADC activity increased. Even though internal pH may be important in polyamine synthesis, Smith (1984) cautioned that interpretations based on association of polylamine content and altered pH in K-deficient tissues is limited by our incomplete understanding of anion cation balance in such tissues. Exposure to NHt also generates excess H+ ions in the tissue, which produce elevated putrescine levels in peas (Klein et a1. 1979; Prierbie et a1. 1978). In grapes, Vitis vinifera cells cultured on NHt medium produced putrescine in proportion of availability of NHt. DFMA, but not DFMO, prevented the increase in putrescine. The putrescine increase was important in the lag phase of subculturing and putrescine production decreased in the exponential phase of cell growth (Triantaphylides et a1. 1990). Based on their own research Slocum and Weinstein (1990) came to the conclusion that putrescine accumulation is an ammonia detoxification mechanism.

B. Chilling Stress Chilling injury develops in many plants of subtropical origin due to exposure to low but nonfreezing temperatures. The relationship between polyamines and chilling injury received considerable attention. The apparent involvement of membrane damage in chilling injury (Raison 1985; Lyons 1973; Wang 1982) and the ability of polyamines to stabilize membranes (Mager 1959; Smith et a1. 1985) have generated the hypothesis that polyamines may playa role in reducing chilling injury (Kramer and Wang 1988; Wang 1988). Correlations between increased resistance to chilling injury and increased polyamine levels have been reported in several species of plants. Chilling injury increased putrescine levels in citrus (McDonald and Kushad 1986) and in zucchini squash (Wang and Ji 1989) but decreased spermidine and spermine levels in zucchini squash (Kramer and Wang 1989). Lipid peroxidation of membranes culminating in fluorescent products seems to be associated with chilling injury (Kramer and Wang 1989). Temperature preconditioning, intermittent warming, or low oxygen storage all significantly increased spermidine and spermine levels and retarded the development of chilling injury in zucchini squash during storage at 2.5°C (Wang 1988; Kramer and Wang 1989). In pepper and avocado fruits (Phelps and McDonald 1990), abnormally high concentrations of spermine and spermidine, such as might occur

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during chilling stress in these chilling sensitive fruits, were detrimental to several oxidase activities, especially to NADH oxidase. The concentrations of polyamines required to affect the various mitochondrial oxidase activities are high enough to question their physiological significance. Furthermore, the strongest inhibitory effect was caused by spermine; putrescine is the polyamine that accumulates the most during stress. The effectiveness of polyamines in protecting membranes corresponds to the number of charges per molecule (Galston and KaurSawhney 1987b). Clearly, spermine and spermidine are more effective in this respect than putrescine. Spermidine and spermine, like Ca 2+, are able to stabilize membranes against lysis (Altman et a1. 1977). In addition, both Ca2+ and spermine prevent a decrease in membrane viscosity associated with senescence of apple tissues (Apelbaum et a1. 1982; Ben-Arie et a1. 1982), which has a profound effect on enzyme activity associate with membranes. However, Ca 2+and spermine or spermidine act somewhat antagonistically; binding to the same sites and the presence of Ca 2+ usually negates the positive effect of polylamines. Thus, it appears that the order of effectiveness of polyamines in protecting membranes against chilling injury is: spermine (tetraamine) > spermine (triamine) > putrescine (diamine). If spermine and spermidine are induced by prechilling treatments, or administered exogenously before chilling (Kramer and Wang 1989; Kramer et a1. 1991), they protect against chilling injury. It must be further noted that Ca2+, a doublecharged cation, is more effective than spermidine and spermine, which have three or four charged groups, respectively.

c.

Other Stresses

Several other stresses cause accumulation of putrescine in plants in general, but examples are few in horticultural plants. Osmotic stress, salt stress, exposure to ozone and other air pollutants, and anaerobiosis all cause putrescine accumulation in agronomic plants. Lovatt (1990) presented data on ammonium accumulation due to low temperature, water deficit, and salinity stress in citrus and summer squash. Assimilation of NH to glutamate and glutamine could acidify the internal pH (NH~+ converted to NHt + H+ and NHt removed) could create a mechanism similar to K-deficiency. The physiological rationale for polyamine accumulation during stress and possible protection of tissues by polyamines against stress is not clear. From disjointed data, it is possible to speculate that spermine and spermidine are able to protect membranes during stress, but they have to be present before the stress is initiated and they usually do not accumulate greatly during stress. In contrast, putrescine accumulates during

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stress. Whether the source of NHt ions required for polyamine synthesis is impaired protein synthesis or protein breakdown is not known.

v. POLYAMINES AND SENESCENCE The hypothesis that polyamines might be related to senescence of plant organs arose from investigations with protoplasts and cereal leaves (Galston and Kaur-Sawhney 1987b). There is no reason to believe that horticultural plants behave differently. In fact polyamines prevented senescence (chlorophyll loss) in radish leaves similarly to their effect on cereals (Altman, 1982). The order of activity among polyamines in preventing chlorophyll loss is similar to their effect in preventing chilling induced stress and is roughly spermine> spermidine> putrescine. The antisenescence activity is being manifested over the range 5 X 10-5 to 10 X 10-3 M. This is the range of concentration in which the polyamines are known to exist in plant tissues and may function as senescence regulators in vivo (Galston and Kaur-Sawhney 1987b). Investigating senescence in horticulturally important plants is more difficult because the interest to prevent senescence usually involves complex organs, such as fruit or large flowers. The interest in polyamine biosynthesis in connection with fruit ripening and senescence has originated from the fact that ethylene and polyamines are antagonistic and they share a common intermediate, Sadenosylmethionine (AdoMet), for their biosynthesis. The alcobaca mutant of tomato (ale), which ripens slowly and has prolonged shelf life, contains three times as much putrescine as the normal cultivar at the ripe stage (Dibble et al. 1987). In general, putrescine levels are high in immature green tomatoes, and decrease in the mature green stage (Davies et al. 1990). Thereafter, putrescine levels in normal fruit remain low, but in the ale fruit they rise to levels similar to that of immature green fruit (Dibble et al. 1988). Similar changes have been also reported in putrescine in another long-keeping cultivar of tomato also (Saftner and Baldi, 1990). Controlled atmosphere storage (CA) involving low oxygen and high carbon dioxide is widely used to prolong the storage life of apples. Low oxygen (10J0 02 at lor 3.5°C) storage induced higher levels of all three polyamines and significantly inhibited the softening of apples at both temperatures compared to apples stored in air (Kramer et al. 1989). The increase in putrescine and spermidine was gradual in CA storage and reached the highest levels within 20 weeks of storage. The increase was higher at 3.5°C than at 1°C. Spermine decreased within four weeks to low levels in air-stored fruit; however, although it also decreased in CA fruit it remained in much higher concentrations than in the air-stored fruit. Fruit

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from CA storage was firmer. Polyamines inhibited polygalacturonase activity in vitro indicating that they may act in vivo in protecting cell walls from degradation (Kramer et al. 1989). In contrast, Miller et al. (1988) showed no increase in fruit firmness due to polyamine treatments. Application of polyamines to mature apples retarded softening and reduced chilling injury without any effect on ethylene biosynthesis. Thus, similarly to the previous study, the conclusion was that polyamines may affect apple softening through the protection of cell walls (Kramer and Wang 1990; Kramer et al. 1991). Exogenously applied polyaminesreduce ethylene production in apple slices (Ben-Arie et al. 1982), orange peel (Even-Chen et al. 1982), and in avocado fruits (Apelbaum et al. 1982). The most potent inhibitor of ethylene synthesis in avocado tissues was spermine which caused a 70% inhibition largely by preventing ACC synthase activity (Apelbaum 1986). The resulting decrease in ethylene production prevented senescence. A decrease in polyamines was noticed in developing and stored pears. Polyamines in pears are relatively high when fruits are young and the polyamine concentration slowly decreases as the fruits enlarge and mature (Toumadje and Richardson 1988). Although there was no perceivable relationship between levels of polyamines and senescence as it was measured by ethylene production, the decrease in polyamines during growth of the fruit paralleled with that reported for avocado fruit (Apelbaum, 1986). In cut carnation flowers, when DMFA and MGBG were used as inhibitors of polyamine synthesis the endogenous levels of polyamines decreased, while ethylene production and the onset of senescence was promoted. In contrast, inhibition of ethylene synthesis by aminooxyacetic acid increased the level of spermine (Roberts et al. 1984) suggesting that in carnations the two biosynthetic pathways compete for AdoMet. During spermidine and spermine biosynthesis one or two molecules of decarboxylated AdoMet is utilized, producing 5'-deoxy-5'-methylthioadenosine (MTA) as a byproduct. MTA is also generated upon conversion of AdoMet to ACC, which in turn is the substrate for ethylene formation. Thus, both ethylene biosynthesis and tri- and tetra-amine biosynthesis use AdoMet as substrate and produce MTA (Kushad 1990). Recycling of MTA to. methionine may be required for AdoMet production, which in turn is required for polyamine and ethylene biosynthesis. The key enzyme in this pathway is MTA nucleosidase. Yang et al. (1990) describe detailed steps for recycling MTA. In avocado (Kushad et al. 1988) and in tomato (Kushad et al. 1985) fruits, MTA nucleosidase activity was relatively high during the early

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stages of fruit development and then declined as the fruit increased in size. The high MTA nucleosidase activity during the early stages of fruit development coincided with an increase in polyamine synthesis. There was a second period of high MTA nucleosidase activity in both fruits before the onset of climacteric rise in ethylene synthesis (Kushad 1990). Comparison of MTA nucleosidase activity in 'Rutgers' and rin tomato cultivars revealed that in 'Rutgers' MTA nucleosidase increased at the breaker stage as expected, although in the nonripening rin fruit no increase in MTA nucleosidase activity was detected in comparable developmental stages (Kusha 1990). Apparently, MTA is prevented from accumulating in tissues over a certain metabolic level because it is highly inhibitory to polyamine and ethylene synthesis. In pea seeds, exogenously applied MTA induced a decline in spermine and spermidine concentrations (Kushad 1990), and in tomato and winter squash MTA applications decreased ACC synthase by 47 and 50%, respectively (Hyodo and Tanaka 1986). MTA inhibition of ACC synthase has been described as uncompetitive (Hyodo and Tanaka 1986). Kushad (1990) argued that MTA is an important regulator of polyamine synthesis. He stated that the only possible way actively dividing cells and tissues undergoing rapid ethylene formation can overcome the inhibitory action of MTA is through a rapid degradation by MTA nucleosidase. This would explain the high level of polyamines in young tissues. It is notable also that high MTA nucleosidase activity in young tissues is also associated with polyamine formation, although, in mature fruits the same enzyme activity seems to be associated with biosynthesis of ethylene.

VI. CONCLUSIONS

Available evidence points strongly to the association of DNA synthesis and polyamine content of tissues. Not only is DNA metabolism able to influence polyamine synthesis but exogenously applied polyamines also influence DNA metabolism and growth. Polyamines are able to influence morphogenesis. Some evidence may indicate that polyamines should be excluded when morphogenetic changes are initiated, but the growth process that follows initiation is positively influenced by polyamines. Thus it may appear that the overall process is enhanced by polyamines. However, the evidence is far from complete on the effect of polyamines on morphogenesis. Stress-induced putrescine accumulates is a general phenomenon in plants. Putrescine accumulation may be a detoxification process for ammonium ions that accumulate during stress. Increase in hydrogen ion

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concentration in stressed tissues may enhance decarboxylase activity and consequently polyamine synthesis. Spennidine and spermine are strongly protective against certain stresses, but they are not involved in responses with all stresses. Polyamines are able to prevent or delay senescence probably by a double action. They protect membranes and maintain macromolecular synthesis, both of which are essential to prevent senescence. The effect of exogenous and endogenous polyamines are not the same. Polyamines are compartmentalized and a high level of endogenous polyamines may not mean that they are effective in influencing certain processes, which may occur outside of the compartment containing polyamines.

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Jarvis, B. C., S. Jasmin, and M. T. Coleman. 1985. RNA and protein metabolism during adventitious root formation in stem cuttings of Phaseolus aureus cultivar Berkin. Physiol. Plant. 64:53-59. Kakkar, R. K., and V. R. Rai. 1987. Effects of spermidine and IAA on carbohydrate metabolism during rhizogenesis in Phaseolus vulgaris. I. Hypocotyl cuttings. Indian J. Expt. BioI. 25:476-478. Kaur-Sawhney, R., A. Chakalammannil, and A. W. Galston. 1989. Effect of inhibitors on polyamine biosynthesis on growth and organization of meristematic centers in petunia protoplast cultures. Plant Sci. 62:123-128. Kaur-Sawhney, R., Y. R. Dai, and A. W. Galston. 1985. Effect of inhibitors of polyamine biosynthesis on gibberellin-induced internode growth in light-grown dwarf peas. Plant Cell Physiol. 27:253-260. Kaur-Sawhney, R., L. M. Shih, T. Cegielska, and A. W. Galston. 1982. Inhibition of protease activity by polyamines. Relevance for control of leaf senescence. FEBS Lett. 145:345-349. Klein, H., A. Pierbe, and H. J. Jager. 1979. Putrescine and spermidine in peas: Effects of nitrogen source and potassium supply. Physiol. Plant. 45:497-499. Kramer, G. F., and C. Y. Wang. 1988. Relationship of polyamine levels to chilling injury in zucchini squash. Plant Physiol. 86 (suppl.): 51. Kramer, G. F., and C. Y. Wang. 1989. Correlation of reduced chilling injury with increased spermine and spermidine levels in zuccnini squash. Physiol. Plant. 76:479-484. Kramer, G. F., and C. Y. Wang. 1990. Effects of chilling and temperature preconditioning on the activity of polyamine biosynthetic enzymes in zucchini squash. J. Plant Physiol. 136:115-119. Kramer, G. F., C. Y. Wang, and W. S. Conway. 1989. Correlation of reduced softening and increased polyamine levels during low oxygen storage of 'McIntosh' apples. J. Am. Soc. Hort. Sci. 114:942-946. Kramer, G. F., C. Y. Wang, and W. S. Conway. 1991. Polyamine application in 'Golden Delicious' and 'McIntosh' apples. J. Am. Soc. Hort. Sci. (in press). Kuehn, G. D., B. Rodriguez-Gray, S. Bagga, and G. C. Phyllips. 1990. Novel occurence of uncommon polyamines in higher plants. Plant Physiol. 94:855-857. Kushad, M. M. 1990. Recycling of 5'-deoxY-5'-methylthioadenosine in plants. p. 50-61 In: H. E. Flores, R. N. Arteca and J. C. Shannon [eds.}, Polyamines and Ethylene: Biochemistry, Physiology and Interactions. Am. Soc. Plant Physiol. Rockville, MD. Kushad, M. M., A. R. Orvos, and G. Yelenowsky. 1990. Relative changes in polyamines during citrus flower development. HortScience 25:946-948. Kushad, M. M., D. G. Richardson, and A. J. Ferro. 1985. 5'-methylthioadenosine nucleosidase and 5'-methylthioribose kinase activities during tomato fruit development and ripening. Plant Physiol. 78:525-529. Kushad, M. M., G. Yelenowsky, and R. Knight. 1988. Interrelationship of polyamine and ethylene biosynthesis during avocado fruit development and ripening. Plant Physiol. 87:463-467. Lovatt, C. J. 1990. Stress alters ammonia and arginine metabolism. p. 166-179 In: H. E. Flores, R. N. Arteca and J. C. Shannon (eds.), Polyamines and Ethylene: Biochemistry, Physiology and Interactions. Am. Soc. Plant Physiol. Rockville, MD. Lyons, J. M. 1973. Chilling injury in plants. Annu. Rev. Plant. Physiol. 24:455-466. Mager, J. 1959. The stabilizing effect of spermidine and related polyamines on bacterial protoplasts. Biochem. Biophys. Acta 36:529-531. McDonald, R. E. and M. M. Kushad. 1986. Accumulation of putrescine during chilling injury of fruits. Plant Physiol. 82:324-326. MengoH, M., N. Bagni, G. Lucca, V. R. Ronchi, and D. Serafini-Fracassani. 1989. Daucus

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carota cell cultures: Polyamines and effect of polyamine biosynthesis inhibitors in the preembrionic phase and different embryo stages. J. Plant Physiol. 134:389-394. Miflin, B. J., and P. J. Lea. 1977. Amino acid metabolism. Annu. Rev. Plant Physiol. 28:299-329. Miller, A. R., C. Velbinger, C. V. Mujer, J. C. Schmid, and D. C. Ferree. 1988. Polyamines as regulatores of apple fruit development. Res. Circ. 295:11-13. Ohio Agr. Res. Develop. Cent. Wooster, OH. Minocha, S. C. 1988. Relationship between polyamine and ethylene biosynthesis in plants and its significance in morphogenesis in cell cultures. p. 601-616 In: V. Zappia and A. E. Pegg (eds.), Progress In Polyamine Research. Plenum Press, New York. Minocha, S. C., C. A. Robie, A. J. Khan, N. S. Papa, A. 1. Samuelsen, and R. Minocha. 1990. Polyamines and ethylene biosynthesis in relation to somatic embryogenesis in carrot (Daucus carota L.) cell cultures. 1990. p. 339-342 In: H. E. Flores, R. N. Arteca, and J. C. Shannon (eds.), Polyamines and Ethylene: Biochemistry, Physiology and Interactions. Am. Soc. Plant Physiol. Rockville, MD. Mizrahi, Y., and Y. M. Heimer. 1982. Increased activity of ornithine decarboxylase in tomato ovaries induced by auxin. Physiol. Plant. 54:367-268. Mizrahi, Y., P. B. Applewhite, and A. W. Galston. 1989. Polyamine binding to proteins in oat and petunia protoplasts. Plant Physiol. 91:738-743. Montague, M. J., J. W. Koppebrink, and E. G. Jaworski. 1978. Polyamine metabolism in embryogenic cells of Daucus carota 1. Changes in intracellular content and rates of synthesis. Plant Physiol. 62:430-433. Montague, M. J., T. A. Armstrong, and E. G. Jaworski. 1979; Polylamine metabolism in embryonoic cells of Daucus carota II. Changes in arginine decarboxylase activity. Plant Physiol. 63:341-345. Murti, K. S., T. A. Smith, and C. Bould. 1971. The relation between the putrescine content and potassium status of black currant leaves. Annu. Bot. N.S. 35:687-695. Nathan, R., A. Altman, and S. P. Monselise. 1984. Changes in activity of polyamine biosynthetic enzymes and in polyamine contents in developing fruit tissues of 'Murcott' mandarin. Scientia Hort. 22:359-364. Nemec, S., and F. L. Meredith. 1981. Amino acid content ofleaves in mycorrhizal and non mycorrhizal citrus rootstock. Annu. Bot. 47:351-358. Olson, A. R., and J. Novak. 1988. Free polyamines in postpollination placentae and ovules of potato. HortScience 23:1042-1044. Ormrod, D. P., and D. W. Beckerson. 1986. Polyamines as antiozonants for tomato. HortScience 21:1070-1071. Palavan, N., and A. W. Galston. 1982. Polyamine biosynthesis and titer during developmental stages of Phaseolus vulgaris. Physiol. Plant. 55:438-444. Phelps, D. C., and R. E. McDonald. 1990. Inhibition of electron transport activities in mithocondria from avocado and pepper fruit by naturally occuring polyamines. Physio!. Plant. 78:15-21. Pistocchi, R, N. Bagni, and J. A. Creus. 1987. Polyamine uptake in carrot cell cultures. Plant Physiol. 84:374-380. Prakash, L., P. John, G. M. Nair, and G. Prathapasenan. 1988. Effect of spermidine and methoxylglyoxal-bis-(guanylhydrazone) (MGBG) on in vitro pollen germination and tube growth in Catharanthus roseus. Annu. Bot. 61:373-375. Prierbie, A .• H. Klein, and H. J. Jager. 1978. Role of polyamines in S02-polluted pea plants. J. Expt. Bot. 29:1045-1050. Rabe, E., and C. J. Lovatt. 1984. De novo arginine biosynthesis in leaves of phosphorusdeficient citrus and Poncirus species. Plant Physiol. 76:747-752. Raison, J. K. 1985. Alteration in the physical properties and thermal responses of

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membrane lipids: Correlation with acclimation to chilling and high temperature. p. 383401 In: J. St.John, E. Berlin, and P. Jackson (eds.), Frontiers of Membrane Research in Agriculture. Rowan and Allenheld, Totowa, NJ. Rajam, M. V. 1989.Restriction of pollen germination and tube growth in lily pollen by inhibitors of polyamine metabolism. Plant. Sci. 59:53-56. Ramakrishna, S., and P. R. Adiga. 1975. Amine levels in Lathyrus sativus seedlings during development. Phytochemistry 14:63.-68. Rastogi, R., and V. K. Sawhney. 1990a. Polyamines and flower development in the male sterile stamenless-2 mutant of tomato (Lycopersicon esculentum Mill.) I. Level of polyamines and their biosynthesis in normal and mutant flowers. Plant Physiol. 93:439445.

Rastogi, R., and V. K. Sawhney. 1990b. Polyamines and flower development in the male sterile stamenless-2 mutant of tomato (Lycopersicon esculentum Mill.) II. Effects of polyamines and their biosynthetic inhibitors on the development of normal and mutant floral buds cultures in-vitro. Plant Physiol. 93:446-452. Roberts, D. R., M. A Walker, J. E. Thompson, and E. B. Dumbroff. 1984. The effects of inhibitors on polyamine and ethylene biosynthesis on senescence, ethylene production and polyamine levels in cut carnation flowers. Plant Cell Physiol. 25:315-322. Robie, C. A, and S. C. Minocha. 1989. Polyamines and somatic embryogenesis in carrot. I. The effect of difluoromethylornithine and difluoromethylarginine. Plant Sci. 65:45-54. Rugini, E., and M. Mencuccini. 1985. Increased yield in the olive with putrescine treatment. HortScience 20:102-103. Rugini, E., G. Bondi, and M. Mencuccini. 1986. Effect of putrescine, L-arginine and cobalt on fruit set, ethylene content and apparent parthenocarpy in olive. Acta Hort. 179:365368. Saftner, R. A, and B. G. Baldi. 1990. Polyamine levels and tomato fruit development: Possible interaction with ethylene. Plant Physiol. 92:547-550. Sawhney, V. K. 1983. Temperature control of male sterility in a tomato mutant. J. Hered. 74:51-54. Serafini-Fracassini, D., S. Del Duca, and D. D'Orazi. 1988. First evidence for polyamine conjugation mediated by an enzymic activity in plants. Plant Physiol. 87:757-761. Serafini-Fracassini, D., S. Del Duca, and P. Torrigiani. 1989. Polyamine conjugation during the cell cycle of Helianthus tuberosus: Nonenzymatic and transglutaminase-like binding activity. Plant Physiol. Biochem. 27:659-668. Sethi. U., A Basu, and S. Guha-Mukherjee. 1988. Control of cell proliferation and differentiation by regulating polyamine biosynthesis in cultures of Brassica and its correlation with glyoxalas I activity. Plant Sci. 56:167-175. Shargool, P. D., J. C. Cain, and G. McKay. 1988. Ornithine biosynthesis and degradation in plant cells. Phytochemistry 27:1571-1574. Shyr, Y. Y., and C. H Kao. 1985. Polyamines and root formation in mung bean hypocotyl cuttings. Bot. Bui. Acad. Sinica. (Taipei) 26:179-184. Slocum, R. D., and L. H. Weinstein. 1990. Stress-induced putrescine accumulation as a mechanism of ammonium detoxification in cereal leaves: p. 157-165 In: H. E. Flores, R. N. Arteca and J. C. Shannon [eds.}, Polyamines and Ethylene: Biochemistry, Physiology and Interactions. Am. Soc. Plant Physioi. Rockville, MD. Slocum, R. D., R. Kaur-Sawhney, andA. W. Galston.1984. The physiology and biochemistry of polyamines in plants. Arch Biochem. Biophys. 235:283-303. Smith, M. A. 1990. The involvement of polyamines in the genetic and gibberellic acid control of internodal growth in peas. p. 101-111 In: H. E. Flores, R. N. Arteca and J. C. Shannon [eds.}, Polyamines and Ethylene: Biochemistry, Physiology and Interactions. Am. Soc. Plant Physiol. Rockville, MD.

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8 Breeding Muscadine Grapes R. G. Goldy· Department of Horticultural Science North Carolina State University Raleigh, North Carolina 27695

I.

II.

III.

IV.

V.

Introduction A. Range and Adaptation B. The Current Industry Germplasm Resources A. Taxonomy B. Preservation Breeding: Intraspecific Hybridization A. History B. Techniques C. Specific Characters Breeding: Intersubgeneric Hybridization A. History B. Cytogenetics C. Specific Characteristics of Euvitis x Muscadinia Hybrids Future Prospects Literature Cited

I. INTRODUCTION

The muscadine grape, Vilis rotundifolia (Michx.), has an interesting and colorful (albeit short) history of cultivation compared to the old world grape, V. vinifera (L.). General reviews of muscadines have been written by Gohdes (1982), Qlien (1990), and Qlien and Hegwood (1990). Ageneral review of grape breeding has been covered by Einset and Pratt (1975). None, however, go into depth on breeding of muscadine grapes, which is the focus of this review. Muscadines comprise a small precentage of world grape production. Qlien (1990)reported 1600 hectare (ha) in muscadine production outof 8. 7 million ha of grapes grown worldwide (Anonymous 1990). Though limited in global importance, muscadines are an important source of income for some and are cultivated and consumed with passion by many in the southeastern United States. From a breeding standpoint muscadines are an interesting species. It is still in the early stages of improvement through either passive or active ·Present address: 86 West Albion Street, Holley, New York, 14470 357

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breeding, with some cultivars being selections from the wild, and others only one to five generations from the wild. Vitis vinifera, however, has been under cultivation for nearly 6000 years (Einset and Pratt 1975) with passive selection having taken place previous to active breeding. Passive selection probably led to development and/or identification of seedlessness, nonshattering (the ability of the fruit to adhere to the pedicel although physiologically mature), uniform ripening, better pigmentation, and greater yield. Therefore, improvement in muscadine grapes allows grape breeders to directly compare active versus passive breeding, and to determine if active breeding programs can greatly reduce the time required to develop superior selections. Another appealing aspect is that muscadines do not have the tremendous name recognition that V. vinifera wine cultivars have, making it easier to introduce wine cultivars to the industry. A. Range and Adaptation

Muscadines are primarily a southeastern U.S. fruit (Qlien and Hegwood 1990). They are native to this area and are found growing in almost any southeastern woodlot and commercial production is limited to this area, with coastal states having the greatest production (Qlien 1990). Muscadine's range is limited to areas not experiencing winter temperatures much below -12°C, and rarely to -18°C (Dearing 1938). However, many vines survived the winter of 1984-1985 in North Carolina when temperatures reached -23°C. These temperature limitations make the natural range for muscadines restricted to southern coastal New Jersey, south to southern Florida, west to eastern Texas, with Virginia, Tennessee, and Arkansas as the northern inland boundaries. Moving south through central Florida V. rotundifolia gradually gives way to V. munsoniana (Simpson ex Munson), a closely related species (Rogers and Mortensen 1979). Muscadines are adapted to a wide range of growing conditions and soil types but prefer well-drained, sandy loam and alluvial soils (Hedrick 1908; Qlien 1990). Based on personal experience, they seem to occupy the same ecosystems as poison ivy (Rhus radicans L.), which causes an irritating skin rash for most people and Smilax (L.) spp., which are thorny, making collection of native vines difficult and potentially uncomfortable. B. The Current Industry The commercial muscadine industry began in North Carolina where the first muscadine grapes were discovered growing along the Cape Fear

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River in 1524 (Morton 1988). The first recorded commercial planting was not until 1985 in Halifax County, North Carolina. This vineyard was bought by Paul Garrett who helped turn North Carolina into the leading wine producing state in the early 1900s (Morton 1988). For more history the reader is referred to Gohdes (1982), Morton (1988) and Olien (1990). The present industry is located in the Coastal Plain of North and South Carolina, Georgia, Mississippi, Alabama, and central and northern Florida, with smaller plantings in the other southeastern states (Olien 1990). Much of the production in these states is processed into wine with smaller sales in fresh fruit and other processed products uuice, jam, and jelly). Most major muscadine cultivars are nonpigmented with the most important being 'Carlos' and 'Magnolia' (Olien 1990), making the industry heavily dependent on a white juiced product. Through its history the muscadine industry has experienced several fluctuations in production and demand. The most recent significant expansion occurred in the 1960s, followed by a contraction in the late 1970s. These expansions and contractions are usually the result of supply and demand economics. The industry is presently experiencing a slight expansion due to increased interest in pasteurized juice, and although static in some production areas, total production has increased slightly to meet the juice demand. Although demand for juice is creating new interest, future growth still appears uncertain at this time.

II. GERMPLASM RESOURCES

A. Taxonomy

Vitis belongs to Vitaceae, order Vitales. Vitaceae is characterized by a climbing habit, terminal buds developing into apparently lateral tendrils, inflorescence opposite a leaf at the node, stamens opposite petals, usually a two-Ioculed ovary with axile placentation, a capitate or discoid stigma, and the fruit a berry (Lawrence 1951). Related genera include Gissus, Parthenocissus, and Ampelopsis. Vitis is the subject of much taxonomic controversy (Comeaux 1984; Comeaux et al. 1987; Moore 1987), and is generally separated into two groupings based on chromosome number. Most authorities follow Bailey (1934) and place the 38-chromosome species into subgenus Euvitis (Planch.), of which V. labrusca (L.) is the best-known North American species, and V. vinifera the best-known old world and commercial species in this subgenus. Following this treatment those Vitis species having 40 chromosomes are placed into subgenus Muscadinia (Planch.), which contains V. rotundifolia. In another treatment, Munson (1909) divided

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Vitis into two sections: Euvitis and Lenticellosis, and then assigned Muscadinia series status within Lenticellosis. Some authorities have also assigned Muscadinia its own status as a genus, making V. rotundifolia equivalent to M. rotundifolia (Small) (Small 1913; Olmo 1976). The desire to place Muscadinia as its own genus is most likely an effort to separate the 38-chromosomed species into their own grouping since other genera in Vitaceae have 40 chromosomes (Olmo 1976). This review follows Bailey's (1934) classification. Only three species are in Muscadinia: (1) V. rotundifolia, (2) v: munsoniana, and (3) V. popenoei (Fennell 1940), compared to more than 50 species in Euvitis (Nesbitt 1974). Of the Muscadinia, only V. rotundifolia is commercially important. Vitis munsoniana and V. popenoei are semitropical to tropical in adaptation (Husmann and Dearing 1916; Fennel 1940), and offer sources of genetic variation available for V. rotundifolia improvement. Vitis munsoniana is in the ancestry of 'Tarheel' (Brooks and Olmo 1972) and 'Noble' (Nesbitt et al. 1974a). The main difference between V. rotundifolia and V. munsoniana is that V. munsoniana has smaller berries but more per cluster, a trait observed in 'Noble' (Goldy and Nesbitt 1985). Vilis munsoniana also breaks bud a month earlier than V. rotundifolia. Lack of substantial differences between v: rotundifolia and V. munsoniana have caused some to speculate V. munsoniana is a semitropical variant of V. rotundifolia. Vitis popenoei has yet to play an important role in muscadine improvement, and its use may be limited for the main muscadine production areas due to its tropical adaptation. Vitis popenoei has been used in the Florida breeding program and may offer some improvement for pigmentation (J. Mortensen, unpublished data). Syamal and Patel (1953) describe an unidentified species (2n = 40) found in a forest in India whose description suggests it is Muscadinia in character, but it may be an Ampelopsis species (J. Mortensen, personal communication). Vitis rotundifolia is a vigorously growing vine often climbing to the top of trees 20-30 m in height. It is described by Comeaux et al. (1987) as follows: Stems vining, glabrous, nodes often banded with red pigmentation; branchlets slightly angled, becoming round, sometimes with aerial roots; pith continuous through nodes; bark gray, adherent on wood several years old, later exfoliating in plates; lenticels conspicuous; growing tips usually glabrous, occasionally pubescent. Leaves cordiform to reniform, very rarely lobed, crenate to dentate, lustrous above, glabrous beneath or with few to many trichomes or veins and in vein axils; petioles glabrous to puberulent; stipules 1 mm long. Tendrils and inflorescences absent every third node; tendrils simple; inflorescences to 10 cm long. Berries with tan, circular lenticels, generally black or purplish, occasionally bronze when ripe, glaucescent, spherical to ellipsoidal, aromatic, sweet, musky flavored, 12-33 mm in diameter. Seeds navicular, brown, 5-8 mm long.

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Besides the cytological differences many morphological features help distinguish V. rotundifolia from Euvitis. The most easily identified traits of V. rotundifolia are adherent bark, simple tendrils, prominent lenticels, and continuous pith (Table 8.1), and it is these traits that are prominent in taxonomic keys (Radford et a1. 1968; Comeaux 1984; Comeaux et a1. 1987). Indigenous muscadines are generally dioecious with pigmented, aromatic berries, which shatter easily. Perfect flowered types were found as early as 1910 by Reimer and Detjen (1910). Husman and Dearing (1913) and Dearing (1917a) report finding three more self-fertile vines and a fifth self-fertile vine, homozygous for this trait was reported by Loomis and Williams (1957). Although most wild vines have pigmented fruit, the oldest and best-known selection, 'Scuppernong', is nonpigmented. Berry adherence to the pedicel of modern cultivars is strong (often too strong). A strong, generally pleasing aroma is typical of most ripe muscadine fruit and is probably the primary means of attracting mammalian seed dispersal agents. In central North Carolina (Wake County) V. rotundifolia generally breaks dormancy 3-4 weeks later than most Euvitis cultivars and blooms later (early to mid-June vs. early to mid-May for Euvitis). This difference is carried over into ripening, with Euvitis generally ripening in August and muscadines in September to October. From a 1987 survey (Goldy et a1. 1989) several native muscadine populations were found to ripen before commercially grown vines (unpublished data). This may be due to the heavier crop load on commercial vines. Unlike many Euvitis, muscadines are difficult to root from dormant cuttings. Attempts at rooting dormant cuttings range from a 2 to 75% success rate (Dearing 1938). As a result, most vines were propagated

Table 8.1. Some easily identifiable morphological traits distinguishing Vitis rotundifolia (subgenus Muscadinia) from members of subgenus Euvitis.

Morphological trait

V. rotundifolia

Euvitis

Bark Bark lenticels

Loose on older wood Lenticels absent

Tendrils Pith

Adherent on older wood Conspicuous on young wood Simple Continuous through node

Fruit lenticels Fruit cluster size Fruit skin Leaf undersurface

Conspicuous Small; 4-10 Thick and tough Shiny

Branched Not continuous through node Not conspicuous Generally larger Thinner. not as tough Dull

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through summer layering (Harman 1943; Cowart and Savage 1944; Goode et aI. 1982). In an important discovery, Sharpe (1954) obtained 100% rooting using soft-wood cuttings placed under mist, and this is how most muscadines are currently propagated. Micropropagation techniques are being investigated for muscadines (Gray and Fisher 1985; Lee and Wetzstein 1990; Sudarsono and Goldy 1991) and will be an important source of plant material in the future.

B. Preservation Vitis rotundifolia is unique among fruit crops on this continent because it is one of the few cultivated fruits completely North American in origin. The U.S. repositories to which grapes are assigned are located at Geneva, New York, and Davis, California (Brooks and Barton 1983). Vilis rotundifolia is assigned to Davis, with the repository presently having 109 Muscadinia accessions (K. Rigert, personal communication). Establishment of a working collection of cultivars and selected breeding lines at a station in the southeastern U.S. would be beneficial to breeders and muscadine researchers. Although workable collections are maintained primarily by publicly supported research scientists, private individuals have collections consisting almost exclusively of cultivars. Scientists using private collections need to authenticate trueness to name of theclone(s) they are using. Nomenclature may not be exact and can be confusing with the same clone having different names, different clones having the same name, or the same clone having different strains (Reimer 1909; Woodroof 1934). Although much germplasm can still be found growing naturally, present breeding programs are somewhat reluctant to exploit it, probably because of the strong desire to release perfect flowered types and reluctance to sacrifice short-term improvement for the sake of maintaining a broad genetic base. Onokpise (1988) indicated that present cultivars do not suffer from inbreeding, and in general are not closely related. Pedigrees used were incomplete and caution needs to be taken when interpreting these findings. Wild germplasm is being severely threatened by human pressures, a situation common to many southeastern plant species (Lyrene 1987). It would be wise for muscadine breeders to determine traits of value, and survey native plants to identify and collect unrelated superior types (as seed and/or cuttings). Such an effort may identify plants superior in yield, fruit qualities (sugar to acid ratios, thin skin, fruit size), cluster size, and seedlessness. Some characters will be hard to predict (i.e., future disease problems), but if the collection is broad and thorough, enough superior types may be included.

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III. BREEDING: INTRASPECIFIC HYBRIDIZATION

A. History Shortcomings inherent to muscadines were recognized by early muscadine growers causing them to experiment with seedling material. The earliest recorded efforts to improve muscadines are from the 1860s (Reimer and Detjen 1914). These first efforts involved collecting and planting open pollinated seed. J. Van Buren of Clarksville, Georgia was a principal advocate, and he himself planted and evaluated several thousand seedlings (Reimer and Detjen 1914). Van Buren was aware of the need for large numbers from which to select as is reflected in his.statement: "Many, doubtless will be inferior to the parents, while some will be equal and others superior in size and flavor." (Reimer and Detjen 1914). The most well-known early breeder was T. V. Munson who demonstrated that V. rotundifolia will hybridize with V. munsoniana and V. lincecumii (Buckley) hybrids. He developed and named several cultivars, but according to Reimer and Detjen (1914), these were little improved over V. rotundifolia. The first public-supported breeding program was initiated in 1908 at Willard, North Carolina as a cooperative effort between the USDA and North Carolina State University. Other state-funded programs began in Georgia in 1909 (Stuckey 1919), University of Florida in 1959 (Balerdi and Mortensen 1969), and a USDA-funded program in Mississippi in 1941. A program has also been recently initiated at Florida A & M University. The Georgia and Florida programs are still in existence; however, the Mississippi program was discontinued in 1965 (Mortensen 1971) and the program in North Carolina was terminated in 1990. Breeding efforts have been conducted outside the southeastern United States, primarily in California (Olmo 1971) and France (Bouquet 1980b), to produce V. vinifera/V. rotundifolia hybrids in order to introgress V. rotundifolia genes into V. vinifera.

The goals of all early breeding efforts were to improve fruit color, and develop types with nonshattering clusters, uniform-ripening within the cluster, dry stem scar, larger and more uniform-sized fruit clusters, and perfect, self-fertile flowers. Significant improvement has been made in these traits, but they still remain important objectives in today's programs. B. Techniques Breeding techniques for muscadines were reported by Detjen (1917a), and more recently Einset and Pratt (1975) have reviewed grape breeding

364

R. G. GOLDY

in general. Techniques have changed little since Detjen's report, and although Einset and Pratt deal primarily with Euvitis the techniques apply to V. rotundifolia. Techniques covered here are taken from different programs in the southeastern U.S. Since muscadines are large vines, essentially all crosses are made in the field. Due to the late bloom time of muscadines there is little chance of spring frosts being a problem. However, since grapes flower and fruit on new growth, frosts can damage vines shortly after they break dormancy. Danger of this is minimized since they break dormancy late, probably a result of natural selection since their range of adaptation is prone to such frosts. Even if late frost does injure shoots, secondary bloom is heavy enough to allow breeders to obtain adequate seed. Various ways have been used to collect and store grape pollen (Einset and Pratt 1975; Galletta 1983; Layne 1983). Detjen (1917a) points out the importance of collecting flowers early in the day and using pollen within 24-30 h. This is still generally true but properly dried pollen can be stored several years at -12°C (Olmo 1942). If the same pollen is used over several days it is best to divide it and take to the field only what will be needed that day. If sufficient flowers are available, an easy collection technique is to collect clusters and rub them through a small-mesh screen. This forces flowers to open and allows anthers and some filaments and calyptras to fall through onto a clean piece of paper placed below the screen; larger debris is sifted out. If desired, filaments and calyptras can be sifted out through smaller screens. Mter drying overnight anthers and pollen are placed in a bottle, labeled, and stored until used. An alternative method is to strike open flower clusters against a piece of glass and scrape pollen into vials using a razor blade. If the seed parent is pistillate, emasculation will be unnecessary. In this case, simply bag clusters intended for pollination prior to flowering to exclude unwanted pollen. Perfect flowered seed parents require emasculation. This is done by selecting flower clusters one or two days before natural opening (these clusters will usually have a few opened flowers, which are removed) and removing calyptras, which usually removes anthers. Several tools have been used; with some practice a pair of pointed, curved forceps does quite well. Mter emasculation the cluster is bagged, protecting it from unwanted pollen. Emasculation is easier on V. rotundifolia than Euvitis. However, V. rotundifolia flowers do not survive emasculation well (Loomis et a1. 1954). Care must be taken not to damage ovary walls or flowers will fall off, wasting much effort. This damage occurs in Euvitis, but does not seem to affect flowers as severely. Ovary damage is observed as darkening of the wall, which appears soon after occurrence.

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BREEDING MUSCADINE GRAPES

365

Flowers should be pollinated one-two days after emasculation, and can be done immediately after emasculation. Pollen should be kept cool in the field. Receptive flowers are identified by a drop of exudate on the stigma. If pollen is abundant it can be transferred using a camel hair brush; if it is limited it is more efficient to use a glass rod. Both instruments eventually become sticky and need occasional cleaning with alcohol. Some workers believe greater success is achieved by pollinating early in the morning. If time is available fruit set can be increased by pollinating on two successive days. However, it may be more efficient to use extra time to pollinate more clusters once than fewer clusters twice. After pollination the bag is replaced and the cross identified by writing directly on the bag. The bag is left in place during the growing season to protect and help locate hybrid clusters at maturity. Fruit containing hybrid seed is harvested at maturity and seed extracted with the aid of enzymes, such as pectinol (Nesbitt et al. 1976), or by forcing seed through a screen separating it from skin and pulp. Seed is dipped in a mild fungicide solution of benomyl and captan and placed in moist medium (sterilized sand works best since it is easily separated from the seed) in polyethylene bags and stratified at 1°C to 4°C for 80 to 100 days (Nesbitt et al. 1976). Seed will germinate poorly and nonuniformly if allowed to dry too much. After stratification seed is removed from the sand by washing through a screen, placed on a mixture of 1 sphagnum:l sand (by volume), placed in the greenhouse under ambient conditions, and watered as needed. Depending on temperature, germination usually begins within 40 days after removal from stratification. When seedlings have attained their first true leaf they are transplanted at a spacing of 8cm in the row and 10cm between rows to a greenhouse bench filled to a depth of 12-14cm with a mixture of 2 pine bark:l peat:l soil:l perlite and steam sterilized in the bench. For each cubic meter of media, add 150g superphospate, 525g dolomitic lime, and 130g hydrated lime. Seedlings are moved from the germination media to the bench media in January and February, allowing 90-days growth under ambient greenhouse conditions. They are watered as needed and fertilized occasionally with a balanced, water-soluble fertilizer. Seedlings are susceptible to fungal diseases and must be inspected often and appropriate steps taken if diseases are identified. Under these conditions seedlings can attain a height of nearly a meter and lignification will begin. Prior to field transplanting, tender growth is removed, leaving the basal four or five buds. Seedlings lifted for field transplantation should retain as much media as possible on the roots. Excellent results have also been obtained using root trainers that separate seedlings into individual "cells" or by transplanting newly germinated seedlings into peatcups. Seedlings are transplanted to a field nursery after the danger of frost is

366

R.G.GOLDY

past. Ideal soil is a well drained, sandy loam that has been fumigated (primarily to aid weed control). Sandy loam is best because it helps fumigant penetration and makes transplanting operations easier. Seedlings are grown for one year in this nursery a t a spacing of O. 3m in the row and 1.3m between rows. In North Carolina, muscadine seedlings are not subjected to a pesticide spray program except for Japanese beetles, allowing for some nursery selection for foliar diseases as well as vigor. After one growing season in the nursery seedlings are cut back to a single shoot having five basal buds and transplanted to vineyard rows while dormant. They are transplanted to fumigated rows (1.2m in the row and 3. 7m between rows) and trained to a double-wire trellis, one wire just above the soil (aiding in training only) and the other at the top of the post. Plants eventually are trained in one direction down the top wire. Some fruiting will occur the third year in the field, and most will fruit by the fourth year. Keeping seedlings through their fifth year should be adequate. By then a majority will have fruited and most selections successfully propagated; therefore most plants can be removed and the fields put back in rotation. The breeder may want to keep selections for additional years to insure successful propagation, check trueness to type, and to use them in breeding prior to propagules coming into production. For further evaluation, selections should be placed in a nonreplicated, three-plant observation trial. Those that continue to appear promising can be moved to a replicated yield trial and dispersed for grower and peer evaluation.

C. Specific Characters 1. Flower type. One of the first traits systematically investigated in muscadines was flower type (Reimer and Detjen 1910, 1914; Detjen 1917b; Loomis et al. 1954; Williams 1954). This interest was botanical, genetic, and economic since 10-15% of the vines in commercial vineyards were staminate pollinizers (Dearing 1938). Having self-fertile cultivars would mean an automatic yield increase. Wild muscadine vines are primarily dioecious, with more being staminate than pistillate (Reimer and Detjen 1910). Until 1946 all fruiting muscadine cultivars were pistillate and required interplanting of pollinizers (Williams 1954). Confusion existedforthe need of a pollinizer for certain vines (Reimer 1909). Work byReimerand Detjen (1910)conclusively indicated leading cultivars of that time were pistillate only. Early work on floral biology identified three flower types: (1) perfect hermaphrodite flowers; (2) staminate or "male" flowers; and (3) imperfect hermaphrodite or "female" flowers (Detjen 1917b). All three have

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distinguishable flower morphology: staminate possess only filaments and anthers and generally have more flowers per cluster; pistillate flowers are morphologically perfect since all flower parts are present but have reflexed filaments and anthers containing nonfunctional pollen; functionally perfect flowers are extremely rare among wild plants since only five have been reported. However, once identified self-fertility was quickly and successfully incorporated and by 1917 Dearing (1917b) states at least 1000 self-fertile seedlings were present in his breeding program. The first report of a perfect-flowered vine was by Reimer (Reimer and Detjen 1910). It was found near Raleigh, North Carolina and was named 'Hope' to commemorate the hope and desire of developing self-fertile vines (Detjen 1917b). Genetics of self-fertility in perfect-flowered types were investigated by Detjen (1917b) and Dearing 1917a, 1917b). Detjen (1917b) intercrossed plants of the three existing flower types: staminate (8), hermaphrodite with reflexed stamens (HR), and hermaphrodite with upright stamens (HU) (i.e., 'Hope'), in all possible combinations (excluding 8 X 8) and evaluated progeny. The cross, HR X 8, produced IHR:18 flowers; HU plants were never obtained. HU X HR produced 1HU:IHR and never 8. Detjen was unable to get ratios from HU X HU ('Hope' self-pollination) due to poor fruit set. No seed set was obtained from HU X 8 or HR X HR; the latter was understandable since HR pollen was nonfunctional. Detjen (1917b) also noticed HU plants were more like 8 than HR in flower morphology. They are similar in number of flowers per cluster, both significantly larger than HR plants, and in HR X HU crosses he could select HU plants prior to flower opening. Also, 8 and HU plants had shorter juvenility. This led him to conclude that 'Hope' was a staminate vine "whose long suppressed pistils have suddenly been regenerated and have recovered the power to function," and the ancestral V. rotundifolia vine was a true and functioning hermaphrodite. Having the mutation occur in staminate flowers would possibly provide an increase in yields due to larger flower clusters. One of the two self-fertile plants (designated HI) used by Dearing (1917a) resulted from a cross between 'Eden' and V. munsoniana, the other (designated H2) from a cross between '8cuppernong' and another V. rotundifolia. Dearing reported a third self-fertile vine (designated H3), but Loomis and Williams (1957) report no further reference to H3 can be found. Dearing's studies were similar to Detjen's except he utilized his HI and H2 vines. By intercrossing the different flower types Dearing found similar progeny ratios, and by self-pollinating H2 he obtained five seedlings, all self-fertile. Dearing's studies do not appear as thorough as Detjen's, nor do they include as many seedlings (232 vs. 1379, respectively).

368

R.G.GOLDY

Detjen's and Dearing's studies apparently progressed no further than to document flower-type ratios obtained from various parental combinations. Later Loomis (1948), Loomis et a1. (1954) and Loomis and Williams (1957) investigated the actual genetics of the trait. Results from Loomis' crosses again gave a 1:1 ratio of parental types in the progeny leading him to conclude inheritance in muscadines is similar to Euvitis as explained by Oberle (1938). This theory is based on sex being controlled by two dominant, linked genes; one controls suppression of ovules and the other development of normal pollen. Oberle's theory and Loomis' conclusion contrasts with Antcliff's (1980) theory of a single set of three alleles. Antcliff did not find support for Loomis' conclusion in the population he studied. However, he did not use any Muscadinia genotypes in his investigation. Some of Loomis' crosses between pistillate and perfect flowered types produced staminate seedlings, something Oberle's theory is not able to explain. According to Loomis, staminate plants may have resulted from "those accidents that plague all plant breeders" (e.g., accidental insect pollination, pollen contamination, mixing of seed). However, he did not want to totally ignore them and states sex inheritance in muscadines may not be exactly the same as that for Euvitis; especially since muscadines have one more pair of chromosomes than Euvitis. Work by Loomis et a1. (1954) found that when self-pollinated, 21 hermaphroditic parents can be placed into two groups: those producing hermaphroditic and pistillate progeny, and those producing hermaphroditic, pistillate, and staminate progeny. The first group produced 3 hermaphroditic:1 pistillate, the second, produced 9 hermaphroditic:3 pistillate:4 staminate. Upon investigating parentage he found that plants producing only hermaphrodite and pistillate progeny had Dearing's H1 plant as the source of hermaphroditism, and the group producing progeny with all three flower types had H2 as the common parent. Therefore, even though H1 and H2 were phenotypically similar they were genotypically distinct in the mutation that led to hermaphroditic flowers. Reviewing Reimer and Detjen's unpublished crossing records, Loomis and Williams (1957) conclude that the hermaphroditism of 'Hope' is genetically similar to Dearing's H2 plant; they theorize that H2 originated as a mutation of a staminate plant. From crossing records it is clear 'Hope', H1, and H2 are heterozygous for hermaphroditic flowers. H1 follows Oberle's (1938) Euvitis, although H2 and 'Hope' do not. Loomis and Williams (1957) reported the first vine genotypically homozygous for hermaphroditic flowers. They also indicate the additional pair of chromosomes and apparent polyploid nature of muscadines complicate inheritance ratios, making it difficult to determine how many genes control flower type in muscadines.

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All perfect flowered muscadines currently recommended for planting in North Carolina (Poling et a1., 1987), and whose ancestries can be easily traced, are derived from H1 or H2, or both, none have 'Hope' as an ancestor (Table 8.2). Of the 17 cultivars listed in Table 8.2, H1 is in the pedigree of seven and H2 is in the pedigree of 15. H1 is the only source of hermaphroditism in 'Tarheel' and its seedling 'Noble'; although H2 is the only source in 10 of the cultivars listed. The first self-fertile cultivars were those released by Dearing in 1946, of which 'Tarheel' is probably the best known. Why it took 30 years to release the first self-fertile cultivar from the time breeding for this trait began is unclear. Such strong emphasis is placed on self-fertility in the North Carolina program that no cultivar is released unless it is self-fertile, and no pistillate cultivar has been released from the North Carolina program since 1946 (Goldy 1987). Pistillate cultivars, however, continue to be of importance and are released to the industry (Lane and Owen 1989). Published results on muscadine flower type ends with Loomis and Williams' 1957 report. New investigations are warranted, especially with the much broader germplasm now available including intercrosses between H1 and H2. Relying on only two sources of hermaphroditism for all of muscadine breeding is not a sound idea for long-term progress. New sources of the trait should be identified and utilized. Table 8.2. Hermaphroditic ancestor and pigmentation of perfect flowered muscadine cultivars recommended for North Carolina.

Cultivar Albemarle Bountiful Burgaw Carlos Chowan Dearing Dixie Doreen Magoon Magnolia Noble Pamlico Regale Roanoke Southland Sterling Tarheel

Hermaphroditic Ancestor HZ HZ HZ Hi HZ HZ HZ HZ HZ H1 H1 HZ Hi Hi HZ H1 H1

&

Hz

&

Hz

& HZ &

HZ

&

HZ

Fruit Color Dark Dark Dark Light Light Light Light Light Dark Light Dark Light Dark Light Dark Light Dark

370

R. G. GOLDY

2. Fruit color. Fruit of wild muscadine is usually pigmented (red to purple or black), with occasional nonpigmented (white or bronze) vines observed. Understandably, fruit color caught the attention of early amateur and professional breeders. Those who simply took open pollinated seed from white fruited vines were discouraged at the low number of white fruited seedlings. This is not surprising with present knowledge of pollination and genetics of pigmentation. Work by Reimer and Detjen (1914) and Stuckey (1919) focused on color relationship and transmission. Recent studies focus on individual skin pigments and pigment relationship to color quality of processed products. Reimer and Detjen (1914) and Stuckey (1919) concluded white fruit color was recessive to red and black; red was recessive to black. This is similar to what Barritt and Einset (1969) reported for Euvitis grapes when ) dominant they found a two-gene system with black fruit color (B and epistatic to red (bbL.) and white (bbrr), and red dominant to white. This along with cross pollination explains why open pollinated seed from white-fruited clones have a preponderance of black-fruited progeny. Vines having pigment in leaves. tendrils. and stems always have pigmented fruit. and vines with bright green vegetation have white fruit. Since color can be selected on vegetation it is possible to select color of staminate vines and possibly seedlings possessing superior pigmented fruit based on vegetative pigments (Goldy et a1. 1987a, 1987b). In North Carolina, most clones introduced from the wild were pigmented. However, since 1946 this has reversed, and most releases from breeding programs have been nonpigmented. This reflects the need for improved white-fruited cultivars, and low interest in black-fruited muscadine. Processors (primarily wineries) do not prefer pigmented muscadines since products from them obtain inferior color with age (Sims and Morris 1986). Much research has gone into eliminating or slowing this process during fermentation and aging, and at identifYing inferior and superior pigments and those clones possessing them. The current industry is dependent on nonpigmented grapes producing white-juiced products. Red wines are produced in limited quantities from muscadines, and a red product can be made by blending white muscadines with pigmented Euvitis grapes (Sistrunk and Morris 1985). Development of superiorpigmented muscadines through breeding appears to be the long-term solution and is an important. attainable goal for muscadine breeders. The first identified muscadine pigment was muscadinin (Brown 1940), later identified as 3,5-diglucoside-3'-O-methyldelphinidin chloride. commonly referred to as petunidin (Ballinger et a1. 1973). Ribereau-Gayon (1959, 1964) determined muscadines had nonacylated 3.5-diglucoside

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forms of malvidin (Mv), peonidin (Pn), petunidin (pt), cyanidin (ey), and delphinidin (Dp). Once reliable identification techniques were available research was initiated to determine which pigments were most stable, how different physical and chemical properties affected them, what could be done to improve product quality, and how pigments change during fruit maturation (Sistrunk and Morris 1982; Sims and Morris 1984, 1985,1986; Lamikanra 1988). Only information relative to breeding will be presented; those readers interested in product quality are referred to Carroll (1985). Diglucoside anthocyanins are generally more susceptible to browning than monoglucoside forms (Robinson et al. 1966). There are differences among the pigments; Dp is the least stable and Mv the most (Hrazdina et al. 1970; Ballinger et al. (1974); Nesbitt et al. (1974b); Simpson et al. 1976 and Flora 1978 found a direct correlation between Mv in skins of fresh berries and good wine color). They found sufficient Mv levels could overcome high Dp levels, and the best-pigmented cultivars were 'Tarheel' and 'Noble'. 'Tarheel' is high in total pigment and Mv, and 'Noble' is high in Mv in comparison to Dp. The survey by Ballinger et al. (1974) found considerable variation in total pigment (ranging from 20-1038mg/100g fresh fruit) and within the pigments (0-251 for Mv, and 8-496mg for Dp). A survey of wild germplasm (Goldy et al. 1989) reported ranges of 0.43-12.32mg/ml for total pigment and 0.8-30.0% of total pigment for Mv and 13.5-68.9% for Dp. Such surveys allow breeders to identify superior types and begin recurrent selection programs to improve color. The survey of Goldy et al. (1989) identified vines possessing 3monoglucoside pigments, something not previously reported. There were three possible reasons for this discovery: (1) greater sensitivity of new analytical techniques, (2) plants may be hybrids with Euvitis, or (3) plants containing monoglucosides were not sampled in previous studies (the most plausible reason). Since ability to produce diglucosides is dominant over monoglucosides (Einset and Pratt 1975), this could be the case. If their numbers are representative of the frequency of monoglucoside producers in the muscadine population, this gives a phenotypic frequency of only 11% (a number that could be missed); especially when sampling among breeding lines, which are more closely related than indiyiduals in wild populations. These plants present the possibility for further investigation into this area. This survey did not find vines that produced acylated pigments. This is unfortunate since acylation enhances pigment stability (Van Buren et al. 1968). It is possible that ability to produce acylated pigments is not present in muscadines, or is at such a low frequency that larger sample sizes are needed for detection. When some of Mortensen's (unpublished data) hybrids between

372

R. G. GOLDY

\Z rotundifolia, V. munsoniana, and V. popenoei were analyzed for pigment quality, some were found to contain relatively high amounts of Mv and low Dp. One hybrid contained 72.6% Mv and 5.1% Dp (measured as percent of total), others were 63.6% Mv and 7.7% Dp, and 56.3% Mv and 2.9% Dp. Although only a few hybrids were analyzed it is possible V. popenoei and V. munsoniana contain better-pigmented types, which could be used to improve V. rotundifolia pigmentation. It should be a fairly easy task to develop clones having superior pigmentation by utilizing natural variation. Discovery of plants having both pigment types makes it possible to combine browning resistance of monoglucosides with color brightness of diglucosides (Robinson et a1. 1966). By developing clones possessing mono- and diglucoside pigments, high Mv and low Dp, and high-total pigments, breeders will make a significant and economically important contribution to the muscadine industry. The color of processed products is a good trait to compare passive versus active selection. For natural selection there is no forseeable reason why it would be advantagous for grapes to contain pigments stable in a processed product. As long as the grape has sufficient pigmentation to attract nonhuman seed dispersal agents there should be no selection pressure for individual pigments. However, when humans began producing wine, it would become obvious that fruit collected from certain clones, or perhaps clones from a given area (populations), produced better-colored wine than did others. Once noticed, superior clones could be propagated for cultivation. In cultivation, selection can continue until the most prized grapes are identified. As evidence of this, many V. vinifera cultivars (pigmented and nonpigmented) have not resulted from active breeding but have been cultivated for centuries and are of unknown origin (Kasimatis et a1. 1980, 1981). Since modern muscadine breeders have a greater knowledge of pigmentation and possess tools to identify and quantify pigments, potential for rapid improvement is great and should be exploited. 3. Pest resistance. Early workers greatly admired the apparent pest resis-

tance of muscadines. However, once cultivated, muscadines proved they had their share of pests. With the current public sentiment against pesticides and the reluctance of chemical companies to register products on minor crops (e.g., such as muscadines), development of genetic resistant cultivars is an important part of the long-term solution. Muscadines vary in response to several economically important grape diseases and insects [fable 8.3). Many evaluations of responses to pests have come through active screening programs, and others have been obtained by years of field observation. The assumed resistance of

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Table 8.3. Relative importance of several grape diseases (Pearson and Goheen 1988) and insects on Vitis rotundifolia. (- =immune, + =little damage, and ++++ =potentially severe damage).

Common name Disease Angular leaf spot Anthracnose Armillaria root rot Bitter rot Black rot

Botrytis bunch rot Cotton root rot Crown gall Dagger nematode Downy mildew Eutypa dieback Fanleaf degeneration Grape rust

Leaf blight Macrophoma rot

Phomopsis cane and leaf spot Pierce's disease Powdery mildew

Causal Organism

Mycosphaeria angulata Jenkins Elsinoe ampelina (de Bary) Shear Armillaria mellea (Vahl:Fr.) Kummer Greeneria uvicola (Berk. & Curt.) Punithalingam Guignardia bidwellii f. sp. muscadinii Luttrell G. bidwellii f. sp. euvitis Luttrell Botryotinia fuckliana (de Bary) Whetzel Phymatotrichum omnivorum (Shear) Dug. Agrobacterium tumefaciens (E. F. Smith & Townsend) Xiphinema index Thorne & Allen Plasmopara viticola (Berk. & Curt.) Ber1. & de Toni Eutypalata (Pers.: Fr.}Tul. & C. Tu1. nepovirus Physopella ampelopsidis (Diet. & Syd.) Cumm. & Ramachar Pseudoeereospora vitis (Lev.) Speg. Botryosphaeria dothidea (Moug. ex Fr.) Ces. & de Not. Phomopsis viticola (Sace.) Sacco Xylella fastidiosa Wells et a1. Uncinula necator (Schw.) Burr.

Relative Importance

++

Source

Clayton 1975 Mortensen 1981 Clayton 1975

+++

Ridings & Clayton 1970

+++

Jabco et a1. 1985

+

Jabco et a1. 1985

+

Smit et a1. 1971 Mortensen 1952

++

Graves et a1. 1988 Olmo 1986 Espino & Nesbitt 1982 Olmo 1986

+

Bouquet 1981

+

Clayton and Ridings 1970 Clayton 1975

+++

Clayton 1975

Olmo 1971

+ +++

Mortensen et a1. 1977 Clayton 1975

continued

374 Table 8.3.

R.G.GOLDY Continued. Relative Importance

Common name

Causal Organism

Ripe rot

Colletotrichum gloeosporioides (Penz.) Penz. & Sacco Meloidogyne spp. (Goeldi) Chitwood Cristulariella moricola (Hino) Redhead

++++

Endopiza viteana Clemens

+

Root-knot nematode Zonate leaf spot Insect Grape berry moth Grape curculio

Erythroneura vulnerata Fitch Vitacea polistiformis (Harris) Aphis illinoisensis Shimer

Japanese beetle Phylloxera

Popillia japonica Newman Daktulosphaira vitifoliae (Fitch)

Daykin & Milholland 1984 Bloodworth et a1. 1980 Clayton 1975

Craponius inaequalis (Say)

Grape leaf hopper Grape root borer Grapevine aphid

Source

++ +++ + +++ +

McGiffen 1985 McGiffen 1985 McGiffen 1985 McGiffen 1985 McGiffen 1985 McGiffen McGiffen 1985

&

Neunzig

&

Neunzig

&

Neunzig

&

Neunzig

&

Neunzig

&

Neunzig Neunzig

&

muscadines to pests (e.g., phylloxera, root borer, and powdery mildew) has not held up. This has led to conflicting reports, such as Bouquet (1980b), indicating that muscadines have good resistance to powdery mildew and Clayton (1975) stating it can be a potentially severe problem. Powdery mildew was not observed on muscadines until the 1960s, and then only on fruit (Clayton 1975). It was then found to be abundant on muscadine leaves in 1973 (Clayton 1975). Other reports have demonstrated the stability of resistance over time, suggesting muscadines may truly be resistant and not just escaping due to lack of exposure to sufficient inoculum. Examples of this are anthracnose and downy mildew. Mortensen (1981) reported that after several years ofvineyardobservationnomuscadinecultivarwas foundto have anthracnose, ap parently even under conditions conducive for infectivity and expression. Muscadines have also been reported to be immune to phylloxera and root-knot and dagger nematodes. A recent report by McGiffen and Neunzig (1985) indicates phylloxera has been found on cultivated V. rotundifolia. Bouquet (1981) reports dagger nematodes do feed on muscadine roots, but populations decline over time. Whether or not

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phylloxera and nematodes will develop into serious problems for muscadines remains to be seen. In North Carolina where a majority of the grapes are processed botrytis bunch rot has not been reported to be a problem (R. D. Milholland, personal communication). However, Smit et al. (1971) report that in Georgia it can be isolated from freshly harvested berries and is one of the main fungal pathogens observed in storage of fresh grapes. Useable variation for relative disease resistance has been observed within the improved muscadine germplasm. Clayton (1975) evaluated 10 cultivars for bitter rot, black rot, angular leaf spot, powdery mildew, ripe rot, and macrophoma rot and found variation for each disease, with 'Albemarle' being least susceptible overall. 'Carlos' and 'Magnolia' (currently the two leading cultivars) were among the most susceptible for all six diseases. Variation in disease susceptibility has also been reported for Pierce's disease (PD) (Hopkins et al. 1974), grapevine fanleaf virus (Walker et al. 1985), and grape rust (Clayton and Ridings 1970). Espino and Nesbitt (1982) report the source of resistance to downy mildew is failure of fungal germ tubes to penetrate stomates, thereby stopping infection. Huang et al. (1986) found PD-resistant muscadines inactivated the disease organism by encapsulating it with a coating consisting of pectic substances, gums, and tannins. Development of disease-resistant cultivars is one of the most important tasks facing muscadine breeders. Resistant material has been identified for most major diseases, and artificial inoculation techniques already exist. It should be a simple matter of intercrossing and screening seedlings through artificial inoculation procedures but they are not routinely practiced. Rather, seedlings are subjected to natural inoculum levels in the field and susceptible types discarded. Currently the most serious insect problem for muscadines is grape root borer, which feeds on cultivated and wild vines and can cause vine death. Chemical controls do exist but timing of application is such that chemical activity is lowest at the time it is most needed. Research into disrupting mating through pheromones is being conducted and appears promising (Johnson et al. 1986; J. R. Meyer, personal communication). The other insect of significance is the Japanese beetle, which feeds on vines during June and July and can cause extensive leaf damage if not controlled. No studies have been reported on variation within muscadine germplasm for insect susceptibility. 4. Yield. Many environmental and genetic factors contribute to yield, and

separating them is difficult in large, long-lived plants, such as grapes. Performance studies have been conducted on suitability of various

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muscadine cultivars to specific locations. These were conducted so recommendations could be made to local growers, with no effort to determine genotype X environment interactions. Onokopise and Mortensen (1988) reported significant differences for sugar levels and pruning weights for six cultivars at two locations. 'Fry' had a wider adaptation for sugar, and 'Noble' and 'Higgins' were narrowly adapted for pruning weights. Care must be taken in drawing conclusions from this study since locations were only 8km apart and data were taken only one year. In general muscadine cultivars have a wide area of adaptation since the same cultivars are important in the major producing states (Olien 1990). What determines planting of a particular cultivar may rely more on industry demand than on adaptability. Two general factors limiting broad adaptability are cold and PD tolerance. Those cultivars developed in southern areas may not be able to survive winters in northern areas of the growing region, although those developed in northern areas where PD pressure is not as great, may not be able to survive the PD pressure present in the South. One would expect yield increases on a per-hectare and per-vine basis would have been realized when perfect-flowered types were developed. This is generally true, although some pistillate vines will outyield some perfect-flowered vines. When Mortensen and Balerdi (1974) evaluated 24 cultivars in central Florida, pistillate types averaged 6.8 tlha compared to 10.4 tlha for perfect-flowered types. The highest yielding cultivar was 'Carlos' (perfect flowered) at 19.3 tlha followed by 'Pride' (pistillate flowered) at 16.8 Vha. The two lowest-yielding vines were two pistillate types, 'Topsail' and 'Scuppernong'(0.7 and 1.1 tlha, respectively). A ninecultivar trial by Nesbitt et a1. (1982a) found similar results with perfectflowered types outyielding pistillate-flowered ones. Mortensen and Harris (1989) report yields of 30 cultivars grown in central Florida ranging from 1.1-19.9 t/ha. Pistillate cultivars ranged from 1.1-11.9 and perfectflowered types from 3.6-19.9 tlha. Yields reported for these studies did not consider yield loss of pistillate cultivars due to the need of staminate pollenizers. A better comparison is between recently developed cultivars with older ones of similar flower types. Comparisons of this type are difficult since studies have not been initiated to directly answer this question. What is clear from most yield trials is that cultivars from breeding programs, regardless of flower type, are higher yielding than cultivars that were wild selections. 'Scuppernong' is notoriously low yielding and will be one of the lowest-yielding cultivars in any planting. Another component contributing to yield is flower cluster size. Interest in increasing flower cluster size, and the resulting fruit cluster size, came from the desire to market whole clusters rather than individual

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fruit. The first studies were by Husmann and Dearing (1913), Reimer and Detjen (1914), Dearing (1917a), and more recently Goldy (1988). Reimer and Detjen (1914) reported considerable variation in number of flowers per cluster in staminate clones, and by using those clones with large flower clusters they could increase fruit cluster size. They do not indicate range in numbers other than clusters having 1-9 flowers were very small and clusters having 90 or more flowers were very large. Of the seedlings observed, 341 were very small and 7 were very large. Using advanced, perfect-flowered selections, Goldy (1988) found vines having individual flower clusters ranging from 44-349 flowers, with clonal averages of 72-202, compared to 24-40, for four-pistillate cultivars observed by Dearing (1917a), and 15--48 for those observed by Husmann and Dearing (1913). Increasing flower cluster size should cause a Yield increase for two reasons (Dearing 1917a). Most obvious is flower number. The other is that pollinators are attracted by odor, and clusters having more flowers will have more odor, increasing their chances of being pollinated. 'Scuppernong's' low yield potential may be due to its small bloom cluster averaging only 24 flowers (Dearing 1917a). Reimer and Detjen (1914) noted that progress could be made in increasing flowers per cluster by using staminate clones having large flower clusters. Husmann and Dearing (1913) state V. munsoniana clones naturally set fruit on a higher percentage of flowers per cluster. It is fortunate that the mutation leading to perfect-flowered types occurred in staminate and not pistillate plants. By occurring in staminate plants it combined the large flower cluster potential of staminate vines with the fruiting ability of pistillate vines. This is probably the reason Goldy (1988) found such high values in the cultivars he evaluated. However, having a large number of flowers does not necessarily guarantee larger fruit clusters since other factors, such as weather during bloom time and number of pollinators, can affect fruit set. This was observed by Reimer and Detjen (1914) and again by Goldy (1988). Goldy found that only 12.25% of the variability in fruit number from six clones could be attributable to flower number. The clone having the highest number of flowers per cluster (202) averaged 21 fruit, although one having 128 flowers per cluster averaged 26 fruit. Reimer and Detjen felt there was a limit of between 25 and 30 berries possible per cluster, with the largest fruit cluster they found having 27 berries. Goldy (1988), however, reported fruit clusters as large as 80 berries. These figures indicate muscadines have fairly low fruit-set potential. Fruit set from Dearing's (1917a) study ranged from 5-16%, Husmann and Dearing (1913) found 10-38%, while Goldy (1988) found 11-23%. In Goldy's study the clone having the highest fruit set had the lowest fruit

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number (16) and flower number (72). Goldy also found flower number to be negatively correlated to fruit set (r = -0.42; P = 0.005). Although no data is presented, Dearing (1917a) states that perfect-flowered clones are capable of setting fruit from 25-50% of their flowers. Reasons for such low fruit set are not clear. Why a plant has the potential of setting 80 fruit on some clusters but averages only 19 warrants further investigation, perhaps from a physiological standpoint. It may be necessary for breeders to concentrate on selecting for high fruit set before further progress in yield can be made for muscadines. Also, the flower and fruit clusters still do not approach the size of those found in Euvitis. The only way to attain this may be by transferring genes for the trait from Euvitis. Due to low fruit set a girdling study was initiated to induce greater fruit set (R. G. Goldy, unpublished data). Initial studies on four cordon Geneva Double Curtain grown vines found for 1/4 vines (one cordon) and for 1Jz vines (two cordons) would survive a 0.33cm girdle, and spurs would not. Preliminary results showed a visual decrease in vegetative growth, and a visual increase in crop load on some vines. Some of the berries were shot berries and did not develop full size. A slightly earlier ripening date was noticed, and vines did not suffer any apparent winter damage. Another factor contributing to yield is berry size. Muscadines are noted for large berries, with wild vines often having fruit 2.5cm in diameter (Reimer 1909). Much variability is present in native and improved germplasm. Goldy et a1. (unpublished data) found berry weights ranging from 1.5-4.7g in 67 wild vines, Mortensen and Balerdi (1974) found ranges of 2.9-9.5g in 48 advanced genotypes, and Mortensen and Harris (1989) found ranges of 2.6-11.5g from 30 cultivars. Early breeding investigations on fruit size by Reimer and Detjen (1914) were complicated because the fruit size potential of staminate clones could not be determined without progeny testing. From five crosses they observed seedlings having "small" to "very large" fruit (they did not define what makes a small or very large berry). Also, when they pollinated 'Scuppernong', 'Thomas'~ and 'James' with the same staminate vine, they found seedlings of 'Scuppernong' ranged from small to very large; seedlings of 'Thomas' had mostly medium-size fruit, and seedlings of 'James' had large to very large fruit. They concluded that berry size could be improved through breeding and was not correlated with fruit cluster size. Their conclusions were supported by Goldy (1988) who found significant variation in fruit size from six clones, ranging from 3.47.2g per fruit, with a correlation of r= 0.41 (p =0.006) of flower number to fruit size. Williams (1954) found 'Duplin', a perfect-flowered cultivar, transmitted larger berry size than other cultivars studied, and Fry (1967) found crosses having 'White Male' in their ancestry transmitted larger

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berry size to their progeny. Williams (1957) noticed many hermaphroditic vines had small berries when pistillate vines from the same cross had larger, a correlation he thought attributed to lower productivity of the pistillate vines. However, when he compared fruit size of pistillate and hermaphroditic vines of similar productivity or weight of seed per berry from the same cross, pistillate vines still had larger berries. His explanation included metaxenia or gene linkage. Muscadine breeders have been successful in transmitting large fruit size, regardless of flower type. 'Fry' is one of the larger-fruited pistillate cultivars, averaging 12.0g per fruit, and 'Nesbitt' is one of the largest-fruited perfect-flowered cultivars with fruit averaging 10.0g. Fry (1963) induced tetraploidy with gamma radiation and increased berry size but other characters were also changed, including: greater propagation difficulty; larger, thicker, and darker leaves; increased cane diameter; shorter internodes; and larger pollen, seed, and fruit. There are no reports on the use of autotetraploidy in muscadine breeding. Goldy (1988) evaluated correlations between five Yield components (flower and fruit number per cluster, fruit cluster and individual fruit weight, and fruit set) and total vine yield. Of the possible 15 correlations only three were higher than r = 0.50: fruit number with cluster weight (r = 0.69; p = 0.0001), fruit number with fruit set (r = 0.57; p = 0.0001), and cluster weight with fruit set (r= 0.52; p =0.0003). No trait was highly correlated to total Yield, indicating to make progress for Yield it would be better to select for Yield directly rather than for components contributing to Yield. No reports were found on photosynthetic rates, efficiencies, or photosynthate partitioning for muscadines.

5. Fruit quality. Fruit quality depends on intended use (fresh or processed) and is a summation of several different factors, including sugar and acid and their ratios, flavor, skin thickness, seed size, pulp texture, adherence to the vine, and uniform ripening. All of these traits were of importance to the first muscadine breeders and continue to be important in current programs. Initial investigations on most of these traits were made by Reimer and Detjen (1914) and Dearing (1917a). Wild muscadines generally have fruit that is easily removed from the vine. This has commercial advantages and disadvantages. If it comes off too easily (shatters) fruit can be removed by a slight breeze or the force of its own weight, causing fruit drop prior to harvest. However, easily removed fruit makes mechanical harvesting easier. An associated trait with shattering is an incomplete abscission zone, allowing juice to leak. coating berries and making them sticky, and allowing for mold growth

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and fermentation. This limits shelf life and is one of the main hindrances to fresh market sales. In studying seedlings produced from 'Scuppernong', 'James', and 'Thomas', Reimer and Detjen (1914) found two classes for shattering: "very poor," which dropped at the slightest touch and "poor," which were a bit more tenacious. They did find vines that were "very tenacious" but they were the exception, and they identified 'Flowers' as a possible source of better adherence. Dearing (1917a) noted 'Flowers' had good adherence and he used male seedlings of 'Flowers' in crosses with cultivars with poor adherence. Williams (1954) found 'Tarheel' gave seedlings with more persistent fruit and seedlings from 'Burgaw' and 'Wallace' shattered equally severely. Sherman and Nevins (1963) found a noticeable difference when studying the abscission zone in 'Thomas' (an abscissing type) and 'Dulcet' (a nonabscissing type). 'Dulcet' did not form a distinct abscission layer and 'Thomas' did. When 'Dulcet' was picked the vascular tissue was retained on the pedicel causing a direct opening in the fruit for leakage and pathogen invasion. Muscadines range from fruit that is easily removed to strong adherence (Balerdi and Mortensen 1974). Current cultivars also vary, but few shatter since this character has been heavily selected against. 'Magnolia' adheres too tightly and results in torn fruit when harvested, making it suitable for processing only, and then quickly before premature fermentation can begin. 'Carlos' is the standard for dry stem scars with reports of as high as 87% (Lane and Bates 1987), and is suitable for onceover mechanical harvest since it retains its fruit well even when ripe (Nesbitt et al. 1970). Balerdi and Mortensen (1974) evaluated 24 cultivars for suitability to mechanical harvest and obtained ratings of 0.1 ('Dulcet') to 12.6 ('Carlos'), with 7.0 or higher adequate for mechanical harvest. They reported a correlation between large berry size and ability to be mechanically harvested. For ease of mechanical harvest breeders should select for low fruit retention force, concentrated ripening, dry pedicel scar, and resistance to berry rot in addition to berry size. Ethephon has been applied to muscadines to enhance abscission and reduce amount of ruptured berries during harvest (Mainland et al. 1977; Lane and Flora 1979; Phatak et al. 1980). Two other traits important for fresh fruit quality are seed size and skin thickness. Muscadines are naturally large seeded and thick skinned, both objectionable traits. Reimer and Detjen (1914) found differences in parental ability to transmit skin thickness. Seedlings from 'James' were thicker skinned, although 'Scuppernong' had some thin-skinned types. Dearing (1917a) found seedlings from 'Eden' had thin skin. Mortensen and Balerdi (1974) found a range of 0.49-1.35 mm for skin thickness from

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48 improved genotypes. Thinner-skinned types are being developed (R. P. Lane, personal communication), which should be more attractive as fresh fruit. There is a danger though, in that muscadines probably have developed thick skin as a defense mechanism against pests, and thinner skin may lead to greater pest problems and cracking during rainy periods. Reimer and Detjen (1914) showed seed size can be affected through breeding. 'James' transmitted larger seed than 'Thomas', and 'Scuppernong' was intermediate. Since seed size and berry size are correlated, breeders would be working on physiologically opposing objectives when trying to increase fruit size while decreasing seed size. For fresh fruit the greatest improvement would be to eliminate seeds. 'Fry Seedless' is the only seedless muscadine reported (patented by Ison's Nursery & Vineyards, Brooks, GA). It appears parthenocarpic rather than stenospermocarpic (D. J. Gray, personal communication), as are most Euvitis seedless grapes, and is male sterile (R. P. Lane, personal communication). This plant appears of no use in breeding, since it cannot be used as a female or a male. As seedlessness in muscadines is extremely rare incorporating seedlessness from Euvitis may be the only means of introducing the trait to muscadines. Reimer and Detjen (1914) and Dearing (1917a) investigated pulp quality and found types ranging from melting to tough and stringy. This is more important for fresh than processed fruit. For fresh fruit a firm but tender pulp that is easily separated from the seeds is desired. High-quality types for this trait have been developed and are recommended for fresh market. One breeding line with a tender skin and a solid pulp similar to V. vinifera has been identified (R. P. Lane, personal communication). Flavor is a difficult trait to measure due to its complex nature and the inherent subjectivity in measuring it. Also, desirable fresh fruit flavor does not always translate into desirable wine flavor. Prior to release and recommendation, muscadines are subjected to sensory evaluation and only those having satisfactory quality for their intended use are introduced (Mortensen and Harris 1988). Two easily measured important components of flavor for fresh and processed grapes are sugar and acid, and their ratio. Muscadines have a relatively low sugar content making it necessary to add sugar prior to fermentation so desired alcohol levels will be reached (Carroll 1985). In a survey of 67 wild clones Goldy et a1. (unpublished data) found fruit having soluble solid levels between 7.4% and 14.0%: pH ranged from 2.8-3.3 and acidity (measured as tartaric acid) from 0.982.85%. For 43 cultivars Armstrong et a1. (1934) reported ranges of 12.821.3% for soluble solids, 2.96-3.42 for pH, and 0.41-1.31% for titratable acidity. Carroll et a1. (1975) and Nesbitt et a1. (1982a, 1982b) found similar

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values for current cultivars. Suitable genetic variation has been found in sugar and acid levels. Early studies were complicated by not being able to directly determine pollen parent phenotype. Williams (1954) found 'Wallace' seedlings from self- and cross-pollinations low in sugar, but crosses with 'Latham', 'Tarheel', and 'Burgaw' generally gave seedlings with higher sugar. Many environmental factors out of the breeders control, however, directly affect these two parameters. The muscadine production area is quite prone to variable weather during the growing season, and to breed a cultivar that would have consistent sugar and acid levels from year to year would be difficult to accomplish. Aroma is an important component of flavor, and it is this trait that distinguishes muscadine products. The muscadine aroma ranges from pleasant to repugnant, with too strong of an aroma unacceptable. Those with a pleasant aroma produce the distinctive "fruitiness" characteristic of muscadine products. An effort has been made to develop neutral muscadines (Lane and Bates 1987), which produce neutral wine. Doing this, however, causes muscadines to lose their uniqueness and puts them in direct competition with V. vinifera wines, something muscadine wineries should avoid. Uneven ripening between and within clusters is common to muscadines. For pick-your-own or fresh-fruit sales, this is beneficial since it prolongs the harvest season. 'Loomis' and 'Nesbitt' are highquality cultivars with extended ripening and are recommended for fresh fruit (Goldy and Nesbitt 1985; Lane and Owen 1989). Uneven ripening presents a problem for wine production. Since most grapes for wine are mechanically harvested by vibration, berries range from badly decayed to hard green. This can be partly controlled by timing of harvest and the experience of the person operating the harvester. Several methods of separating harvested grapes into ripeness categories have been developed and include: vibration (Hamann and Carroll 1971); density (Lanier and Morris 1979); light (Ballinger et a1. 1978), but none are currently used commercially. Carroll et a1. (1978) sorted grapes into four ripeness classes, made wine from each class, and compared it to wine made from unsorted grapes. They found unsorted, underripe, and overripe grapes produced wine inferior to the two optimum ripeness classes. This points out the need to strongly select for even ripening types. This has been a goal from the initiation of muscadine breeding programs, and continues to be important but there are no genetic studies of this trait. 6. Other traits. Other traits have been investigated in muscadines but are

no longer important, such as breeding for improved staminate vines

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(Dearing 1917a). Important traits for male vines were profuseness of bloom, foliar disease resistance, long bloom period, and proper bloom time. Most growers who have pistillate vines currently use hermaphroditic vines as pollinizers, eliminating the need for staminate vines. Boyle and Hsu (1990) identified a sediment sometimes observed in standing muscadine juice as ellagic acid, which varied from 1.6-23.1 ug/mL ('Higgins' and 'Hunt', respectively) in the 11 cultivars they evaluated. Since ellagic acid has been indicated as a naturally occurring inhibitor of carcinogenesis (Daniel et a1. 1989), breeders may want to increase its content rather than decrease it in muscadine juice. There are no genetic studies on time of bloom and time of ripening. Wide differences in ripening time do exist, from early September to midOctober for central North Carolina (Poling et a1. 1987). Differences in bloom time, however, are not that great and differences in maturity appear to be more related to rate of fruit development than to bloom time. If earlier blooming V. rotundifolia genotypes could be developed, growers could have an earlier and longer season, better utilizing labor and equipment and processors would not handle such large volumes in such a short time, possibly increasing product quality. Cyanamide applications have been used to break dormancy in V. vinifera grapes (Shulman et a1. 1983, 1986; Zelleke and Kliewer 1989). In most years V. rotundifolia could bloom 2-3 weeks earlier and still escape spring frosts. Cyanamide applied in the early spring to dormant muscadines inhibited budbreak (R. G. Goldy and M. Mainland, unpublished data), a typical reaction if the plant has fulfilled dormancy and is in the quiescent stage (Fuchigami and Nee 1987). This indicates budbreak in muscadines is possibly controlled more by heat unit than chill unit accumulation, helping to explain why muscadines grow well in North Carolina when exposed to approximately 1500 chilling h and in central Florida where they get 150 h. Several growth regulators have been used to break dormancy in woody plant species (young 1987) and should be investigated on muscadines to determine if they have a beneficial effect. IV. BREEDING: INTERSUBGENERIC HYBRIDIZATION A. History

Production of Euvitis X Muscadinia hybrids has been of intense interest for amateur and professional breeders for many years. The desire in making these crosses is to combine adaptability, pest resistance (rootstock and scion), fruit size, and ease of harvest of the muscadine with fruit quality of Euvitis. The first reported effort to produce these hybrids

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was that of Wylie (1871). Wylie's seedlings were highly sterile and therefore probable hybrids; however, none of the plants from his work survive. Later, Millardet (1901) pollinated several different V. vinifera with V. rotundifolia and reported several seedlings thought to be hybrids but which resembled the female parent entirely. Munson, in 1891, collected open-pollinated seed from a white-fruited, pistillate muscadine, which he thought had been pollinated by a nearby Euvitis plant (Detjen 1919a). From 50 seedlings he selected and named two, 'La Salle' and 'San Jacinto'. He named four additional clones, which he also reported as hybrids. Detjen (1919b) demonstrated that Millardet's and Munson's "hybrids" were not hybrids but self- or accidental pollinations. Since there were V. munsoniana staminate vines in the vicinity, 'La Salle' and 'San Jacinto' are likely V. rotundifolia X V. munsoniana hybrids. Efforts of these early workers were complicated due to differences in bloom time between most Euvitis and Muscadinia, and lack of knowledge and/or ability to store pollen. Vitis rotundifolia blooms after most Euvitis species when grown in the same location. The logical way to make hybridizations, therefore, is to use Euvitis as the male and Muscadinia as the female, storing pollen for only a short period of time. This also allowed use of pistillate muscadine clones, making timeconsuming emasculation unnecessary. This is what most early breeders did; however, it was later discovered the reciprocal cross had much greater success (Detjen 1919a). Differences in bloom time can be overcome so V. rotundifolia can be used as the male. Vitis rotundifolia vines can be brought into a greenhouse and forced to flower early, pollen can be stored for 11 months between bloom time of the parents, or pollen can be obtained from plants grown further south, since in normal years V. rotundifolia bloom in central Florida is about the same time as most Euvitis bloom in North Carolina. All these techniques have proven successful (Goldy et a1. 1988). Other techniques are the use of late-blooming Euvitis and the forcing of secondary flowering on Euvitis. The first scientific effort investigating Euvitis X Muscadinia hybridization was initiated in 1911 by Dearing (1917a) and Detjen (1919a). Dearing followed methods of previous breeders and always used pistillate muscadines as females. He also used only three pure Euvitis species and two Euvitis species hybrids. Detjen was critical of Dearing's limited species numbers and his failure to perform reciprocal crosses. Dearing, however, did obtain true hybrids and found crosses with 'Thomas' more successful than other pistillate clones he used. Dearing stated that hybrids can be produced in quantity using V. rotundifolia as the female, a statement not substantiated by Detjen (1919a) and others (Patel and Olmo 1955).

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Detjen's research again appears to be more thorough than that of Dearing. His studies were better planned, much broader in scope, and it is his work that produced the first documented, true Euvitis X V. rotundifolia hybrid (Detjen 1919a). Detjen used 'Scuppernong' as the female and applied pollen from V. labrusca, V. aestivalis (Michx.), V. cinerea (Engelm. ex Millardet), and V. bourquiniana (Munson). Fruit and seed were only obtained from V. bourquiniana and they concluded that 'Scuppernong' hybridized poorly with these species. Detjen (1919b) investigated previously reported hybrids produced by other workers and found many were not hybrids but resulted from selfpollinations or were totally Muscadinia in character. He stated that true hybrids between V. rotundifolia and Euvitis are intermediate in character (others indicated V. rotundifolia characteristics predominated in hybrids) and almost sterile. In a companion study (Detjen 1919a) Detjen came to the following conclusions, which still stand largely unrefuted: 1. Vitis rotundifolia will hybridize with V. munsoniana and some species of Euvitis, namely: V. vinifera, V. bourquiniana, V. labrusca, V. cordifolia (Michx.), and V. aestivalis, and with the species hybrids 'Winchell' and 'Concord'. 2. It is doubtful V. rotundifolia will hybridize with all species of Vitis. 3. Our efforts so far indicate V. rotundifolia will not hybridize either way with Parthenocissus quinquefolia (Planch.), P. tricuspidata (planch.), or Ampelopsis heterophylla (Sieb. and Zucco var. Elegans (Koch). 4. Vitis rotundifolia will hybridize with its own F1 hybrids with other species of Vilis. 5. Vitis rotundifolia when used as the male parent will hybridize quite readily with some species of Euvitis, but when used as the female parent it will hybridize only rarely. Some combinations seem more congenial for hybridization than do others.

Detjen noted that even with great care in emasculation and pollination, seed lots can have a mixture of hybrid and selfed seedlings. He attributes this to bud-pollination prior to emasculation and cautions against making conclusions without considering this phenomenon. Fortunately hybrid seedlings are intermediate in character and can be distinguished from selfed seedlings based on leaves, tendrils, pith, lenticels, bark, wood, flower cluster, fruit cluster, flavor, pulp, skin, and the brush left on the pedicel (Patel and Olmo 1955), and on their stem characteristics (Williams 1923). It is also possible to identify hybrids using electrophoretic analyses of leaf tissues (Chaparro et a1. 1989). In North Carolina flowering time of Euvitis XV. rotundifolia hybrids is intermediate to the parental species. An exceptional case occurs in Florida where flowering of hybrids occurs after V. rotundifolia U. A. Mortensen, personal communication). Two conclusions can be made from the early research: (1) hybrids can be obtained with difficulty between Euvitis and Muscadinia, even if

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Euvitis is used as the female and (2) hybrids, when obtained, are highly female and male sterile. This is a discouraging condition, and probably lead to the abandonment of early efforts. However, to quote Detjen (1919a): "The hybridizer must not be easily discouraged when positive results are not immediately forthcoming. He must try and try again until success follows his efforts." Although interest and research continued in Euvitis X Muscadinia hybrids, little was published between 1925 and 1955. Snyder (1937) listed several hybrids under evaluation by the USDA at the Willard N.C. station. Two hybrids produced by Dearing and Detjen have taken on historical prominence (Mortensen 1971). One, NC 6-15, resulted from a cross between an open-pollinated 'Malaga' seedling (V. vinifera) and an hermaphroditic V. rotundifolia (Detjen 1919b). The other, NC B4-50, is a cross between a female V. rotundifolia and 'Black Morocco' (V. vinifera) (Dearing 1917a). These two vines were mostly sterile and unimpressive. However, in the 1950s Dunstan (1962a) pollinated NC 6-15 with pollen from several Euvitis clones and obtained seed and seedlings. Most of the seedlings were weak but some (those having 2n = 38) did show heterosis and had normal ovule fertility. Dunstan backcrossed one of these seedlings to Euvitis and Muscadinia and obtained fruitful seedlings segregating at the diploid level (Dunstan 1962b, 1964). NC B4-50 is in the ancestry of 'Farrer 30', which produced fertile diploid seedlings when Fry (1964) used it to pollinate Muscadinia female cultivars. Although the initial cross is difficult to obtain and F1 's are highly sterile, these difficulties can be overcome by determining the right parental combination and having enough patience and persistence. Goldy et aI. (1989) found that success could possibly be enhanced if hybrid embryos were rescued six weeks after pollination, or at veraison. Recent investigations on Euvitis and Muscadinia crosses began in the late 1940s directed by H. P. Olmo at the University of California. Olmo coauthored several papers on cytogenetics (to be discussed in the next section) of these hybrids as diploids, allotetraploids, and diploid backcrosses (Patel and Olmo 1955; Jelenkovic and Olmo 1968, 1969a, 1969b), and was involved in investigating the use of these hybrids (Davidis and almo 1964; Firoozabady and almo 1982a, 1982b, 1987; almo 1971, 1986). Investigations also continued at North Carolina under the direction of C. F. Williams and later W. B. Nesbitt. Patel and almo (1955) did similar investigations to those of Detjen (1919a) and obtained similar results: hybrid seedlings were obtained using Euvitis as a female and V. rotundifolia as the male, but reciprocal crosses failed. They also observed that the Euvitis used as the female was important to obtain larger numbers of viable seed. They found F2-7 best, 'Hunisa' worst, and 'Almeria' intermediate (all V. vinifera). Hybrids

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obtained were vigorous, almost completely male and female sterile, and morphological characteristics of V. rotundifolia were dominant. Patel and Olmo (1955) investigated failure of V. rotundifolia X V. vinifera crosses. Vitis vinifera pollen germinated better on V. rotundifolia stigmas than did V. rotundifolia pollen on V. vinifera stigmas. Vitis vinifera pollen tubes could be observed growing down the entire length of V. rotundifolia styles and penetrating the ovule within 36 h. However, it appeared fertilization did not occur since no ovary enlargement was observed, and pollinated flowers abscised at the same time as nonpollinated. They were unable to determine the exact cause, but theorized it may be due to the production of an inhibiting substance in the cytoplasm of the V. rotundifolia embryo sac. Jelenkovic and Olmo (1968) report on a fertile F1 population of plants obtained by crossing F2-35 (V. vinifera) with 'Trayshed' (V. rotundifolia). Seedlings were more fertile as females than as males with berry set ranging from 2.4-22.4% and ovule set from 0.6-5.9%, values not much below commercial V. vinifera. Seedperberryrangedfrom 1.0-1.2, values well below those of standard V. vinifera cultivars. These results contrasted greatly with previous reports and added further to the importance of trying several different parental combinations. They underscore the importance the female V. vinifera parent plays in crossing success. These F1's were reciprocally crossable to V. vinifera, but were only good as females with V. rotundifolia, and some V. rotundifolia characters were dominant. From breeding tests they were able to determine that the incompatibility of V. rotundifolia X V. vinifera was not cytoplasmic but under nuclear control. A breeding program to transfer the desirable traits from Muscadinia to Euvitis was initiated in France (Bouquet 1980b). Bouquet (1980b) reached the same conclusions as others concerning the direction of the cross, ovule set, and importance of the female genotype. He found hybrids to be quite sterile, with 70% not producing berries when pollinated, and the remaining 30% being highly sterile «0.1 0J0 ovule set) or only partially fertile (>0.1% 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

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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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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.

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

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).

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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,

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

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).

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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

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'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.

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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

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

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

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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

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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

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(1987) provided supporting evidence for an important role of glutamate dehydrogenase. However, further work is required to resolve the contribution of GDH in the overall ammonia-assimilating capacity of plants.

C. Synthesis of Amino Acids Amino acids are the first products of ammonia assimilation, are the building blocks of proteins, and playa significant role in the regulation of metabolism, transport, and storage of nitrogen. There are groups or families of amino acids derived from one precursor (Durzan and Steward 1983).

The synthesis of amino acids in grapevine leaves was studied following administration of 14C-HZC03 to grapevine leaves for one minute during anthesis; serine, aspartic acid, glutamic acid, alanine, and glycine contained 900/0 ofthe radioactivity (Bertasvili 1967). Also, following administration of 14COz to immature grapes, serine, glycine, f3-alanine, and aspargic acid were highly labeled (Paynaud and Ribereau-Gayon 1971). Furthermore, labeled arginine was metabolized by grapevine protoplasts to ornithine, aspartic acid, glutamic acid, citrulline, and probably glutamine and lysine (Theodoropoulos and Roubelakis-Angelakis 1989) (Fig. 9.5). Several enzymes involved in amino acid metabolism have been studied

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Migration , em

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.

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

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,

NITROGEN METABOLISM IN GRAPEVINE

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

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

-0o

a:::

9467 -

43 -

30-

21.1-

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.

NITROGEN METABOLISM IN GRAPEVINE

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

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

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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~

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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

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

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.

NITROGEN METABOLISM IN GRAPEVINE

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

::t

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Z ..J

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50 7

9

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13

15

17

19

AGE (WEEKS)

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

430

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

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.

NITROGEN METABOLISM IN GRAPEVINE

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 <1 <1 97 905 24 44 <1

293

327

220 1080

46

2

130

3 548

0 173

6 92

22

11

11

64 61 15 16

7 20 16 1

57

5

29 <1 <1 29 17 28 <1 <1 122 <1 270 25 86 58 51 39

11

3490 Y 490 Y 210 Y 24 48 64 Castor and Archer (1956)

266 69 258 0 1 6 LafonLafourcade and Peynaud (19S9)

510

1250 180 240

5 51 18

Kliewer (1968); and Nassar and Kliewer (1966)

Z'Aglianico', 'Cabernet Sauvignon', 'French Colombard', 'Sauvignon Blanc', and 'Tannat'. Y'French Colombard' only. x'The 18 cultivars included 'Alicante Bouschet', 'Black Corinth', 'Cardinal', 'CabernetSauvignon', 'Carignane', 'Chardonnay', 'Chenin Blanc', 'Flora', 'Gewurztraminer', 'Malbec', 'Muscat Hamburg', 'Palomino'. 'Perlette', 'Pinot Noir', 'Thompson Seedless', and 'Tokay'.

9.

NITROGEN METABOLISM IN GRAPEVINE

433

Seedless' fruits was positively correlated with total leaf area per vine. Furthermore, the nitrogen content of fruits from scions grown on 'Rupestris du Lot' rootstock was considerably greater than when 'Richter 99' was used as the rootstock. Fruits from vines with light crop loads generally have higher levels of nitrogen, arginine, proline, and total free amino acids than vines with heavy crops (Kliewer and Ough 1970). Ammonia and the amino nitrogen of must, especially arginine, phenylalanine, histidine, valine, and glutamic acid, are largely assimilated by yeast during fermentation. Lysine, proline, and glycine are much less easily assimilated. Although present in very small amounts as compared to human daily requirements, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine are required by man. Koch and Sajak (1959) found several soluble proteins in the juice of grapes, which differed somewhat between cultivars. The amounts increased during ripening and reached maximum quantities before full maturity; then they decreased up to harvest. Whether grapes for wine are harvested at the peak of protein content or later may affect the clouding of wine. Greater amounts of soluble proteins were formed during warm seasons than during cool seasons. The water soluble high molecular weight nitrogenous substances are believed to be low molecular weight polypeptides and proteins. The complete list of amino acids involved and their peptide linkage is unknown. The juice contains only about one-fifth of the total nitrogen, the remaining four-fifths being in the skin and seeds (Peynaud and Ribereau-Gayon 1971). These authors also reported that there is some release of nitrogenous compounds from the seeds into the pulp of the mature grape and that about half the nitrogen content of the juice of ripe grapes is due to free amino acids.

v. STORAGE AND REALLOCATION OF NITROGEN A. Nitrogen Reserve Partitioning Although several attempts had been made to determine the partitioning and use of nitrogen reserves in grapevine, our knowledge was far from complete due to the difficulty in distinguishing between stored and newly absorbed nitrogen, and to the problems associated with the excavation of whole vines. Recently appreciable information on the fate of 15N was obtained in South African studies (Conradie 1980,1983.1986. 1990. 1991a, b) using 'Chenin Blanc' potted vines and in a California study using field-grown 'Thompson Seedless' grapevines (Williams 1991). The N storage forms consist of an insoluble (which is usually larger)

434

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

and a soluble. Arginine is the principal storage form of soluble N accounting for more than 60% of the total soluble N in roots and woody tissues (Kliewer and Cook 1974; Schaller et al. 1989). Aspartic is another significant soluble form of N (Balasubrahmanyam 1978). Shoots and leaves have a high N turnover and seem to act as intermediate N reservoirs between root and berry (Conradie 1986). Nitrogen allocation in pot-grown grapevines in South Africa (Conradie 1986, 1991a,b) corresponded to that found infield trials for different cultivars in Australia (Alexander 1957), France (Lafon et al. 1965), Germany (Lohnertz 1988), and the United States (Williams 1987a,b). These results suggest that the partitioning of N between vegetative growth and ripened clusters is genetically determined (Conradie 1991a). Also comparable is the absolute amount of N required for the production of a crop of a specific size. Each ton of grapes removes 1.39 kg-1.93 kg of N (Conradie 1980; Koblet and Perret 1990). In general, the N distribution of a balanced vine at harvest should be of the following order: 26% in the permanent structure (roots, trunk, canes), 41% in the vegetative growth, and 33% in the clusters (Conradie 1991a).

B. Nitrogen Nutrition Cycle The annual N-nutrition cycle of the grapevine follows a set pattern and can be divided into four distinct growth phases: budbreak to the end of bloom (I), end of bloom to veraison (II), veraison to harvest (III), and postharvest (IV) (Conradie 1991a,b). The translocation and storage of N absorbed during the first half of phase II (regarded as spring absorbed), during the second half of phase II (regarded as summer absorbed) and during phase IV (autumn) were investigated in three different trials through 15N-Iabeling of two-year-old Chenin blanc in sand culture. After labeling, whole vines were sampled regularly during the rest of the second and the first part of the third growing season. Up to veraison (end of phase II) the amount of spring-absorbed N in the leaves remained practically constant at ca 36%, while ca 20% of the labeled N from the shoots and the permanent structure was translocated to the clusters. At veraison the distribution of spring-absorbed N was similar to that of summer-absorbed N (Conradie 1991a,b).. Between veraison and harvest (phase III), 34%, 35%, and 20% of the amount of the labeled N accumulated during phase II in the permanent structure, shoots, and leaves, respectively, were translocated presumably to the clusters. It is suggested that these figures can be regarded as realistic estimates of N turnover during this phase. At harvest, spring- and summer-absorbed N was again distributed equally, (Le., 14% in the permanent structure, 41% in the vegetative growth, and 45% in the clusters). Nitrogen absorbed during

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phase IV was largely accumulated in the roots and older wood, and at the start of the third season the permanent structure contained 24%, 21%, and 65% of the labeled N applied during the previous spring, summer, and autumn, respectively. These reserves, accumulated during the second season, were utilized equally in the third season with about one quarter, amounting to 18% of the demand of the new growth, being translocated up to the end of phase 1. Newly absorbed N and reserve N were allocated to all the new organs in equal ratios (Conradie 1991a,b). The factors. which affect the storage and utilization of reserve N by grapevines. are yield, nutrient status, soil temperature, soil type, and pruning type (Conradie 1991a,b). In the Californian study, about 33% of the N found in the current season's shoot and cluster biomass was from N reserves in the trunk and root systems. This was equivalent to an average of 22.5 g N vine-1 remobilized from these two organs. The greater amount of N came from the root system. The proportion of fertilizer N found in any organ of the vine was never greater than 12% of the total N in that organ. Approximately 60% of the nitrogen required for new vegetative and reproductive growth of 'Thompson Seedless' vines was from the soil nitrogen (Williams 1991). C. Nitrogen Allocation Modeling Wermelinger and Baumgartner (1990) and Wermelinger et al. (1991) have proposed a model for N distribution in grapevine. This model, VIMO-model (vine model), is a dynamic, stochastic population model that includes the life spans of the plant parts and the variables, daily solar radiation, and minimum and maximum temperatures. The daily demand for N is proportional to the demand for carbohydrates. and the core ofthe model is the metabolic pool concept. The allocation of the daily available C and N from the pool to sinks is (in decreasing order) fruit, vegetative organs. and reserves (Wermelinger 1991). Also, Gutierrez et al. (1985), Williams (1987a,b), Williams and Smith (1985) and Conradie (1986) have suggested that the dynamics of uptake of nitrogen and its partitioning in the vine are correlated to the total amount of dry mass produced by the vine and. therefore, the amount of nitrogen required for vines in a particular vineyard may be predicted. VI. TRANSLOCATION OF NITROGENOUS COMPOUNDS A. Xylem Transport of Nitrogen Transport in the xylem is a one-way passive transport driven by the water transpiration stream with active or diffusive loading and unloading

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of the solutes (Bidwell 1979). It appears to be subjected to a circadian rhythm. Although nitrogen solutes frequently are the major components of dry matter in xylem extracts, second only to carbohydrate in the phloem (Pate 1973, 1980, 1983), relatively little of the total nitrogen is transported as nitrate or ammonia. Most nitrate is reduced by nitrate and nitrite reductases to nitrite and then to ammonia, before being incorporated into organic forms. In some plants, e.g., cocklebur (Xanthium strumarium) and cotton (Gossypium), nitrate accounts for up to 95% of the xylem nitrogen transported from the root (Pate 1980). Plants of this type have little nitrate reductase activity in the root but high activity in leaves (Kirkby and Mengel 1962). Xylem nitrogen in plant species with active root nitrate reductase is almost all in an organic form. Generally, root nitrate reductase is high in woody species (Kirkby and Mengel 1962). A range of nitrogen compounds is found in xylem extracts including amides (glutamine, asparagine, and substituted amidesJ, amino acids, and alkaloids (Pate 1980). Usually only one or two compounds predominate and they have a low carbon-nitrogen ratio. Grapevine xylem composition is not clearly defined. A large range in nitrogen solute concentrations are reported in root pressure exudates (Andersen and Brodbeck 1989a,b: Roubelakis-Angelakis and Kliewer 1979; Marangoni et a1. 1986; Trione and Almela Pons 1972; Trione et al. 1972; Wormall 1924). Sampling time, species, cultivar, plant nutrition, environment, and sampling site on the plant (Pate 1980; Ferguson 1980), and possibly artifacts due to sample preparation, can explain the large concentration range found. Table 9.2 lists estimates of the broad xylem Table 9.2. exudates.

Comparison of nitrogen fraction concentration reported for grape xylem

Concentration (mM - nitrogen basisf Total soluble nitrogen

Ammonia nitrogen

Nitrate nitrogen

Amides and free amino acidsY

4.6- 9.7

1.1-0.6

0.1-0.9

3.4- 8.3

6.0-12.7

0.4-2.1

0.1-0.2

5.6-10.6

1.6- 5.9

0.1-0.3

0.05-0.6

1.4- 5.0

Source Andersen and Brodbeck (1989a) Andersen and Brodbeck (1989b) Roubelakis-Angelakis and Kliewer (1979)

Low to high total nitrogen samples from each source. fraction estimated as remainder from total nitrogen less ammonia and nitrate nitrogen fractions. Z

YAmino/amide

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nitrogen constituents for V. vinifera and V. rotundifolia. RoubelakisAngelakis and Kliewer (1979) generally found low concentrations in Vitis vinifera. They noted a big difference between years but his was based on only one sampling time per year. Potentially, sample time differences relative to vine physiological development could explain the year to year difference. Andersen and Brodbeck (1989a,b) samples of V. rotundifolia xylem sap had intermediate concentrations of amino acids and other nitrogenous constituents. Glutamine accounted for slightly over half the total soluble nitrogen in the bleeding sap of 'Thompson Seedless' grapevines. "Normal" nitrogen solute concentration in xylem extracts for V. vinifera are not established. Nitrate nitrogen accounts for about 2-15% of the total soluble nitrogen in grape xylem extracts (Table 9.2), which indicates that grapes have significant root nitrate reductase activity. Nitrate reductase activity is present in grape leaves (Perez and Kliewer 1982; Smart 1991), but it has not been compared to root nitrate activity. Pate (1973) compared the relative proportion of nitrate nitrogen to total nitrogen in many plant xylem extracts. The relative proportion of about 2-25% total nitrogen as nitrate in grapes is similar to plants where most nitrate reduction occurs in the roots. Plants with most nitrate reduction in leaves or shoots have greater than 50% nitrate nitrogen in xylem extracts. Removal of amino acids and amides by the rising sap from the roots into aerial parts has been studied in five rootstocks (Bilyk and Stoer 1975). Rootstocks differed in the removal rate of individual amino acids, especially glutamine. In 'Rupestris du Lot' injecting water in the soil to a depth of 50 cm at 50 liter/plant stimulated sap flow and glutamic acid, alanine, and lysine synthesis.

B. Phloem Transport of Nitrogen The transport of N in the phloem seems to follow the bulk flow theory for carbohydrate movement (Bidwell 1979). Phloem exudates usually contain 10-20 times the xylem concentration of nitrogen solutes (Pate 1980). Although nitrate concentration maybe high in the xylem, it is often absent or much lower in phloem. In his review paper, Pate (1983) stressed the importance of phloem translocation for supplying nitrogen to fruit. In lupin (Lupin us albus), he estimated that 89% of the fruit nitrogen is supplied by the phloem. The importance of phloem transport for ammonia is not clear. Reports are inconsistent about the relative ammonium concentration in xylem and phloem extracts. It has proved difficult or impossible to obtain pure phloem extracts in many plants. The nitrogen solute composition of grapevine phloem has not been reported.

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VII. DIAGNOSIS OF NITROGENOUS STATUS

Soil analysis has not been used in the United States to diagnose the nitrogen needs of vineyards for over 50 years; but this method has been widely used in Europe, especially in Germany (Muller, 1990). The nitrogen content of soils is influenced by soil type, rooting depth, nutrient interactions, and nutrient availability and has not proven to be a reliable means of estimating the nitrogen requirements of grapevines in California (Cook and Kishaba 1956). Three foliar methods have been mainly used to estimate the nitrogen status of grapevines; namely the concentration of total nitrogen in leaf blades and/or petioles, nitrate nitrogen in petioles, and arginine in mature fruits and/or dormant cane tissue. The level of total nitrogen in both petioles and leaf blades of grapevines has been extensively used to estimate the nitrogen status of grapevines, especially in Europe and South Mrica (Kliewer 1991). The concentration of total nitrogen, as well as other nitrogenous compounds in grapevines, depends on the type of tissue sample, leaf age or position of leaf on shoot, the physiological stage of vine growth, the presence or absence of fruit clusters, the species and cultivar of Vitis, and geographical location. Therefore, each of these factors must be standardized to obtain meaningful results. The concentration of total nitrogen in blades ranges from a high of 4-6% soon after budbreak to a low of 1-1.5% at leaf fall. Generally, levels of total N in leaf blades of < 1.5-1.8% between flowering and veraison indicate deficiency. The main problem of using total N to estimate the nitrogen status of grapevines is the relatively small range between deficiency and sufficiency. Unless sampling is very carefully done and well replicated, deficiency situations may be easily missed. Ulrich (1942) was the first to use the level of nitrate in leaf petioles to indicate the nitrogen status of grapevines. He found that the range of nitrate in leaf petioles of nitrogen-fertilized and unfertilized vines varied as much as ten-fold in contrast to total N, which varied less than 50%. Cook and Kishaba (1956), Christensen (1969) and Christensen et al. (1978) have extensively investigated the use of nitrate levels in petioles of 'Thompson Seedless' grapevines for predicting nitrogen needs and developed critical deficiency ranges for this variety grown in the San Joaquin Valley of California. They showed that bloomtime petiole nitrate-nitrogen levels of <350 ppm indicate deficiency, 500-2000 ppm indicate sufficiency, and >3000 ppm indicate excessive. Nitrate levels in petioles of fully expanded leaves fluctuate greatly during the growing season, but generally are highest seven to ten days before bloom; these levels decline rapidly through the bloom period and reach a relatively stable level from about four weeks after bloom through the fruit ripening

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period. Cultivar, tissue age, rootstock, irrigation, light, temperature, covercrop, and fertilization with potassium are known to influence the nitrate level in grape leaves. How each of these factors influences the nitrate level in grapevines has been recently reviewed (Kliewer 1991). The petiole nitrate test has been very successful for indicating the nitrogen status of 'Thompson Seedless' vines under the relatively uniform soil and climate conditions of the San Joaquin Valley. However, in the coastal valleys of California and in many other countries, where climate is more variable, the petiole nitrate level was not correlated to crop yield of grapevines to a high degree. Considerable evidence has accumulated over the past 30 years, indicating that many perennial woody plants, including grapevines, store much of their reserve nitrogen as low-molecular-weight soluble nitrogen compounds (especially arginine) in the bark and wood of roots, trunk, and stems (Kliewer, 1991). In grapevines, fruits of many cultivars also accumulate relatively large amounts of arginine during the period of fruit ripening (Kliewer 1969, 1970). Kliewer and Cook (1974) and Juhasz et al. (1984) found a close positive correlation between the level of nitrogen fertilization and the arginine concentration of grape berry juice at harvest, which consistently gave a higher degree of correlation than petiole nitrate, total nitrogen of leaves, and arginine in dormant canes with rates of nitrogen fertilization. Assay of arginine level in juice of grapes at harvest offers a convenient and relatively inexpensive way of estimating the nitrogen needs of vineyards. Since samples of grapes are normally taken for determination of total soluble solids when fruits come into a winery or for indication of table grape ripeness, these same juice samples can be used for determination of arginine and thus eliminate the tedious task of sampling vineyards and drying and grinding tissue for extraction. Berry juice samples only need to be diluted with distilled water and chemically analyzed for arginine. However, the concentration of arginine in fruits of different grape cultivars may differ greatly; therefore critical deficiency and sufficiency levels must be determined for each cultivar. In high arginine-type cultivars, arginine levels <400 mg/liter in mature fruit generally indicates deficiency, whereas in lowarginine type cultivars, the critical level is about 200 mg/liter or less. Recently, it was suggested that the amount of nitrogen required by grapevines may be determined if the total amount of dry mass produced per vine is known (Alexander 1957; Conradie 1980; Gutierrez et al. 1985; Wermelinger, 1991; Williams 1987a,b; Williams and Smith 1985; Williams et al. 1985). Dry matter production of different parts of the vine may be predicted by several methods (Gutierrez et al. 1985; Williams and Smith 1985; Williams 1987a). Therefore if the dynamics of nitrogen uptake and partitioning within the vine are known, the amount of

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nitrogen required for vines in a particular vineyard may be predicted. Williams (1987b) calculated the amounts of nitrogen in various parts of 'Thompson Seedless' grapevines grown in the Fresno area. He found that a vineyard with 1121 vines/ha and producing 25 kg fruit/vine used about 82 kg N of which 30 kg N was in the fruit. He concluded that, since only the fruit was removed from the vineyard, an annual application of 30 kg N/ha should be sufficient to replace the N removed from the vineyard in the form of fruit. This of course presumes that all the N in leaves and prunings is returned to the soil and eventually utilized by the vine.

VIII. FUTURE RESEARCH DIRECTIONS Each process in the uptake, distribution, reduction, and assimilation of nitrogen into organic forms by plants seems to be at least partially under genetic control. Therefore, understanding the physiology and biochemistry of nitrogen metabolism in each plant species and identification of the involved process(es) will provide the necessary information to increase the nitrogen assimilating capacity and to minimize excessive fertilization which can have a hazardous impact on ground water and the environment in general. Although an appreciable amount of information on aspects of nitrogen metabolism in Vilis spp. has been accumulated over the past three decades, there are still important aspects to be studied; among them are the elucidation of uptake mechanisms of nitrate, ammonium ions, and other nitrogenous substances at the cellular, tissue, and whole plant level and their metabolic fate and use for growth and development. Of special importance is the achievement of purification, characterization, and definition of subcellular localization, tissue distribution, and regulatory mechanism(s) of the enzymes, which mediate the reactions of ammonia assimilation in Vitis spp. Determination of the relative importance of the GDH and GS/GOGAT pathways could greatly contribute to further understanding nitrogen metabolism in Vilis spp. Also, cDNA cloning of the pertinent enzymes, characterization of their genes, and the use of new biotechnologies may be considered as one of the main routes for improving efficacy of nitrogen use in Vitis. Other aspects of grapevine physiology that require elucidation include the role of polyamines and a better understanding of protein synthesis, storage, and recycling.

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Subject Index

A

Anatomy and morphology, heliconia,

Grape muscadine breeding, 357-405 nitrogen metabolism, 407-452

5-13

Asexual embryogenesis, 258-259, 337-339

H

Heliconia, 1-55 B

Bulb, root physiology, 57-88

c

In vitro thin cell layer morphogenesis, 239264

Cell culture, woody legumes, 265-332

D Disease, turnip mosaic virus, 199-238 F Floricultural crops, heliconia, 1-55 Flower and flowering, thin cell layer morphogenesis, 239-256 Fruit crops grape nitrogen metabolism, 407-452 muscadine grape breeding, 357-405

G Genetics and breeding muscadine grapes, 357-405 potato tuberization, 121-124 woody legume tissue and cell culture, 311-314 Geophyte. See Bulb; Tuber

woody legume culture, 265-332 L

Leguminosae, in vitro, 265-332 N

Nitrogen fixation in woody legumes, 322-323 metabolism in grapevine, 407-452

o Ornamental plants flowering bulb roots, 57-88 heliconia, 1-55 p

Physiology heliconia, 5-13 453

454

SUBJECT INDEX

nitrogen metabolism in grapevine,

s

407-452

polyamines, 333-356 potato tuberization, 89-188 roots of flowering bulbs, 57-88 thin cell layer morphogenesis, 239264

Pollination, heliconia, 13-15 Polyamines, 333-356 Potato, tuberization, 89-198 Propagation, woody legumes in vitro, 265-332

R

Root, physiology of bulbs, 57-88 Root and tuber crops, potato tuberization, 89-188

Secondary metabolites, woody legumes, 314-322 T

Tuber, potato, 89-188 Tuber and root crops. See Root and tuber crops Tulip. See Bulb Turnip Mosaic Virus, 199-238

v Vegetable crop, potato tuberization, 89-188

Virus, turnip mosaic, 199-238

Cumulative Subject Index (Volumes 1-14)

A

Abscisic acid cold hardiness, 11:65 dormancy, 7:275-277 rose senescence, 9:66 stress, 4:249-250 Abscission anatomy and histochemistry, 1:172203

flower and petals, ~:104-107 regulation, 7:415-416 rose, 9:63-64 Acclimatization foliage plants, 6:119-154 herbaceous plants, 6:379-395 micropropagation, 9:278-281, 316317

Actinidia, 6:4-12 Adzuki bean, genetics, 2:373 Agaricus, 6:85-118 Agrobacterium tumefaciens, 3:34 Air pollution, 8:1-42 Almond, in vitro culture, 9:313 Alocasia, 8:46, 57, see also Aroids Alternate bearing chemical thinning, 1:285-289 fruit crops, 4:128-173 pistachio, 3:387-388 Aluminum deficiency and toxicity symptoms in fruits and nuts, 2:154 Ericaceae, 10:195-196 Amorphophallus, 8:46, 57, see also Aroids Anatomy and morphology apple flower and fruit, 10:273-308 apple tree, 12:265-305 asparagus, 12:71 cassava, 13:106-112

embryogenesis, 1:4-21, 35-40 fig, 12:420-424 fruit abscission, 1:172-203 fruit storage, 1 :314 ginseng, 9:198-201 grape flower, 13:315-337 grape seedlessness, 11:160-164 heliconia, 14:5-13 kiwifruit, 6: 13-50 orchid, 5:281-283 navel orange, 8:132-133 pecan flower, 8:217-255 petal senescence, 1:212-216 pollution injury, 8:15 Androgenesis, woody species, 10:171173

Angiosperms, embryogenesis, 1:1-78 Anthurium. See also Aroids, ornamental fertilization, 5:334-335 Antitranspirants, 7:334 cold hardiness. 11:65 Apical meristem, cryopreservation. 6:357-372

Apple alternate bearing, 4:136-137 anatomy and morphology of flower and fruit. 10:273-309 bitter pit. 11:289-355 bioregula tion. 10:309-401 CA storage. 1:303-306 chemical thinning, 1:270-300 fertilization. 1:105 fire blight control, 1:423-474 flower induction, 4:174-203 fruiting, 11:229-287 in vitro, 5:241-243; 9:319-321 light, 2:240-248 maturity indices, 13:407-432 nitrogen metabolism, 4:204-246 455

CUMULATIVE SUBJECT INDEX

456

replant disease, 2:3 root distribution, 2 :453-456 stock-scion relationships, 3:315-375 summer pruning, 9:351-375 tree morphology and anatomy,

CA storage, 1:352-353 fluid drilling of seed, 3:21 resistance to bacterial pathogens, 3:28-58

Bedding plants, fertilization, 1 :99-100;

12:265-305

vegetative growth, 11:229-287 watercore, 6:189-251 yield, 1:397-424 Apricot, CA storage, 1:309 Aroids edible, 8:43-99; 12:166-170 ornamental, 10:1-33 Arsenic, deficiency and toxicity symptoms in fruits and nuts, 2:154 Artichoke, CA storage, 1:349-350 Asexual embryogenesis, 1:1-78; 2:268-310; 3:214-314; 7:163168,171-173,176-177,184,185187, 187-188, 189; 10:153-181; 14:258-259, 337-339

Asparagus CA storage, 1:350-351 fluid drilling of seed, 3:21 postharvest biology, 12:69-155 Auxin dormancy, 7:273-274 petal senescence, 11:31 Avocado flowering, 8:257-289 fruit development, 10:230-238 fruit ripening, 10:238-259 Azalea, fertilization, 5:335-337 B

Babaco, in vitro culture, 7:178 Bacteria diseases of fig, 12:447-451 ice nucleating, 7:210-212; 11:69-71 pathogens of bean, 3:28-58 tree short life, 2:46-47 wilt of bean, 3:46-47 Bacteriocides, fire blight, 1:450-459 Bacteriophage, fire blight control, 1:449-450

Banana CA storage, 1:311-312 fertilization, 1:105 in vitro culture, 7:178-180 Bean

5:337-341

Beet CA storage, 1:353 fluid drilling of seed, 3:18-19 Begonia (Rieger), fertilization, 1:104 Biochemistry, petal senescence, 11:15-43

Biennial bearing. See Alternate bearing Bioregulation. See also Growth substances apple and pear, 10:309-401 Bird damage, 6:277-278 Bitter pit in apple, 11:289-355 Blueberry developmental physiology, 13:339405

nutrition, 10:183-227 Branching, lateral apple, 10:328-330 pear, 10:328-330 Boron deficiency and toxicity symptoms in fruits and nuts, 2:151-152 foliar application, 6:328 nutrition, 5:327-328 pine bark media, 9:119-122 Brassicaceae, in vitro, 5:232-235 Breeding. See Genetics and breeding Broccoli, CA storage, 1:354-355 Brussels sprouts, CA storage, 1:355 Bulb. See also Tulip root physiology, 14:57-88

c CA storage. See ControIledatmosphere storage Cabbage CA storage, 1:355-359 fertilization, 1:117-118 Caladium. See Aroids, ornamental Calciole, nutrition, 10:183-227 Calcifuge, nutrition, 10:183-227 Calcium bitter pit, 11:289-355

CUMULATIVE SUBJECT INDEX

cell wall, 5:203-205 container growing, 9:84-85 deficiency and toxicity symptoms in fruits and nuts, 2:148-149 Ericaceae nutrition, 10:196-197 foliar application, 6:328-329 fruit softening, 10:107-152 nutrition, 5:322-323 pine bark media, 9:116-117 tipburn, disorder, 4:50-57 Calmodulin, 10:132-134, 137-138 Carbohydrate fig, 12:436-437 kiwifruit partitioning, 12:318-324 metabolism, 7:69-108 partitioning, 7:69-108 petal senescence, 11:19-20 reserves in deciduous fruit trees, 10:403-430

Carbon dioxide, enrichment, 7:345398, 544-545

Carnation, fertilization, 1:100; 5:341345

Carrot CA storage, 1:362-366 fluid drilling of seed, 3:13-14 Caryophyllaceae, in vitro, 5:237-239 Cassava, 12:158-166; 13:105-129 Cauliflower, CA storage, 1:359-362 Celeriac, CA storage, 1:366-367 Celery CA storage, 1:366-367 fluid drilling of seed, 3:14 Cell culture, 3:214-314 woody legumes, 14:265-332 Cell membrane calcium, 10:126-140 petal senescence, 11:20-26 Cell wall calcium, 10:109-122 hydrolases, 5:169-219 ice spread, 13:245-246 tomato, 13:70-71 Chelates, 9:169-171 Cherry, CA storage, 1:308 Chestnut blight, 8:281-336 in vitro culture, 9:311-312 Chicory, CA storage, 1:379 Chilling injury, 4:260-261 pistachio, 3:388-389

457

Chlorine deficiency and toxicity symptoms in fruits and nuts, 2:153 nutrition, 5:239 Chlorosis, iron deficiency induced, 9:133-186

Chrysanthemum fertilization, 1:100101; 5:345-352

Citrus alternate bearing, 4:141-144 asexual embryogenesis, 7:163-168 CA storage, 1:312-313 chlorosis, 9:166-168 cold hardiness, 7:201-238 fertilization, 1:105 flowering, 12:349-408 honey bee pollination, 9:247-248 in vitro culture, 7:161-170 navel orange, 8:129-179 nitrogen metabolism, 8:181 rootstock, 1:237-269 Cloche (tunnel), 7:356-357 Coconut palm asexual embryogenesis, 7:184 in vitro culture, 7:183-185 Cold hardiness, 2:33-34 apple and pear bioregulation, 10:374-375

citrus, 7:201-238 factors affecting, 11:55-56 herbaceous plants, 6:373-417 injury, 2:26-27 nutrition, 3:144-171 pruning, 8:356-357 Colocasia, 8:45, 55-56, see also Aroids Common blight of bean, 3:45-46 Compositae, in vitro, 5:235-237 Container production, nursery crops, 9:75-101

Controlled environment agriculture, 7:534-545, see also Greenhouse and greenhouse crops; Hydroponic culture; Protected crops Controlled-atmosphere (CA) storage asparagus, 12:76-77, 127-130 flowers, 3:98, 10:52-55 fruit quality, 8:101-127 fruits, 1:301-336; 4:259-260 pathogens, 3:412-461 seeds, 2:134-135 tulip, 5:105 vegetable quality, 8:101-127

458

CUMULATIVE SUBJECT INDEX

vegetables, 1:337-394; 4:259-260 Copper deficiency and toxicity symptoms in fruits and nuts, 2:153 foliar application, 6:329-330 nutrition, 5:326-327 pine bark media, 9:122-123 Corynebacterium flaccumfaciens, 3:33,46

Cowpea genetics, 2:317-348 U.S. production 12:197-222 Cranberry, fertilization, 1:106 Cryphonectria parasitica. See Endothia parasitica Cryopreservation apical meristems, 6:357-372 cold hardiness, 11:65-66 Crytosperma, 8:47, 58, see also Aroids Cucumber, CA storage, 1:367-368 Cytokinin cold hardiness, 11:65 dormancy, 7:272-273 floral promoter, 4:112-113 grape root, 5:150, 153-156 lettuce tipburn, 4:57-58 petal senescence, 11:30-31 rose senescence, 9:66

cowpea, 12:210-213 fig, 12:447-479 flooding, 13:288-299 hydroponic crops, 7:530-534 lettuce, 2:187-197 mycorrhizal fungi, 3:182-185 ornamental aroids, 10:18 root, 5 :29-31 stress, 4:261-262 sweet potato, 12:173-175 tulip, 5:63, 92 turnip mosaic virus, 14:199-238 yam (Dioscorea), 12:181-183 Disorder, see also Postharvest physiology bitterpit, 11:289-355 fig, 12:477-479 pear fruit, 11:357-411 watercore, 6:189-251; 11:385-387 Dormancy, 2:27-30 blueberry, 13:362-370 release in fruit trees, 7:239-300 tulip, 5:93 Drip irrigation, 4:1-48 Drought resistance, 4:250-251 cassava, 13:114-115 Dwarfing apple, 3:315-375 apple mutants, 12:297-298 by virus, 3:404-405

D

Date palm asexual embryogenesis, 7:185-187 in vitro culture, 7:185-187 Daylength. See Photoperiod Deficiency symptoms, in fruit and nut crops, 2:145-154 Defoliation, apple and pear bioregulation, 10:326-328 'Delicious' apple, 1:397-424 Dieffenbachia. See Aroids, ornamental Dioscorea. See Yam Disease and air pollution, 8:25 aroids, 8:67-69; 10:18; 12:168-169 bacterial, of bean, 3:28-58 cassava, 12:163-164 control by virus, 3:399-403 controlled-atmosphere storage, 3:412-461

E

Easter lily, fertilization, 5:352-355 Embryogenesis. See Asexual embryogenesis Endothia parasitica, 8:291-336 Energy efficiency, in greenhouses, 1:141-171; 9:1-52

Environment air pollution, 8:20-22 controlled for agriculture, 7: 534-545 controlled for energy efficiency, 1:141-171; 9:1-52

embryogenesis, 1:22, 43-44 fruit set, 1:411-412 ginseng, 9:211-226 greenhouse management, 9:32-38 navel orange, 8:138-140 nutrient film technique, 5:13-26 Epipremnum. See Aroids, ornamental

459

CUMULATIVE SUBJECT INDEX

Erwinia a mylovora, 1:423-474 lathyri, 3:34 Essential elements foliar nutrition, 6:287-355 pine bark media, 9:103-131 plant nutrition 5:318-330 soil testing, 7:1-68 Ethylene apple bioregulation, 10:366-369 avocado, 10:239-241 CA storage, 1:317-319, 348 cut flower storage, 10:44-46 dormancy, 7:277-279 flower longevity, 3:66-75 kiwifruit respiration, 6:47-48 petal senescence, 11:16-19, 27-30 rose senescence, 9:65-66 F

Flooding fruit crops, 13:257-313 Floricultural crops, see also individual crops fertilization, 1:98-104 growth regulation, 7:399-481 heliconia, 14:1-55 postharvest physiology and senescence, 1:204-236; 3:59143; 10:35-62; 11:15-43

Florigen, 4:94-98 Flower and flowering alternate bearing, 4:149 apple anatomy and morphology. 10:277-283

apple bioregulation, 10:344-348 aroids. ornamental. 10:19-24 avocado, 8:257-289 blueberry development. 13:354-378 citrus, 12: 349-408 control, 4:159-160 fig, 12:424-429

Fertilization and fertilizer anthurium, 5:334-335 azalea, 5:335-337 bedding plants, 5:337-341 blueberry, 10: 183-227 carnation, 5:341-345 chrysanthemum, 5:345-352 controlled release, 1:79-139; 5:347348

Easter lily, 5:352-355 Ericaceae, 10:183-227 foliage plants, 5:367-380 foliar, 6:287-355 geranium, 5:355-357 greenhouse crops, 5:317-403 lettuce, 2:175 nitrogen, 2 :401-404 orchid, 5:357-358 poinsettia, 5:358-360 rose, 5:361-363 snapdragon, 5:363-364 soil testing, 7:1-68 trickle irrigation, 4:28-31 tulip, 5:364-366 Vaccinium, 10:183-227 Fig industry, 12:409-490 ripening, 4:258-259 Filbert, in vitro culture, 9:313-314 Fire blight, 1:423-474

grape anatomy and morphology, 13:354-378

honey bee pollination. 9:239-243 induction, 4:174-203; 254-256 initiation, 4:152-153 in vitro, 4:106-127 kiwifruit, 6:21-35; 12:316-318 orchid, 5:297-300 pear bioregulation, 10:344-348 pecan, 8:217-255 perennial fruit crops, 12:233-264 phase change, 7:109-155 photoperiod, 4:66-105 pistachio, 3:378-387 postharvest physiology, 1:204-236; 3:59-143; 10:35-62; 11:15-43

pruning, 8:359-362 raspberry. 11:187-188 regulation in floriculture, 7:416-424 rhododendron, 12:1-42 rose, 9:60-66 senescence, 1:204-236; 3:59-143; 10:35-62; 11:15-43

sugars, 4:114 thin cell layer morphogenesis, 14:239-256

tulip, 5:57-59 Fluid drilling, 3:1-58 Foliage plants acclimatization, 6:119-154

460

CUMULATIVE SUBJECT INDEX

fertilization, 1:102-103; 5:367-380 Foliar nutrition, 6:287-355 Freeze protection. See Frost, protection Frost apple fruit set, 1 :407-408 citrus, 7:201-238 protection, 11:45-109 Fruit abscission, 1:172-203 apple anatomy and morphology, 10:283-297

apple apple apple apple

bioregulation, 10:348-374 bitter pit, 11:289-355 maturity indices, 13:407-432 ripening and quality, 10:361-

374

avocado development and ripening, 10:229-271

blueberry development, 13:378-390 CA storage and quality, 8:101-127 diseases in CA storage, 3:412-461 drop, apple and pear, 10:359-361 fig, 12:424-429 kiwifruit, 6:35-48; 12:316-318 maturity indices, 13:407-432 navel orange, 8:129-179 nectarine, postharvest, 11:413-452 peach, postharvest, 11:413-452 pear, bioregulation, 10:348-374 pear, fruit disorders, 11:357-411 pear maturity indices, 13:407-432 pear ripening and quality, 10:361374

pistachio, 3:382-391 quality and pruning, 8:365-367 ripening, 5:190-205 set, 1:397-424; 4:153-154 set in navel oranges, 8:140-142 size and thinning, 1:293-294; 4:161 softening, 5:109-219; 10:107-152 thinning, apple and pear, 10:353-359 tomato parthenocarpy, 6:65-84 tomato ripening, 13:67-103 Fruit crops alternate bearing, 4: 128-173 apple bitter pit, 11:289-355 apple growth, 11:229-287 apple maturity indices, 13:407-432 avocado flowering, 8:257-289 blueberry developmental physiology, 13:339-405

blueberry nutrition, 10:183-227 carbohydrate reserves, 10:403-430 CA storage, 1:301-336 CA storage diseases, 3:412-461 chlorosis, 9:161-165 citrus cold hardiness, 7:201-238 citrus flowering, 12:349-408 dormancy release, 7:239-300 Ericaceae nutrition, 10:183-227 fertilization, 1:104-106 fig, industry, 12:409-490 fireblight, 11:423-474 flowering, 12:223-264 foliar nutrition, 6:287-355 frost control, 11:45-109 grape flower anatomy and morphology, 13:315-337 grape nitrogen metabolism, 14:407452

grape root, 5:127-168 grape seedlessness, 11:164-176 honey bee pollination, 9:244-250, 254-256

in vitro culture, 7:157-200; 9:273349

kiwifruit, 6:1-64; 12:307-347 muscadine grape breeding, 14:357405

navel orange, 8:129-179 nectarine postharvest, 11:413-452 nutritional ranges, 2:143-164 orange, navel, 8:129-179 orchard floor management, 9:377430

peach postharvest, 11:413-452 pear fruit disorders, 11:357-411 pear maturity indices, 13:407-432 pecan flowering, 8:217-255 photosynthesis, 11:111-157 pruning, 8:339-380 raspberry, 11:185-228 roots, 2:453-457 short life and replant problem, 2:1116

summer pruning, 9:351-375 Vaccinium nutrition, 10:183-227 water status, 7:301-344 Fungi fig, 12:451-474 mushroom, 6:85-118 mycorrhizal, 3:172-213; 10:211-212 pathogens in postharvest storage,

461

CUMULATIVE SUBJECT INDEX

3:412-461

Fungicide, and apple fruit set, 1:416 G

Garlic, CA storage, 1:375 Genetic variation alternate bearing, 4:146-150 photoperiodic responses, 4:82 pollution injury, 8:16-19 Genetics and breeding aroids (edible), 8:72-75; 12:169 aroids (ornamental), 10:18-25 bean, bacterial resistance, 3:28-58 cassava, 12:164 chestnut blight resistance, 8:313-321 citrus cold hardiness, 7:221-223 embryogenesis, 1 :23 fig, 12:432-433 fire blight resistance, 1:435-436 flower longevity, 1:208-209 ginseng, 9:197-198 in vitro techniques, 9:318-324 lettuce, 2:185-187 muscadine grapes, 14:357-405 mushroom, 6:100-111 navel orange, 8:150-156 nitrogen nutrition, 2:410-411 plant regeneration, 3:278-283 pollution insensitivity, 8:18-19 potato tuberization, 14:121-124 rhododendron, 12:54-59 sweet potato, 12:175 tomato parthenocarpy, 6:69-70 tomato ripening, 13: 77-98 tree short life, 2:66-70 Vigna, 2:311-394 woody legume tissue and cell culture, 14:311-314 yam (Dioscorea), 12:183 Geophyte. See Bulb; Tuber Geranium, fertilization, 5:355-357 Germination, seed, 2:117-141, 173174

Germplasm preservation cryopreservation, 6:357-372 in vitro, 5:261-264; 9:324-325 Gibberellin cold hardiness, 11:63 dormancy, 7:270-271 floral promoter, 4:114

grape root, 5:150-151 Ginseng, 9:187-236 Girdling, 4:251-252 Grafting phase change, 7:136-137, 141-142 rose, 9:56-57 Grape CA storage, 1:308 chlorosis, 9:165-166 flower anatomy and morphology, 13:315-337

muscadine breeding, 14:357-405 nitrogen metabolism, 14:407-452 pollen morphology, 13:331-332 root, 5: 127-168 seedlessness, 11:159-187 sex determination, 13:329-331 Greenhouse and greenhouse crops carbon dioxide, 7:357-360, 544-545 energy efficiency, 1:141-171; 9:1-52 growth substances, 7:399-481 nutrition and fertilization, 5 :317-403 pest management, 13:1-66 Growth regulators. See Growth substances Growth substances, 2:60-66, See also Abscisic acid; Auxin; Cytokinin; Ethylene; Gibberellin apple bioregulation, 10:309-401 apple dwarfing, 3:315-375 apple fruit set, 1:417 apple thinning, 1:270-300 aroids, ornamental, 10:14-18 avocado fruit development, 10:229243

CA storage in vegetables, 1:346-348 cell cultures, 3:214-314 cold hardiness 7:223-225; 11:58-66 dormancy, 7:270-279 embryogenesis, 1:41-43; 2:277-281 floriculture, 7:399-481 flower induction, 4:190-195 flower storage, 10:46-51 ginseng, 9:226 grape seedlessness, 11:177-180 in vitro flowering, 4:112-115 meristem and shoot-tip culture, 5:221-227

navel oranges, 8:146-147 pear bioregulation, 10:309-401 petal senescence, 3:76-78 phase change, 7:137-138, 142-143

CUMULATIVE SUBJECT INDEX

462

raspberry, 11:196-197 regulation, 11:1-14 rose, 9:53-73 seedlessness in grape, 11:177-180 triazole, 10:63-105

apple propagation, 10:325-326 aroids, ornamental, 10:13-14 cassava propagation, 13:121-123 cold acclimation, 6:382 cryopreservation, 6:357-372 embryogenesis, 1:1-78; 2:268-310;

H

flowering, 4:106-127 pear propagation, 10:325-326 phase change, 7:144-145 propagation, 3:214-314; 5:221-277;

7:157-200; 10:153-181

Halo blight of beans, 3:44-45 Hardiness, 4:250-251 Harvest flower stage, 1:211-212 index, 7:72-74 lettuce, 2:176-181 Hazelnut. See Filbert Heliconia, 14:1-55 Herbaceous plants, subzero stress, 6:373-417

Histochemistry flower induction, 4:177-179 fruit abscission, 1:172-203 Histology, flower induction, 4: 179184, see also Anatomy and morphology Honey bee, 9:237-272 Horseradish, CA storage, 1:368 Hydrolases, 5:169-219 Hydroponic culture, 5:1-44; 7:483-558 Hypovirulence, in Endothia parasitica, 8:299-310

7:157-200; 9:57-58, 273-349

thin cell layer morphogenesis, 14:239-264

woody legume culture, 14:265-332 Iron deficiency and toxicity symptoms in fruits and nuts, 2:150 deficiency chlorosis, 9:133-186 Ericaceae nutrition, 10:193-195 foliar application, 6:330 nutrition, 5:324-325 pine bark media, 9:123 Irrigation drip or trickle, 4:1-48 frost control, 11:76-82 fruit trees, 7:331-332 grape root growth, 5:140-141 lettuce industry, 2:175 navel orange, 8:161-162 root growth, 2:464-465

J

I

Ice, formation and spread in tissues, 13:215-255

Ice-nucleating bacteria, 7:210-212; 13:230-235

Insects and mites aroids, 8: 65-66 avocado pollination, 8:275-277 fig, 12:442-447 hydroponic crops, 7:530-534 integrated pest management, 13:166

lettuce, 2:197-198 ornamental aroids, 10:18 tree short life, 2:52 tulip, 5:63, 92 Integrated pest management greenhouse crops, 13:1-66 In vitro

Juvenility, 4:111-112 pecan, 8:245-247 tulip, 5:62-63 woody plants, 7:109-155 K

Kale, fluid drilling of seed, 3:21 Kiwifruit botany, 6:1-64 vine growth, 12:307-347 L

Lamps, for plant growth, 2:514-531 Leaves

CUMULATIVE SUBJECT INDEX

apple morphology, 12:283-288 flower induction, 1:188-189 Leek CA storage, 1:375 fertilization, 1 :118 Leguminosae, in vitro, 5:227-229; 14:265-332

Lemon, rootstock, 1:244-246, see also Citrus Lettuce CA storage, 1:369-371 fertilization, 1 :118 fluid drilling of seed, 3:14-17 industry, 2:164-207 tipburn, 4:49-65 Light fertilization, greenhouse crops, 5:33(}-331

fruit set, 1:412-413 lamps, 2:514-531 nitrogen nutrition, 2:406-407 orchards, 2:208-267 ornamental aroids, 10:4-6 photoperiod, 4:6~105 photosynthesis, 11:117-121 plant growth, 2:491-537 M

Magnesium container growing, 9:84-85 deficiency and toxicity symptoms in fruits and nuts, 2:148 Ericaceae nutrition, 10:19~198 foliar application, 6:331 nutrition, 5:323 pine bark media, 9:117-119 Mandarin, rootstock, 1:25(}-252 Manganese deficiency and toxicity symptoms in fruits and nuts, 2:15(}-151 Ericaceae nutrition, 10:189-193 foliar application, 6:331 nutrition, 5:235-326 pine bark media, 9:123-124 Mango alternate bearing, 4:145-146 asexual embryogenesis, 7:171-173 CA storage, 1:313 in vitro culture, 7:171-173 Media

463

fertilization, greenhouse crops, 5:333

pine bark, 9:103-131 Meristem culture, 5:221-277 Metabolism flower, 1:219-223 nitrogen in citrus, 8:181-215 seed, 2:117-141 Micronutrients container growing, 9:85-87 pine bark media, 9:119-124 Micropropagation, see also In vitro, propagation nuts, 9:273-349 rose, 9:57-58 temperate fruits, 9:273-349 tropical fruits and palms, 7:157-200 Microtus. See Vole Moisture, and seed storage, 2:125-132 Molybdenum nutrition, 5:328-329 Monocot, in vitro, 5:253-257 Monstera. See Aroids, ornamental Morphology navel orange, 8:132-133 orchid, 5:283-286 pecan flowering, 8:217-243 Moth bean, genetics, 2:373-374 Mung bean, genetics, 2: 348-364 Mushroom CA storage, 1:371-372 spawn, 6:85-118 Muskmelon, fertilization, 1:118-119 Mycoplasma-like organisms, tree short life, 2:5(}-51 Mycorrhizae container growing, 9:93 Ericaceae, 10:211-212 fungi, 3:172-213 grape root, 5:145-146 N

Navel orange, 8:129-179 Nectarine CA storage, 1:309-310 postharvest physiology, 11:413-452 Nematodes aroids, 8:66 fig, 12:475-477 lettuce, 2:197-198 tree short life, 2:49-50 NFT (nutrient film technique), 5:1-44

464

CUMULATIVE SUBJECT INDEX

Nitrogen CA storage, 8:116-117 container growing, 9:80-82 deficiency and toxicity symptoms in fruits and nuts, 2:146 Ericaceae nutrition, 10:198-202 fixation in woody legumes, 14:322-

ginseng, 9:209-211 greenhouse crops, 5:317-403 kiwifruit, 12:325-332 mycorrhizal fungi, 3:185-191 navel orange, 8:162-166 nitrogen in apple, 4:204-246 nutrient film techniques, 5:18-21,

323

foliar application, 6:332 in embryogenesis, 2:273-275 metabolism in apple, 4:204-246 metabolism in citrus, 8:181-215 metabolism in grapevine, 14:407-

31-53

ornamental aroids, 10:7-14 pine bark media, 9:103-131 raspberry, 11:194-195 slow-release fertilizers, 1:79-139

452

nutrition, 2:395, 423; 5:319-320 pine bark media, 9:108-112 trickle irrigation, 4:29-30 Nursery crops fertilization, 1:106-112 nutrition, 9:75-101 Nut crops chestnut blight, 8:291-336 fertilization, 1:106 honey bee pollination, 9:250-251 in vitro culture, 9:273-349 nutritional ranges, 2:143-164 pistachio culture, 3:376-396 Nutrient concentration in fruit and nut crops, 2:154-162

film technique, 5:1-44 foliar-applied, 6: 287-355 media, for asexual embryogenesis, 2:273-281

media, for organogenesis, 3:214-314 plant and tissue analysis, 7:30-56 solutions, 7:524-530 uptake, in trickle irrigation, 4:30-31 Nutrition (human) aroids, 8:79-84 CA storage, 8:101-127 Nutrition (plant) air pollution, 8:22-23, 26 blueberry, 10:183-227 calcifuge, 10:183-227 cold hardiness, 3:144-171 container nursery crops, 9:75-101 embryogenesis, 1:40-41 Ericaceae, 10:183-227 fire blight, 1:438-441 foliar, 6:287-355 fruit and nut crops, 2:143-164

o Oil palm asexual embryogenesis, 7:187-188 in vitro culture, 7:187-188 Okra, CA storage, 1:372-373 Olive, alternate bearing, 4:140-141 Onion CA storage, 1:373-375 fluid drilling of seed, 3:17-18 Orange, see also Citrus alternate bearing, 4:143-144 sour, rootstock, 1:242-244 sweet, rootstock, 1:252-253 trifoliate, rootstock, 1 :247-250 Orchard and orchard systems floor management, 9:377-430 light, 2:208-267 root growth, 2:469-470 water, 7:301-344 Orchid fertilization, 5:357-358 physiology, 5:279-315 Organogenesis, 3:214-314, see also In vitro; Tissue culture Ornamental plants chlorosis, 9:168-169 fertilization, 1 :98-104, 106-116 flowering bulb roots, 14:57-88 foliage acclimatization, 6:119-154 heliconia, 14:1-55 rhododendron, 12:1-42 p

Paclobutrazol. See Triazole Papaya

465

CUMULATIVE SUBJECT INDEX

asexual embryogenesis, 7:176-177 CA storage, 1:314 in vitro culture, 7:175-178 Parsley CA storage, 1:375 drilling of seed, 3:13-14 Parsnip, fluid drilling of seed, 3:13-14 Parthenocarpy, tomato, 6:65-84 Passion fruit, in vitro culture, 7:180181

Pathogen elimination, in vitro, 5:257261

Peach CA storage, 1:309-310 postharvest physiology, 11:413-452 short life, 2:4 . summer pruning, 9:351-375 Peach palm (Pejibaye) in vitro culture, 7:187-188 Pear bioregulation, 10:309-401 CA storage, 1:306-308 decline, 2:11 fruit disorders, 11:357-411 fire blight control, 1:423-474 in vitro, 9:321 maturity indices, 13:407-432 root distribution, 2:456 short life, 2:6 Pecan alternate bearing, 4:139-140 fertilization, 1:106 flowering, 8:217-255 in vitro culture, 9:314-315 Pejibaye, in vitro culture, 7:189 Pepper (Capsicum) CA storage, 1:375-376 fertilization, 1 :119 fluid drilling in seed, 3:20 Persimmon CA storage, 1:314 quality, 4:259 Pest control aroids (edible), 12:168-169 aroids (ornamental), 10:18 cassava, 12:163-164 cowpea, 12:210-213 fig, 12:442-477 fire blight, 1:423-474 ginseng, 9:227-229 greenhouse management, 13:1-66 hydroponics, 7: 530-534

sweet potato, 12:173-175 vertebrate, 6:253-285 yam (Dioscorea), 12:181-183 Petal senescence, 11:15-43 pH container growing, 9:87-88 fertilization greenhouse crops, 5:332-333

pine bark media, 9:114-117 soil testing, 7:8-12, 19-23 Phase change, 7:109-155 Phenology apple, 11:231-237 raspberry, 11:186-190 Philodendron. See Aroids, ornamental Phosphorus container growing, 9:82-84 deficiency and toxicity symptoms in fruits and nuts, 2:146-147 nutrition, 5:320-321 pine bark media, 9:112-113 trickle irrigation, 4:30 Photoperiod, 4:66-105, 116-117 Photosynthesis cassava, 13:112-114 efficiency, 7:71-72; 10:378 fruit crops, 11:111-157 ginseng, 9:223-226 light, 2:237-238 Physiology, see also Postharvest physiology bitter pit, 11:289-355 blueberry development, 13:339-405 calcium, 10:107-152 carbohydrate metabolism, 7:69-108 cassava, 13:105-129 citrus cold hardiness, 7:201-238 cut flower, 1:204-236; 3:59-143; 10:35-62

dormancy, 7:239-300 embryogenesis, 1:21-23; 2:268-310 fruit ripening, 13:67-103 flowering, 4:106-127 fruit softening, 10:107-152 ginseng, 9:211-213 heliconia, 14:5-13 juvenility, 7:109-155 nitrogen metabolism in grapevine, 14:407-452

nutritional quality and CA storage, 8:118-120

orchid, 5: 279-315

CUMULATIVE SUBJECT INDEX

466

petal senescence, 11:15-43 pollution injury, 8:12-16 polyamines, 14:333-356 potato tuberization, 14:89-188 pruning, 8:339-380 raspberry, 11:190-199 regulation, 11:1-14 root pruning, 6:158-171 roots of flowering bulbs, 14:57-88 rose, 9:3-53 seed, 2:117-141 subzero stress, 6:373-417 summer pruning, 9:351-375 thin cell layer morphogenesis, 14:239-264

tomato fruit ripening, 13:67-103 tomato parthenocarpy, 6:71-74 triazole, 10:63-105 tulip, 5:45-125 watercore, 6:189-251 Phytohormones. See Growth substances Phytotoxins, 2:53-56 Pigmentation flower, 1:216--219 rose, 9:64-65 Pinching, by chemicals, 7:453-461 Pineapple CA storage, 1:314 in vitro culture, 7:181-182 Pine bark, potting media, 9:103-131 Pistachio alternate bearing, 4:137-139 culture, 3:376--393 in vitro culture, 9:315 Plantain, in vitro culture, 7:178-180 Plant protection, short life, 2:79-84 Plum, CA storage, 1:309 Poinsettia, fertilization, 1:103-104; 5:358-360

Pollination apple, 1:402-404 avocado, 8:272-283 embryogenesis, 1:21-22 fig, 12:426-429 fruit crops, 12:223-264 fruit set, 4:153-154 ginseng, 9:201-202 grape, 13:331-332 heliconia, 14:13-15 honey bee, 9:237-272 kiwifruit, 6:32-35

navel orange, 8:145-146 orchid, 5:300-302 petal senescence, 11:33-35 protection, 7:463-464 rhododendron, 12:1-67 Pollution, 8: 1-42 Polyamines, 14:333-356 Polygalacturonase, 13:67-103 Postharvest physiology apple bitter pit, 11:289-355 apple maturity indices, 13:407-432 aroids, 8:84-86 asparagus, 12:69-155 CA storage and quality, 8: 101-127 cut flower, 1:204-236; 3:59-143; 10:35-62

foliage plants, 6:119-154 fruit, 1:301-336 fruit softening, 10:107-152 lettuce, 2:181-185 navel orange, 8:166-172 nectarine, 11:413-452 pathogens, 3:412-461 peach, 11:413-452 pear disorders, 11:357-411 pear maturity indices, 13:407-432 petal senescence, 11:15-43 seed, 2:117-141 tomato fruit ripening, 13:67-103 vegetables, 1: 337-394 watercore, 6:189-251; 11:385-387 Potassium container growing, 9:84 deficiency and toxicity symptoms in fruits and nuts, 2:147-148 foliar application, 6:331-332 nutrition, 5:321-322 pine bark media, 9:113-114 trickle irrigation, 4:29 Potato CA storage, 1:376-378 fertilization, 1:120-121 tuberization, 14:89-198 Propagation, see also In vitro apple, 10:324-326; 12:288-295 aroids, ornamental, 10:12-13 cassava, 13:120-123 floricultural crops, 7:461-462 ginseng, 9:206-209 orchid, 5:291-297 pear, 10:324-326 rose, 9:54-58

CUMULATIVE SUBJECT INDEX

tropical fruit, palms 7:157-200 woody legumes in vitro, 14:265-332 Protected crops, carbon dioxide, 7:345-398

Protoplast culture, woody species, 10:171-173

Pruning, 4:161; 8:339-380 apple, 9:351-375 apple training, 1:414 chemical, 7:453-461 cold hardiness, 11:56 fire blight, 1:441-442 light interception, 2:250-251 peach, 9:351-375 phase change, 7:143-144 root, 6:155-188 Prunus, see also Almond; Cherry; Nectarine; Peach; Plum in vitro, 5:243-244; 9:322 root distribution, 2:456 Pseudomonas phaseolicola, 3:32-33, 39, 44-45 solanacearum, 3:33 syringae, 3:33, 40; 7:210-212 R

Rabbit, 6:275-276 Radish, fertilization, 1:121 Raspberry productivity, 11:185-228 Rejuvenation rose, 9:59-60 woody plants, 7:109-155 Replant problem, deciduous fruit trees, 2:1-116 Respiration asparagus postharvest, 12:72-77 fruit in CA storage, 1:315-316 kiwifruit, 6:47-48 vegetables in CA storage, 1:341-346 Rhizobium, 3:34, 41 Rhododendron, 12:1-67 Rice bean, genetics, 2:375-376 Root apple, 12:269-272 diseases, 5:29-31 environment, nutrient film technique, 5:13-26 Ericaceae, 10:202-209 grape, 5:127-168 kiwifruit, 12:310-313

467

physiology of bulbs, 14:57-a8 pruning, 6:155-188 raspberry, 11:190 rose, 9:57 tree crops, 2:424-490 Root and tuber crops aroids, 8:43-99; 12:166-170 cassava, 12:158-166 minor crops, 12:184-188 potato tuberization, 14:89-188 sweet potato, 12:170-176 yam (Dioscorea), 12:177-184 Rootstocks alternate bearing, 4:148 apple, 1:405-407; 12:295-297 citrus, 1:237-269 cold hardiness, 11:57-58 fire blight, 1 :432-435 light interception, 2:249-250 navel orange, 8:156-161 root systems, 2:471-474 stress, 4:253-254 tree short life, 2:70-75 Rosaceae, in vitro, 5:239-248 Rose fertilization, 1:104; 5:361-363 growth substances, 9:3-53 in vitro, 5:244-248

s Salinity, 4:22-27 air pollution, 8:25-26 Secondary metabolites, woody legumes, 14:314-322 Scoring, and fruit set, 1:416-417 Seed abortion, 1 :293-294 apple anatomy and morphology, 10:285-286

conditioning, 13:131-181 environmental influences on size and composition, 13:183-213 flower induction, 4:190-195 fluid drilling, 3:1-58 grape seedlessness, 11:159-184 kiwifruit, 6:48-50 lettuce, 2:166-174 rose propagation, 9:54-55 vegetable, 3:1-58 viability and storage, 2:117-141

CUMULATIVE SUBJECT INDEX

468

Senescence cut flower, 1:204-236; 3:59-143; 10:35-62

petal, 11:15-43 rose, 9:65-66 Sensory quality CA storage, 8:101-127 Shoot-tip culture, 5:221-277, see also Micropropagation Short life problem, fruit crops, 2:1-116 Small fruit, CA storage, 1:308 Snapdragon fertilization, 5:363-364 Sodium, deficiency and toxicity symptoms in fruits and nuts, 2:153-

allocation, 7:74-94 flowering, 4:114 Sulfur deficiency and toxicity symptoms in fruits and nuts, 2:154 nutrition, 5:323-324 Sweet potato culture, 12:170-176 fertilization, 1:121 Symptoms, deficiency and toxicity symptoms in fruits and nuts, 2:145-154

Syngonium. See Aroids, ornamental

154

Soil grape root growth, 5:141-144 management and root growth, 2:465-469

orchard floor management, 9:377430

plant relations, trickle irrigation, 4:18-21

stress, 4:151-152 testing, 7:1-68; 9:88-90 Soilless culture, 5:1-44 Solanaceae, in vitro, 5:229-232 Somatic embryogenesis. See Asexual embryogenesis Spathiphyllum. See Aroids, ornamental Stem, apple morphology, 12:272-283 Storage, see also Postharvest physiology, Controlledatmosphere (CA) storage cut flower, 3:96-100; 10:35-62 rose plants, 9:58-59 seed, 2:117-141 Strawberry fertilization, 1:106 in vitro, 5:239-241 Stress benefits of, 4:247-271 climatic, 4:150-151 flooding, 13:257-313 petal, 11:32-33 plant, 2:34-37 protection, 7:463-466 subzero temperature, 6:373-417 Sugar beet, fluid drilling of seed, 3:1819

Sugar, see also Carbohydrate

T

Taro. See Aroids, edible Temperature apple fruit set, 1:408-411 CA storage of vegetables, 1:340-341 cut flower storage, 10:40-43 cryopreservation, 6:357-372 fertilization, greenhouse crops, 5:331-332

fire blight forecasting, 1:456-459 interaction with photoperiod, 4:8081

navel orange, 8:142 nutrient film technique, 5:21-24 photosynthesis, 11:121-124 plant growth, 2:36-37 seed storage, 2:132-133 subzero stress, 6:373-417 Thinning, apple, 1:270-300 Tipburn, in lettuce, 4:49-65 Tissue, see also In vitro culture, 1:1-78; 2:268-310; 3:214314; 4:106-127; 5:221-277; 6:357-372; 7:157-200; 8:75-78; 9:273-349; 10:153-181 dwarfing, 3:347-348 nutrient analysis, 7:52-56; 9:90

Tomato CA storage, 1:380-386 fertilization, 1:121-123 fluid drilling of seed, 3:19-20 fruit ripening, 13:67-103 galacturonase, 13:67-103 parthenocarpy, 6:65-84 Toxicity symptoms in fruit and nut crops, 2:145-154

CUMULATIVE SUBJECT INDEX

Transport, cut flowers, 3:100-104 Tree decline, 2:1-116 Triazole, 10:63-105 Trickle irrigation, 4:1-48 Tuber, potato, 14:89-188 Tuber and root crops. See Root and tuber crops Tulip. See Bulb fertilization, 5:364-366 physiology, 5:45-125 Tunnel (cloche), 7:356-357 Turfgrass, fertilization, 1:112-117 Turnip, fertilization 1 :123-124 Turnip Mosaic Virus, 14:199-238

u Urd bean, genetics, 2:364-373 Urea, foliar application, 6:332

v Vaccinium, 10:185-187, see also

Blueberry; Cranberry Vase solutions, 3:82-95; 10:46-51 Vegetable crops aroids, 8:43-99; 12:166-170 asparagus postharvest, 12:69-155 cassava, 12:158-166; 13 :105-129 CA storage, 1:337-394 CA storage and quality, 8:101-127 CA storage diseases, 3:412-461 fertilization, 1:117-124 fluid drilling of seeds, 3:1-58 greenhouse pest management, 13:166 honey bee pollination, 9:251-254 hydroponics, 7:483-558 minor root and tubers, 12:184-188 mushroom spawn, 6:85-118 potato tuberization, 14:89-188 sweet potato, 12:170-176 tomato fruit ripening, 13:67-103 tomato parthenocarpy, 6:65-84 yam (Dioscorea), 12:177-184 Vernalization, 4:117 Vertebrate pests, 6:253-285 Vigna. See also Cowpea genetics, 2:311-394 U.S. production 12:197-222 Virus

469

benefits in horticulture, 3:394-411 elimination, 7:157-200; 9:318 fig, 12:474-475 tree short life, 2:50-51 turnip mosaic, 14:199-238 Vole, 6:254-274

w Walnut, in vitro culture, 9:312 Water relations cut flower, 3:61-66 fertilization, greenhouse crops, 5:332 fruit trees, 7:301-344 kiwifruit, 12:332-339 light in orchards, 2:248-249 photosynthesis, 11:124-131 trickle irrigation, 4: 1-48 Watercore, 6:189-251 pear, 11:385-387 Watermelon, fertilization, 1:124 Weed control, ginseng, 9:228-229 Weeds lettuce research, 2:198 virus, 3:403 Woodchuck, 6:276-277 Woody species, somatic embryogenesis, 10:153-181

x Xanthomonas phaseoli, 3:29-32, 41, 45-46 Xanthosoma, 8:45-46, 56-57, see also Aroids y

Yam (Dioscorea), 12:177-184 Yield determinants, 7:70-74; 97-99

z Zantedeschia. See Aroids, ornamental Zinc deficiency and toxicity symptoms in fruits and nuts, 2:151 foliar application, 6:332, 336 nutrition, 5:326 pine bark media, 9:124

Cumulative Contributor Index (Volumes 1-14)

Aldwinckle, H. S., 1:423 Anderson, P. C., 13:257 Ashworth, E. N., 13:215-255 Asokan, M. P., 8:43 Atkinson, D., 2:424 Aung, L. H., 5:45 Bailey, W. G., 9:187 Baird, L. A. M., 1:172 Barden, I. A., 9:351 Barker, A. V., 2:411 Bass, L. N., 2:117 Beer, S. V., 1:423 Bennett, A. B., 13:67 Benschop, M., 5:45 . Blanpied, G. D., 7:xi Borochov, A., 11:15 Bower, J. P., 10:229 Bradley, G. A., 14:xiii Broschat, T. K., 14:1 Buban, T., 4:174 Bukovac, M. J., 11:1 Burke, M. J., 11:xiii Buwalda, J. G., 12:307 Byers, R. E., 6:253 Caldas, L. S., 2:568 Campbell, L. E., 2:524 Carter, I. V., 3:144 Cathey, H. M., 2:524 Chambers, R. I., 13:1 Chin, C. K., 5:221 Cohen, M., 3:394 Collier, G. F., 4:49 Collins, W. L., 7:483 Compton, M. E., 14:239 Conover, C. A., 5:317; 6:119 Coyne, D. P., 3:28 Crane, J. C., 3:376 470

Criley, R. A., 14:1 Cutting, J. G., 10:229 Daie, I., 7:69 Dale, A., 11:185 Darnell, R. L., 13:339 Davenport, T. L., 8:257; 12:349 Davies, F. S., 8:129 Davis, T. D., 10:63 DeGrandi-Hoffman, G., 9:237 De Hertogh, A. A., 5:45; 14:57 DellaPenna, D., 13:67 Dennis, F. G., Jr., 1:395 Doud, S. L., 2:1 Dunavent, M. G., 9:103 Early, I. D., 13:339 Elfving, D. C., 4:1; 11:229 El-Goorani, M. A., 3:412 Esan, E. B., 1:1 Evans, D. A., 3:214 Ewing, E. E., 14:89 Faust, M., 2:vii, 142; 4:174; 6:287, 14:333 Fenner, M., 13:183 Ferguson, A. R., 6:1 Ferguson, I. B., 11:289 FergJ,lson, L., 12:409 Ferree, D. C., 6:155 Fery, R. L., 2:311; 12:157 Fischer, R. L., 13:67 Flick, C. E., 3:214 Flore, J. A., 11:111 Forshey, C. G., 11:229 Geisler, D., 6:155 Geneve, R. L., 14:265 George, W. L., Jr., 6:65

CUMULATIVE CONTRIBUTOR INDEX

Gerrath, J. M., 13:315 Giovannoni, J. J., 13:67 Glenn, G. M., 10:107 Goldschmidt, E. K, 4:128 Goldy, R. G., 14:357 Goszczynska, D. M., 10:35 Graves, C. J., 5:1 Gray, D., 3:1 Grierson, W., 4:247 Griffen, G.J., 8:291 Grodzinski, B., 7:345 Hackett, W. P. 7:109 Halevy, A. H., 1:204; 3:59 Helzer, N. L., 13:1 Hendrix, J. W., 3:172 Henny, R. J., 10:1 Hogue, E. J., 9:377 Huber, D. J., 5:169 Hutchinson, J. F., 9:273 Isenberg, F. M. R., 1:337 Iwakiri, B. T., 3:376 Jackson, J. K, 2:208 Janick, J., 1:ix; 8:xi Jensen, M. H., 7:483 Joiner, J. N., 5:317 Jones, H. G., 7:301 Jones, J. B., Jr., 7:1 Kang, S.-M., 4:204 Kato, T., 8:181 Kawa, L., 14:57 Kawada, K., 4:247 Kelly, J. F., 10:ix Khan, A. A., 13:131 Kierman, J., 3:172 King, G. A., 11:413 Kingston, C. M., 13:407-432 Kliewer, W. M., 14:407 Knox, R. B., 12:1 Kofranek, A. M., 8:xi Korcak, R. F., 9:133; 10:183 Krezdorn, A. H., l:vii Lakso, A. N. 7:301; 11:111 Lang, G. A., 13:339 Larsen, R. P., 9:xi Larson, R. A., 7:399 Ledbetter, C. A., 11:159

471

Li, P. H., 6:373 Lill, R. K, 11:413 Lipton, W. J., 12:69 Litz, R. K, 7:157 Lockard, R. G., 3:315 Loescher, W. H., 6:198 Lorenz, O. A., 1:79 Maraffa, S. B., 2:268 Marini, R. P., 9:351 Marlow, G. C., 6:189 Maronek, D. M., 3:172 Martin, G. G., 13:339 Mayak, S., 1:204; 3:59 Maynard, D. N., 1:79 Merkle, S. A., 14:265 Michailides, T. J., 12:409 Mika, A., 8:339 Miller, S. S., 10:309 Mills, H. A., 9:103 Molnar, J. M., 9:1 Monk, G. J., 9:1 Moore, G. A., 7:157 Mor, Y., 9:53 Mills, H. A., 2:411 Monselise, S. P., 4:128 Murashige, T., 1:1 Neilsen, G. H., 9:377 Niemiera, A. X., 9:75 Ogden, R. J., 9:103 O'Donoghue, E. M., 11:413 O'Hair, S. K., 8:43; 12:157 Oliveira, C. M., 10:403 Ormrod, D. P., 8:1 Palser, B. F., 12:1 Pellett, H. M., 3:144 Ploetz, R. C., 13:257 Pokorny, F. A., 9:103 Poole, R. T., 5:317; 6:119 Poovaiah, B. W., 10:107 Porter, M. A., 7:345 Pratt, C., 10:273; 12:265 Preece, J. E., 14:265 Priestley, C. A., 10:403 Proctor, J. T. A., 9:187 Raese, J. T., 11:357 Ramming, D. W., 11:159

472

Reddy, A. S. N., 10:107 Reid, M., 12:xiii Richards, D., 5:127 Rieger, M., 11:45 Roubelakis-Angelakis, K. A., 14:407 Rouse, J. L., 12:1 Rudnicki, R. M., 10:35 Ryder, E. J., 2:164; 3:vii Sachs, R., 12:xiii Sakai, A., 6:357 Salisbury, F. B., 4:66 San Antonio, J. P., 6:85 Sankhla, N., 10:63 Saure, M. C., 7:239 Schaffer, B., 13:257 Schneider, G. W., 3:315 Schuster, M. L., 3:28 Scorza, R., 4:106 Scott, J. W., 6:65 Sedgley, M., 12:223 Sharp, W. R., 2:268; 3:214 Shattuck, V. I., 14:199 Shear, C. B., 2:142 Sheehan, T. J., 5:279 Shorey, H. H., 12:409 Smith, G. S., 12:307 Smock, R. M., 1:301 Sommer, N. F., 3:412 Sondahl, M. R., 2:268 Sopp, P. 1.,13:1 Soule, J., 4:247 Sparks, D., 8:217 Splittstoesser, W. E., 6:25; 13:105 Srinivasan, C., 7:157 Steffens, G. L., 10:63 Stevens, M. A., 4:vii

CUMULATIVE SUBJECT INDEX

Stroik, P. C., 14:89 Stutte, G. W., 13:339 Styer, D. J., 5:221 Sunderland, K. D., 13:1 Swanson, B., 12:xiii Swietlik, D., 6:287 Syvertsen, J. P., 7:301 Tibbitts, T. W., 4:49 Tisserat, B., 1:1 Titus, J. S., 4:204 Trigiano, R. N., 14:265 Tunya, G. 0., 13:105 Veilleux, R. E., 14:239 Wang, S. Y., 14:333 Wann, S. R., 10:153 Watkins, C. B., 11:289 Webster, B. D., 1:172; 13:xi Weichmann, J., 8:101 Wetzstein, H. Y., 8:217 Whitaker, T. W., 2:164 White, J. W., 1:141 Williams, E. G., 12:1 Williams, M. W., 1:270 Wittwer, S. H., 6:xi Woodson, W. R., 11:15 Wright, R. D., 9:75 Wutscher, H. K., 1:237 Yadava, U. L, 2:1 Yelenosky, G., 7:201 Zieslin, N., 9:53 Zimmerman, R. H., 5:vii; 9:273 Zucconi, F., 11:1