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 se