Horticultural Reviews (Volume 18)

HORTICULTURAL REVIEWS Volume 18

Horticultural Reviews is sponsored by: American Society for Horticultural Science

Editorial Board, VoluDle 18 Edward N. Ashworth Toyoki Kozai Mark P. Widrlechner

HORTICULTURAL REVIEWS Volume 18

edited by

Jules Janick Purdue University

John Wiley & Sons, Inc. NEW YORK / CHICHESTER / BRISBANE / TORONTO /SINGAPORE /WEINHEIM

This text is printed on acid-free paper. Copyright © 1997 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., 605 Third Avenue, New York, NY 10158-0012.

This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold with the understanding that the publisher is not engaged in rendering legal, accounting, or other professional services. If legal advice or other expert assistance is required, the services of a competent professional person should be sought. Library of Congress Catalog Card Number 79-642829 ISBN 0-471-57334-5 ISSN 0163-7851 Printed in the United States of America 10 9 8 7 6 5 4 3 2 1

Contents List of Contributors Dedication 1.

xiii

Water Relations of Cut Flowers Wouter G. van Doorn I. II. III. IV. V. VI. VII. VIII. IX. X.

Introduction Water Relations and Petal Development Xylem Anatomy Transpiration and Stomatal Opening Water Uptake, Water Potential, and Turgor Vascular Occlusion of Flowers Placed Directly in Water Vascular Occlusion in Dry-Stored Flowers Evaluation of the Causes of Vascular Blockage Relationships Between Water Stress, Hormonal Control of Flower Opening, and Senescence Conelusions Literature Cited

2. Tissure Culture of Ornamental Flowering Bulbs (Geophytes) Kiu- Wean Kim and A. A. De Hertogh I. II. III. IV. V. VI.

ix

Introduction Micropropagation Virus Elimination Breeding and Genetic Improvement Genera Reviewed Conelusions Literature Cited

1

2 4

6 7 13

16 46 56 63 65 68

87

88 89 113 119

124 146 147 v

vi

CONTENTS

3. Desiccation-Tolerance of Plant Tissues: A Mechanistic Overview Melvin J. Oliver and J. Derek Bewley I. II. III. IV. V. 4.

Physiology of Light Tolerance in Plants Barbara Demmig-Adams, William W. Adams III, and Stephen C. Grace I. II. III. IV.

5.

Introduction Processes Involved in Leaf Acclimation Role of the Xanthophyll Cycle in Photoprotective Energy Dissipation Concluding Remarks Literature Cited

172 176 195 196 203 204 215

216 217 226 239 241

Acquired Resistance to Disease in Plants Ray Hammerschmidt and Jennifer Smith Becker

247

Introduction ExampIes of Acquired Resistance Mechanisms of Resistance The Systemic Signal for Resistance Acquired Resistance and Disease Control Summary Literature Cited

248 249 255 266 272 278 279

Cacti as Crops Yosef Mizrahi, Avinoam Nerd, and Park S. Nobel

291

Introduction Biological Characteristics of Cacti Cacti as Animal Feed Cacti as Vegetables Cacti as Fruit Crops Cacti as Industrial Crops

292 294 297 299 302 309

I. II. III. IV. V. VI. 6.

Introduction Vegetative Tissues Pollen Seeds Closing Remarks Literature Cited

171

I. II. III. IV. V. VI.

CONTENTS

VII. 7.

vii

Future Prospects Literature Cited

Reproductive Biology of Cactus Fruit Crops Avinoam Nerd and Yosef Mizrahi I. II. III. IV. V. VI.

Introduction Cultivated Species Flowers Pollination Requirements Fruit Development Concluding Remarks Literature Cited Subject Index Cumulative Subject Index Cumulative Contributor Index

312 315 321

322 323 325 331 335 341 342 347 349 375

Contributors William W. Adams III, Department of Environmental, Population, and Organismic Biology, University of Colorado, Boulder, Colorado 80309-0334 Jennifer Smith Becker, Department of Plant Pathology, University of California, Riverside, California 92521 J. Derek Bewley, Department of Botany, University of Guelph, Guelph, Ontario N1G2W1, Canada A. A. De Hertogh, Department of Horticultural Science, North Carolina State University, Raleigh, North Carolina 27695-7609 Barbara Demmig-Adams, Department of Environmental, Population, and Organismic Biology, University of Colorado, Boulder, Colorado 80309-0334 Stephen C. Grace, Department of Environmental, Population, and Organismic Biology, University of Colorado, Boulder, Colorado 80309-0034 Ray Hammerschmidt, Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan 48824 Kiu-Weon Kim, Department of Horticultural Science, Yeungnam University, Kyongsan 712-749, Korea Yosef Mizrahi, Department of Life Sciences, Institutes for Applied Research, Ben-Gurion University of the Negev, Beer-Sheva, Israel 84105 Avinoam Nerd, Department of Life Sciences, Institutes for Applied Research, Ben-Gurion University of the Negev, Beer-Sheva, Israel 84105 Park S. Nobel, Department of Biology, University of California, Los Angeles, California 90024-1606 Melvin J. Oliver, Plant Stress Unit, Cropping Systems Research Laboratory, United States Department of Agriculture, Agricultural Research Service, Box 215, Route 3, Lubbock, Texas 79401 Harvey Quamme, Agriculture Canada Research Centre, Summerland, British Columbia, Canada Wouter G. van Doorn, Agrotechnological Research Institute (ATODLO), P.O. Box 17, 6700 AA Wageningen, The Netherlands ix

HORTICULTURAL REVIEWS Volume 18

Norman E. Looney

Dedication: Norman E. Looney Harvey Quamme Agriculture Canada Research Centre Summerland, British Columbia, Canada

Norm Looney is a dedicated scientist who has a passionate interest in horticulture research. He well deserves the honor of this dedication for his many research achievements and leadership in forwarding the cause of horticulture science over the past 35 years. Norm was born in Adrian, Oregon, in 1938. He completed his Bachelor of Science degree with honors in agriculture education at Washington State University (1960) followed in 1966 by a doctoral degree with a major in horticulture and minors in botany and biochemistry. His doctorate research revealed a close relationship between chlorophyllous activity and the ripening of apple and banana fruit. After completing his doctorate, Norm was persuaded to come north and accept a research position with the Canadian Department of Agriculture at the Summerland Research Station. Norm has since remained at Summerland except for brief sojourns on national and international work transfer. He has adapted well to his new country. In fact, he has become a more ardent Canadian than most nativeborn citizens. One of Norm's first research tasks at Summerland was to establish the relationship of light distribution in the canopy of apple trees and fruit color. This research has become the theoretical basis for use of the form of the central tree training and pruning system that is popular in northwestern United States and in British Columbia and is often cited in the field of canopy management. The main focus of Norm's research, however, has been on understanding the mode of action of growth regulators and their application to fruit production. A few highlights indicate the scope of this research. Early on he discovered that daminozide inhibits apple ripening through its influence on ethylene ripening and developed the technology for its use in orchards. The discovery that the growth regulator ethephon combined with fenoprop advanced fruit ripenxiii

xiv

H. QUAMME

ing had an important influence on the Canadian 'McIntosh' apple industry. His growth regulator research was extended to stone fruits. He found that applications of gibberellic acid increase firmness of cherries and make them more resistant to rain-induced splitting. In collaboration with J. M. Lee he showed that a heritable compact habit of apple displayed higher cytokinin activity but not abcissic acid, gibberellic acid, or auxin levels. This latter requirement is consistent with media requirements in tissue culture. Recently, he has turned his attention to the use of natural occurring growth regulators and their application to fruit growing. In collaboration with the research team lead by R. P. Pharis, University of Alberta, Calgary, he demonstrated that certain members of the gibberellin family increase flowering in apples. Norm has also continued his interest in postharvest fruit physiology at Summerland. In cooperation with O. 1. Lau he demonstrated that British Columbia-grown 'Golden Delicious' apples are more prone to damage from prestorage high CO 2 treatment than those from Washington State and that this was related to greater free water on the fruit. These observations led to the development of "rapid CA" as a fruit storage procedure in North America. Work on the effects of temperature on ripening was further extended with M. Knee while on work transfer to East MaIling. The contribution made by Norm to the development of chemical thinning practices range from determining influence of spray volume, cultivar, and a host of other variables on effectiveness of growth regulators to reduce fruit set and control biennial bearing. The development of effective chemical thinning practices has had a great impact on the economics of fruit growing in British Columbia and other regions. As head of the tree fruit production section at The Summerland Research Centre for over a decade, Norm has provided leadership and helped to develop the section into a well-coordinated, productive research group. He has been an active participant on grower education committees and industry development projects. As member of the Agrologist Institute of Canada and the Canadian Society of Horticulture Science (CSHS), he has actively promoted Canadian horticultural research. He is a Fellow in the American Society of Horticultural Science (ASHS) and has served as program chairman for the joint ASHS-CSHS meeting held in Vancouver in 1984 as well as numerous other ASHS committees. He acted for one term as associate editor of the ASHS journal. As chairman of the International Society for Horticultural Science (ISHS) Growth Regulators Work-

DEDICATION

xv

ing Group in Fruit, he organized a symposia held at Summerland in 1986. Recently, he was appointed ISHS council member responsible for the Fruit Section. He initiated and organized a successful bid to have CSHS host the International Congress 2002 in Toronto. All these endeavors are testimony to Norm's leadership capabilities and organizing talents. Norm is genuinely interested in people and is at ease with strangers. His participation in a discussion group-scientific or socialensures lively conversation. Locally, he is at the hub of a network of friends and acquaintances who frequent Theo's Restaurant in Penticton. This network gives him insight into local politics and society. His frequent travels have allowed him to establish a worldwide network of friends and associates; some have become collaborators in one or more of his many research projects. He is a generous and gracious host to the many people who have visited his home on Lake Okanagan. In addition to being a talented research scientist, Norm is a gifted musician. Local operatic societies and choirs seek his participation. When persuaded, he can demonstrate that he is a fine pianist. Norm also is an avid bird watcher and hiker. Another of his pet projects is the orchard just below his home. Peach and prune production is his specialty. As he has one of the few prune orchards in the neighborhood, he is affectionately referred to as the "Prune King of Summerland." In modern times the flow of cross-border emigration has been in favor of the United States. Norm Looney is one of those energetic, talented Americans that chose to buck the trend and settle in Canada. The Canadian horticultural industry and Canadian society have gained much from his move here.

1 Water Relations of Cut Flowers Wouter G. van Doorn *

Agrotechnological Research Institute (ATO-DLO) P.O. Box 17,6700 AA The Netherlands

I. II. III. IV.

V. VI.

Introduction Water Relations and Petal Development Xylem Anatomy Transpiration and Stomatal Opening A. Stomatal Transpiration 1. Stomata on the Perianth, Stamens, and Gynoecia 2. Stomatal Reaction in Leaves of Cut Flowers 3. Effect of the Boundary Layer on Transpiration 4. Effects of Solutes in Vase Water B. Cuticular Transpiration Water Uptake, Water Potential, and Turgor Vascular Occlusion of Flowers Placed Directly in Water A. Deposition of Lignin, Suberin, and Tannin B. Deposition of Gum in Conduits by Xylem Cells 1. Acacia 2. Alnus glutinosa, Amelanchier spicata, and Dahlia variabilis 3. Prunus 4. Rosa C. Exudation of Latex and Other Substances at the Cut Surface 1. Euphorbia 2. Heliconia 3. Narcissus 4. Prunus

* This review is dedicated to J.F.T. Aarts at Boxmeer, The Netherlands, who significantly contributed to our understanding of the physiology of cut flowers. Sincere thanks are due to Henk de Stigter, Dominic Durkin, Jeremy Harbinson, Harmannus Harkema, Michael Reid, and Ernst Woltering for critically reading the manuscript, and to Michael Blanke for providing information on cuticular surfaces of petals.

Horticultural Reviews, Volume 18, Edited by Jules Janick ISBN 0-471-57334-5 © 1997 John Wiley & Sons, Inc. 1

2

W. G. VAN DOORN D.

Tyloses 1. Prunus 2. Rosa 3. Syringa E. Microbial Growth 1. Correlation Between Microbial Growth and Vascular Occlusion 2. Microscopical Evidence 3. Role of Yeasts, Filamentous Fungi, and Bacteria 4. Identification of Bacteria and Fungi 5. Effects of Antimicrobial Compounds 6. Inclusion of Bacteria and Their Products in Vase Water 7. Mode of Action of Bacteria F. Cavitation VII. Vascular Occlusion in Dry-Stored Flowers A. Aspired Air: The Lumen Pathway and the Cell Wall Pathway for Water B. Cavitation C. Deposition of Material and Tylose Formation D. Microbial growth VIII. Evaluation of the Causes of Vascular Blockage A. Astilbe and Bouvardia B. Dendranthema (chrysanthemum) C. Gerbera D. Gypsophila E. Rosa F. Syringa G. Ferns H. Other Cut Foliage IX. Relationships Between Water Stress, Hormonal Control of Flower Opening, and Senescence X. Conclusions Literature Cited

When I have pluck'd thy rose, I cannot give it vital growth again, It needs must wither. I'll smell thee on the tree. Shakespeare (Othello, V. ii 13-15)

I. INTRODUCTION

The vase life of some cut flowers is limited by a disturbance in hormonal regulation (Borochov and Woodson 1989), whereas in many other flowers the limiting factor is water stress. The water relations of cut flowers have been briefly reviewed by Halevy and Mayak (1981), and those of intact plants have been covered in depth in sev-

1.

WATER RELATIONS OF CUT FLOWERS

3

eral monographs (e.g., Slatyer [1977], Nobel [1991], and Kramer and Boyer [1995] and in some recent symposia (Smith and Griffiths 1993). Early physiological studies on cut flowers dealt with their water relations. Sachs (1870, p. 575; 1887, p. 211), for example, noted that sunflowers (Helianthus annuus), freshly cut and placed in water, wilted rapidly. When a pressure of about 10 cm of mercury (13.3 kPa) was applied on the water, wilting shoots recovered within 30 min. De Vries (1873) extended these observations to flowers of Helianthus tuberosus and concluded that during the short exposure of the cut end to air, a blockage developed in the lowermost part of the stem, which could be overcome by placing the stems in warm water or by recutting them under water. Early workers also observed that some shoots exuding latex or mucilaginous material upon cutting often take up little water, even at considerably increased hydrostatic pressure (Moll 1880). The life of uncut flowers is terminated either by color changes, flower closure, petal wilting, or petal abscission. When flower shoots are cut and placed in water, symptoms of natural senescence are often not observed, but symptoms of water stress, such as premature wilting of the flowers and leaves, are expressed. Examples of flowers that show water stress are roses, Gypsophila, Astilbe, Bouvardia, and Acacia; other flowers, such as tulips, Freesia, and Iris, do not show this early water stress. As compared to cut flowers, less is known about the water relations of cut greens. Foliage cut from a wide range of monocotyledonous and dicotyledonous Angiosperms are in use by the floral trade. Several Gymnosperm species (e.g., Abies, Cedrus, Pinus, and Juniperus), a number of ferns (e.g., Rumohra adiantiformis and Pteris spp.), and a club moss (Lycopodium taxifolia) are also in common use. Most of the cut foliage has apparently been selected against problems in water relations, since their vase life is usually long (Maync et al. 1985; Bazzocchi et al. 1987; Broschat and Donselman 1987; Vaughan 1988). Cut foliage is included in this review because the responses of water transport and the development of water stress in nonflowering stems are essentially the same as in those with flowers. This paper describes (1) the water relations of petals both on stems attached to the plant, and on stems that have been cut and placed in water, (2) the xylem anatomy, (3) the characteristics of stomatal and cuticular transpiration of cut stems, (4) water uptake, and the maintenance of water potential and turgor, (5) the various causes for the observed reduction in water flow, and (6) the relationships between water stress and hormonal regulation.

4

W. G. VAN DOORN

In the terminology of flower parts, a distinction is made between those flowers bearing variously colored petals and green sepals, and flowers in which no clear distinction can be made between these two whorls (e.g., tulip, iris). In the latter group, the members of both whorls are called tepals. In this review, the tepals will be referred to as petals, for simplicity. Furthermore, cultivars will be referred to by their trade name, not by their officially registered cultivar name. According to international convention the trade names are neither placed between quotation marks nor indicated by the prefix cv. In roses, for example, Sonia (the trade name) is equivalent to 'Sweet Promise,' which may also be written as cv. Sweet Promise. When the trade name differs from the official cultivar name, the latter is usually not well known (e.g., in roses Samantha = 'Jacanth-PL', and Frisco = 'Korflapei'). II. WATER RELATIONS AND PETAL DEVELOPMENT

Growth can be due to an increase in cell number and to an increase in cell size. Visible petal growth is mainly based on cell expansion, which requires two cooperative processes. First, the cell wall must be able to expand. This requires mechanical changes in the wall and/ or synthesis of new wall material. Second, water must enter the cell. A physical driving force, based on accumulation of osmotically active substances, has to be established before water will enter. In narcissus petals, for example, cell expansion is related to a drop in the levels of sucrose and an increase in the concentration of reducing sugars (Nichols 1976). Petal growth in cut roses is related to a decline in starch content and a concomitant increase in reducing sugars (Evans and Reid 1986, 1988; van Doorn et al. 1991c). An increase in amylolytic activity has been observed during the opening of rose flowers (Hammond 1982). Other common osmotica, including inorganic ions, organic acids, and amino acids, may also accumulate to drive water influx (Acock and Nichols 1979; Winkenbach 1970). The petal cells often increas9 their volume by a factor of 10 in a relatively short period. The process of cell expansion in the petals is influenced by various growth regulators, studied in detail in Pharbitis (= Ipomoea) nil (Convulvulaceae) and the corolla of the ray flowers of Gaillardia grandiflora (Asteraceae). The results suggested that petal growth in both species is promoted by gibberellins and is inhibited by endogenous

1.

WATER RELATIONS OF CUT FLOWERS

5

ethylene, whose synthesis is partially controlled by endogenous indoleacetic acid (IAA). Changes in the concentrations of these hormones, and probably in the sensitivity of the cells to them, determine the rate of growth (Koning 1984,1986; Raab and Koning 1987a,b; 1988). The growth of Pharbitis nil petals was also regulated by phytochrome, which accounted for the rapid expansion after 10 h of uninterrupted darkness (Koning 1986). Rose flowers also showed a diurnal rhythm of growth and opening; when placed in a 12-h light/ dark cycle, growth started a few hours before the light period (Evans and Reid 1986), suggesting regulation by phytochrome. Depending on the species, growth and opening of flowers is also associated with light intensity, temperature, and, in a few flowers, relative humidity (Goldsmith and Hafenrichter 1932). Adverse water relations may inhibit petal growth. The petals of roses that had been stored dry prior to placement in water, for example, did not reach the same size as those of unstressed controls. After prolonged dry storage, the petals did not grow at all (Halevy and Mayak 1975). Disruption of water uptake will inhibit growth directly, and when the hormonal balance is disturbed, it may also be inhibited indirectly (see Section IX). Flower opening generally includes a change in petal orientation. In tulips, for example, the reversible opening and closing movements are due to osmotic changes in special cells at the petal base (Goldsmith and Hafenrichter 1932). The opening and closing movements of Pharbitis nil flowers are due to differential growth and to asymmetric turgor changes in a specific group of inner epidermal cells. at the midribs (Phillips and Kende 1980; Takimoto and Kaihara 1984). In other species the movements may be due to differential growth at the upper and lower sides of the petals (Schrempf 1977; Reid and Evans 1986). Petal movement, therefore, can also be adversely affected by a water deficit. When the petals are fully expanded they remain turgid for a period of time, varying from a few hours to several months, depending on the species (Stead and van Doorn 1994). In several species petal life is terminated by abscission; the abscised petals being fully or almost fully turgid. The cells in abscising petals may show a small loss of solutes prior to abscission, and hence show a small increase in cell leakiness (Stead and Moore 1983). In other species the first symptom of petal senescence is wilting or withering, usually preceded by a dramatic increase in leakage of inorganic ions, organic acids, reducing sugars, amino acids, and anthocyanins. The cause of

6

W. G. VAN DOORN

leakage is unknown, but may relate to loss of semipermeability of the tonoplast and the plasma membrane (Bieleski and Reid 1992; Hanson and Kende 1975; Kende and Hanson 1977; Mayak et al. 1977; Nichols 1968a; Suttle and Kende 1980; van Meeteren 1979).

III. XYLEM ANATOMY The water-conducting tissue of plants consists of cells that have elongated and subsequently died. In general, the conduction of water in nonvascular plants occurs in fibers and tracheids, whereas in vascular plants it occurs in fibers, tracheids, and vessels. Fibers and tracheids consist of one cell only. Fibers are usually 1000 to 2000 11m long and have a diameter of less than 50 11m. Tracheids are generally shorter than the fibers and are also less than 50 11m wide (Esau 1965). Vessels consist of a series of stacked vessel members that are open at the upper and lower sides, except at the vessel ends where they are open at one side only. Vessel members are usually much shorter than fibers and tracheids and vary in length from about 200 to more than 1000 11m; their diameter varies from less than 50 11m to more than 200 11m. Vessel length varies within each species, but the majority of vessels are short with an exponentially declining number in the higher length categories. Longest vessels are up to several decimeters in herbaceous plants, and up to 10 m in tall trees. Because the rate of water transport in cylinders is proportional to the fourth power of radius, most water transport in vascular plants occurs in the wide vessels (Zimmermann 1983). Water flow between vessels, tracheids, and fibers is through pits that contain a (physical) membrane consisting of a network of cellulose microfibrils (O'Brien and Thimann 1969; Butterfield and Meylan 1982). The pit membrane contains small pores, whose diarneter varies, depending on the species. Average effective radii in some gymnosperms ranged from 16 to 28 nm (Stamm 1935), and in several softwood species from 40 to 110 nm (Stamm 1952; Stamm and Wagner 1961). In petioles of alfalfa it was about 100 nm (Van Alfen et al. 1983). The xylem also contains ray cells, often filled with reserve carbohydrates, and paratracheal parenchyma cells. Depending on the species, ray cells can be involved in deposition of gums into the lumen of the xylem conduits, and in the formation of tyloses in these lumina (see Sections VLB and VLD).

1.

WATER RELATIONS OF CUT FLOWERS

7

IV. TRANSPIRATION AND STOMATAL OPENING As may be expected, flowers with a relatively small leaf area, such as carnations, lose much less water per stem and unit time than those with relatively high leaf area, such as lilies or roses. A water deficit will develop only when the rate of water uptake is lower than the rate of transpiration, hence the onset of water stress can be delayed by reducing the rate of transpiration. Because water loss occurs much more rapidly through open stomata than through the cuticle, the presence of functional or nonfunctional stomata and the reaction of stomata to a developing water stress, as well as their reopening after the stress has subsided, are relevant to the vase life of cut flowers. A. Stomatal Transpiration 1. Stomata on the Perianth, Stamens, and Gynoecia. Most cut flowers include a stem, leaves, and one or more flowers, and most flowers bear sepals and petals, stamens, and pistils. Stomata are usually present in all green epidermal tissues, such as in leaves and sepals, and sometimes in the epidermis of nongreen parts, such as petals. Esau (1965) and Fahn (1974) noted that stomata can be often be found in petals. According to Esau (1965) these stomata are sometimes nonfunctional, but Fahn (1974) considered them always nonfunctional. Troll (1959) reported that petals of commercial chrysanthemum (Den dran thema grandiflora, formerly called Chrysanthemum morifolium) contained stomata. The petals of Aranda have also been found to contain stomata (Hew et al. 1987). In a survey of some other cut flower species no stomata were found in the petals of roses (Rosa hybrida) , carnations (Dianthus caryophyllus), Delphinium, Dianthus barbatus, Gerbera jamesonii, and most cultivars of Cymbidium (Table 1.1). Stomata occurred in Lilium and Tulipa. Depending on the species, the stomata occurred on the adaxial or the abaxial side of the petals, or on both sides. In four Cymbidium cultivars no stomata were found along the pedicels and the stem nor on the petals. Stomata were present on the abaxial side of the petals in flowers of King Arthur Cymbidium (Table 1.1). The petals of this cultivar, contrary to most others, contain chlorophyll, as do the guard cells of the stomata. Chlorophyll is not a prerequisite for guard cell functioning: leaves of Paphiopedilum orchids have functional stomata with nonchlorophyllous guard cells (Nelson and Mayo 1975).

W. G. VAN DOORN

8

Table 1.1. Presence of stomata on the petals of some commercial cut flowers.

Genus

Aranda

Cultivar Christine

Wendy Scott

Cymbidium

Alexalban; Sirius; Tapestry King Arthur

Den dran thema (chrysanthemum)

Cultivar not known Reagan; Cassa

Dianthus caryophyllus Gerbera

White Sim; Scania Mickey; Liesbeth; Tamara Enchantment

Lilium Rosa

Tulip a

Lady Seton Golden Wave Sonia; Madelon Ilona; Motrea Frisco; Cara Mia Apeldoorn; Frappant

Number of stomata

Reference

38 per cm 2 abaxial side 40 per cm 2 abaxial side 45 per cm 2 abaxial side 45 per cm 2 abaxial side None

Hew et al. 1987

Some, abaxial side only Some

M. G. J. Mensink (pers.comm.1993) Troll 1959

Hew et al. 1987

::::: 20 per cm 2 , adaxial side only None None : : : 10 per cm 2 , adaxial side only None Stubbs and Francis 1971 None Mayak and Halevy 1974 None None None Both on inner and outer whorl: ::::: 500 per cm 2 outer whorl, adaxial ::::: 100 per cm 2 outer whorl, abaxial ::::: 100 per cm 2 inner whorl, adaxial ::::: 10 per cm 2 inner whorl, abaxial

Note. Data have not previously been published, unless otherwise indicated. Abaxial side = underside; adaxial side = upperside.

It remains to be established whether the stomata on the petals of cut flowers function like those on the leaves. In Aranda (Hew et al. 1987) the stomata on petals apparently remained closed, and in avocado (Persea americana) petals, the stomata remained open even when those on the leaves closed (Blanke and Lovatt 1993). Stomata are also usually present on the stamens and on the gynoecia of flowers (Esau 1965). They have also been found on the necta-

1.

WATER RELATIONS OF CUT FLOWERS

9

ries, but here their function is not primarily in gas exchange; the nectar is exuded through the stomatal opening. The stomata on nectaries apparently stay open (Zer and Fahn 1992; Figueirido and Pais 1992). 2. Stomatal Reaction in Leaves of Cut Flowers. Stomata on leaves normally react to light, the water potential of the tissue, the hormonal balance, the carbon dioxide concentration, and in a number of plants to the relative humidity of the air (Meidner and Mansfield 1968; Losch and Tenhunen 1981; Nobel 1991). In plants growing in the light, stomata usually open early in the morning and may partially close before noon as the water potential in the plant drops, after which they may remain closed or reopen again in the afternoon (Tenhunen et al. 1987). In intact plants, stomatal opening is often delayed after a period of reduced water supply (Raschke 1989), and this is apparently also true for cut flowers. When Sonia or Frisco roses were held dry (by placing individual stems on the laboratory bench) for 3 h and then placed in water, the stomata reopened; when the stems were held dry for 24 h and subsequently placed in water they did not reopen during the next 2 days of observation (E. de Koning, unpublished). Rapid stomatal closure in water-stressed plants has been attributed to accumulation of abscisic acid (ABA) and its derivatives (Aspinall 1980). Slow stomatal reopening after water stress is perhaps related to slow ABA degradation (Loveys and Kriedemann 1973; Reid and Wample 1985), although the evidence for this hypothesis seems weak (Raschke 1989). Stomatal opening may also be regulated by cytokinin levels. Exogenous cytokinins are known to induce stomatal opening and enhance wilting (Livne and Vaadia 1972). The inclusion of kinetin in the vase solution of leafy rose stems increased the transpiration rate and stomatal opening. However, kinetin delayed wilting both in leafy and leafless rose flowers, which was attributed to effects other than on stomata (Mayak and Halevy 1974). When cut roses are placed in water directly after harvest, their leaf stomata show a rhythm of opening and closing that is similar to their diurnal rhythm before cutting. This rhythm is even maintained for several days when the flowering shoot is continuously held in darkness or in the light (Mayak et al. 1974; van Doorn et al. 1989). Increasing the day length in the greenhouse by artificial light, now commonplace in Western Europe, may result in an increase in the duration of stomatal opening of cut roses in vases and this may lead to higher water loss and hence aggravation of water stress (Slootweg

10

W. G. VAN DOORN

and van Meeteren 1991). Similarly, the stomata on leaves of diploid or triploid rose cultivars (Donnelly and Skelton 1989) lack a closing mechanism, resulting in excessive transpiration. 3. Effect of the Boundary Layer on Transpiration. One of the factors affecting the rate of transpiration is the layer of still air on the surface. The thickness of this boundary layer decreases as the windspeed increases, but the effect of wind will be reduced by the presence of hairs on the surface. Upon precooling with forced air the flowers may lose considerable amounts of water, whereas flowers placed indoors in water are usually not subject to rapid air movement. Nobel (1991) pointed out that under such conditions, provided that the stomata are fully open, the resistance of the boundary layer may be the limiting factor for the rate of transpiration. 4. Effects of Solutes in Vase Water. The rate of transpiration de-

pends on the gradient between the water potential of the air (which at 20°C and 50% RH is about -100 MPa) and that of the solution in which the stems are placed. The water potential of deionized water is about zero, but will be lowered by dissolved chemicals. Glucose and fructose, for example, are often used in preservative solutions at 10-20 gIL. Shortly after harvest, cut flowers may also be placed in sugar pulsing solutions in which the sucrose concentration may be up to 200 gIL, which at 20°C results in a water potential of the solution of -1.55 MPa (15.5 bars). In a gradient of about 100 MPa the effect of such a decrease in water potential is relatively small. Apart from an effect on water potential, some solutes also increase solution viscosity, which could lead to reduced uptake. An aqueous solution of 20% sucrose, for example, has a viscosity that is about double that of pure water. Sugars in the solution are often reported to decrease transpiration. Sugars usually result in increased bacterial growth, which may lead to stomatal closure as a result of a water deficit, an effect not fully realized in most reports. Even when an antimicrobial compound was included, its effect was usually not fully checked. Dissolving 50 giL of glucose or sucrose in the vase solution has been reported to reduce the rate of transpiration in cut flowers of Nigella damascena, Latbyrus odoratus, and Petunia sp. by 40-60%, in chrysanthemum by 20%, in Gaillardia grandiflora by 10%, and in Antirrbinum by 7% (Arnold 1931). When Mattbiola incana flowers were placed in water with an antimicrobial compound and 10 gIL sucrose, no decrease in water uptake and no stomatal closure was found, whereas

1.

WATER RELATIONS OF CUT FLOWERS

11

the stomata of shoots placed in 40 giL sucrose completely closed within 2 days (Aarts 1957a). Inclusion of sucrose or glucose in the water also decreased the rate of transpiration in roses, which was attributed to stomatal closure (Marousky 1969, 1972; de Stigter 1980a,b; Venkatarayappa et al. 1981). Sucrose in the water similarly reduced the rate of water uptake of rose flowers that had been held dry for some hours, that is, in flowering stems in which the stomata had already closed. In this experiment, water uptake was determined during the first hours after dry storage, using a freshly prepared sucrose solution, hence a bacterial effect was apparently excluded (Harkema and Boom 1983). This result suggests that one of the reasons for reduced transpiration is a reduction in water uptake rather than stomatal closure. A solution of 40 giL sucrose was found to reduce the flow rate in isolated 5-cm stem segments of roses to almost one-third of that in controls, in an experiment that apparently excluded bacterial effects (Durkin 1979a). Hydroxyquinoline compounds, often used as antimicrobial agents, are also reported to result in stomatal closure. Stoddart and Miller (1962) placed the petioles of chrysanthemum leaves in an aqueous solution of 2000 mg/L hydroxyquinoline sulfate (HQS) and found a reduction of stomatal aperture. This concentration is 10 times higher than that used for vase water in the flower trade (about 200 mg/L), and the solution had a pH of about 1. In a standard assay in which tobacco leaf disks were floated on an aqueous solution of HQS, the concentration to produce 50% stomatal closure was 1 mM, that is, 388 mg/L (Zelitch 1963). If we assume adequate wetting of the surface, the stomata in this test were apparently in direct contact with the chemical, but it is not clear how much of the 8-hydroxyquinoline compound would reach the stomata, either in this test or in cut flowers. Others have not been able to show that hydroxyquinoline citrate (HQC) in the vase solution reduced stomatal opening. In cut chrysanthemum flowers, for example, no effect was found, at least at concentrations up to 250 mg/L (Gay and Nichols 1977). Aluminum compounds are also often included in the water to inhibit microbial growth, and were found to reduce the rate of transpiration in cut rose flowers (Schnabl and Ziegler 1974). Floating epidermis strips on a solution containing 1 mM aluminum sulfate resulted in stomatal closure (Schnabl and Ziegler 1974; 1975; Schnabl 1976). It has been suggested, therefore, that aluminum sulfate reduces the transpiration of cut flowers by decreasing stomatal conductance (Schnabl and Ziegler 1975), but this has not yet been critically evaluated by measuring stomatal conductance.

12

W. G. VAN DOORN

Exogenous ABA is very effective in decreasing stomatal opening. Wilting of cut rose flowers was delayed when the stems were held in an aqueous ABA solution at 1 mg/L, or when they had been pulsed for 1 day with 10 mg/L (Kohl and Rundle 1972). Adding 10 mg/L to the vase solution extended the vase life of Chamelaucium uncinatum, the Geraldton wax flower (Joyce et al. 1993). In chrysanthemum the inclusion of 10 mg/L ABA also resulted in stomatal closure (Gay and Nichols 1977). ABA may also have other effects, for example, increasing the rate of senescence (Abeles et al. 1992). ABA extended longevity of cut rose flowers held at relatively high evaporative demand, due to stomatal closure, but accelerated senescence of roses held under relatively low evaporative demand (Halevy et al. 1974).

B. Cuticular Transpiration

When leaves are cut and held in air, a biphasic weight loss is usually observed. The first phase (rapid weight loss), lasting 1-2 h, apparently relates to stomatal transpiration. Once the stomata are closed, the second phase (slow weight loss) is due to cuticular transpiration (Kramer 1983). Similarly, flowers that lack stomata on any part of the flowering stem, such as most cultivars of Cymbidium, lose considerably less water per unit fresh weight than other cut flowers (Harkema and van Doorn 1985). In some cut flowers, considerable water loss may still occur after stomatal closure, however. This water is apparently mainly lost through the flowers. For example, in Astilbe, with numerous small flowers, the leaves and stem accounted for only 40% of total water loss (E. Ch. Sytsema-Kalkman, personal communication, 1993). The lower (abaxial) surface of the petals of cut flowers is usually smooth, but the upper (adaxial) surface is generally very uneven, because the epidermal cells are often strongly convex or may bear papillae. Strongly bulged cells have been described in Viola and Nasturtium (Esau 1965), Rosa (Stubbs and Francis 1971), Nicotiana alata (Eveling 1984), Antirrhinum (Robards 1970), Diosma alba (Troughton and Sampson 1973), Lobularia maritima (Troughton and Donaldson 1972), and Fragaria (Blanke 1991). This unevenness increases the surface area and in several of these species the papillae are covered with ridges, which increases the surface even more. To what degree the increase in surface has consequences for the rate of transpiration by the petals is as yet unknown. Blanke (1991) found that the petal surface of the strawberry flower had no cuticle. The presence of petal cuticle has apparently not been

1.

WATER RELATIONS OF CUT FLOWERS

13

evaluated in cut flowers, except for roses, where the cuticle has been described and is suggested to contribute to petal iridescence (Martin and Juniper 1970). Water transport through cuticles is generally low, the permeability coefficient for cuticle water transport being of the same magnitude as that of polypropylene plastics (Schonherr 1982). For a given cuticle the rate of transpiration will relate to its thickness. When several species were compared, the rate of transpiration was not dependent of thickness (Kamp 1930; Martin and Juniper 1970; Schonherr 1982), but rather on chemical composition, the presence of pores and crevices, the presence of epicuticular wax, and the form of surface structures (Martin and Juniper 1970; Schonherr 1982). The permeability coefficient of the epidermis of petals that lack a cuticle has not been reported, but may be higher than in petals with a cuticle. V. WATER UPTAKE, WATER POTENTIAL, AND TURGOR The rate of water uptake will depend, among other factors, on the transpirational pull and on the temperature and composition of the solution. Temperature has an effect on solution viscosity. Rehydration in dry-stored stems may increase with water temperature (Holle 1916), although using water of more than 40°C for more than a few hours usually results in a short vase life. The use of low-temperature water for rehydration has also been described (Carow 1981, Durkin 1979b; Stamps 1986; van Meeteren 1989). Some reports indicate that the ionic composition of the vase solution is a determinant of the rate of water uptake. Sacalis (1974) found that removing the ions from tap water improved the rate of water uptake and delayed wilting in cut rose flowers. Tap water is often alkaline and it has been suggested that water uptake is reduced in such hard water (Sacalis 1993), although this seems contradicted in a few other reports. Aarts (l957a) showed that a 0.1-0.2% solution of calcium nitrate increased the rate of water flow through stem segments, and in roses higher alkalinity of the water was related to a longer vase life (Crossmann 1968). A decrease of solution pH to well below 7 (Aarts 1957a; Durkin 1979a,b; Conrado et al. 1980) clearly promotes flower water uptake. Similarly, water uptake is increased by the inclusion of surfactants in the solution (Mertens 1944; Durkin 1980). Effects of acid solutions and surfactants are discussed in detail in Section VII. The rate of water uptake of freshly cut flowers may initially be high when the plant has a low water potential at cutting. The rate of

14

W. G. VAN DOORN

uptake will reach a steady state corresponding to the rate of transpiration, but, depending on the species, the rate of uptake may subsequently decrease. In Rosa, Bouvardia, Astilbe, Zantedeschia, some Dendranthema (chrysanthemum) cultivars, Polianthes tuberosa (tuberose), and several species from Australia, such as Anigozanthos (kangaroo paw), Chamelaucium, Banksia, Grevillea, Thryptomene, Leptospermum, and Telopea (Mayak et al. 1974; Tjia and Funnell 1986; Faragher 1989; Naidu and Reid 1989), to mention a few, the rate of water uptake rapidly declines to low values. In other cut flowers, such as Heliconia, there is little water uptake even shortly after cutting and placement in water (Donselman and Broschat 1986; KaIpo et al. 1989). During vase life the rate of transpiration also declines but tends to be higher than the water uptake rate. This results in a negative water balance (= rate of uptake - rate of transpiration), a decrease in water potential, and in stomatal closure (de Stigter 1980a,b). Even the rate of cuticular transpiration may eventually be higher than the rate of water uptake, leading to a further drop in water potential. The water potential \jIw of a cell comprises several components:

where \jilt is the osmotic potential, \jIp the pressure potential (turgor), and \jim the matrix potential. These values are negative, except \jIp' It is usually assumed that the matrix potential is constant in the range of positive to zero pressure potential. The above equation can then also be written

where p is the hydrostatic (turgor) pressure, which can be rewritten as

where s is the solute content in osmoles, R the gas constant, T the absolute temperature, and V the volume. When the rate of water uptake remains lower than the rate of transpiration' the flowers, the leaves, or both, may show turgor loss. The rate of change of the turgor p with a change in water potential depends on the elasticity of the cell walls (expressed as modulus of elasticity e) and on the osmotic potential:

1.

WATER RELATIONS OF CUT FLOWERS

15

When water stress occurs gradually, some plants are able to increase the solute content per cell, a process called osmotic adjustment, thereby partially or completely preventing the drop of turgor. The molecules involved, probably in different ratios in the various cellular compartments, include inorganic and organic ions, soluble carbohydrates, and amino acids (Turner and Jones 1980; Hanson and Hitz 1982; Morgan 1984). In intact Gladiolus plants grown in the open in Israel the water content of the leaves decreased in the late morning, and the osmotic potential dropped in proportion to the amount of water lost, indicating no osmotic adjustment (Halevy 1960). When rose plants grown in a greenhouse were subjected to a gradual water stress, the leaves also showed no osmotic adjustment. The stress did increase the modulus of elasticity (Auge et a1. 1990). In other cut flowers the possible role of cell wall elasticity or osmotic adjustment in the maintenance of turgor has apparently not been assessed. The presence of a high concentration of solutes in the petals cells may delay loss of turgor when a water deficit develops. Aarts (1957a) found that the inclusion of sucrose in the vase solution of Dahlia flowers prevented early wilting, and the same was observed in gladioli, after a 24-h pulse with a sucrose solution (Halevey and Mayak 1974). In these experiments, the rate of transpiration was not affected. In Matthiola incana, stems placed in water showed early wilting of the lowermost, oldest flowers, whereas the young flowers stayed turgid. The osmotic potential of petals of old and young flowers were initially the same, but during vase life the youngest flowers maintained their osmotic potential, whereas the osmolarity in the older flowers dropped. The inclusion of 3% sucrose in the vase solution had no effect on the osmotic potential of the young flowers but prevented the drop in osmolarity in the old ones (Aarts 1957a). In some chrysanthemum cultivars the petals have a lower osmotic potential than the leaves. This may explain why leaves wilt at moderate water stress while the petals do not. Feeding with sucrose resulted in a sharp decrease in osmotic potential, more so in petals than in leaves (Halevy and Mayak 1974; Halevy 1976). Acock and Nichols (1979) showed that the inclusion of sucrose in the vase solution of carnation flowers increased the concentrations of glucose and fructose in the petals, as compared to controls, and Paulin (1980)

16

W. G. VAN DOORN

demonstrated similar accumulation of reducing sugars in petals of roses fed with sucrose. Free proline has been found to accumulate in a number of waterstressed tissues (Hanson and Hitz 1982) and in petals of cut roses placed in water (Schnabl and Ziegler 1974). Accumulation of free proline in the petals was delayed by inclusion of an antimicrobial agent in the vase solution (Schnabl and Ziegler 1974). However, feeding cut rose flowers with an aqueous proline solution, in which the proline molecule was negatively charged in order to increase its mobility in the xylem, did not delay wilting (Tonecki et a1. 1989). VI. VASCULAR OCCLUSION OF FLOWERS PLACED DIRECTLY IN WATER

Premature loss of turgor in many species of cut flowers has been found to be due to an occlusion in the water conducting system. Flowers of Zantedeschia aethiopica and Z. elliotiana, for example, decreased in fresh weight early in vase life, and occlusions were observed in the xylem of the scapes (Tjia and Funnell 1986). In most flowers recutting of the basal part of the stem under water restores the rate of water uptake. It was concluded, therefore, that the main blockage was present at the lower end of the stems, that is, on the cut surface, and/or inside the xylem elements (de Vries 1873; Aarts 1957a). In several cut flowers the rate of water uptake decreases considerably even after stomatal closure. The blockage, therefore, apparently involves a large number of xylem conduits. Experiments with saw cuts in trees have shown that the architecture of the xylem allows the water to flow around areas that have been blocked, since all conduits have lateral connections (Zimmermann 1983). In cut roses no reduction in the rate of water uptake was found after using a razor blade to block as much as two-thirds of the stem cross-sectional area. When two-thirds of the transverse stem area was obstructed, the rate of water flow in the few vessels that remained unblocked had increased (van Doorn et a1. 1989). These results indicate that the reduction of water uptake, at least in cut rose flowers, must be the result of occlusion in a large majority of xylem conduits. The occlusion in stems placed in water directly after harvest may relate to a reaction from the stem. Several authors suggest that the occlusion is part of a wound reaction, a defense mechanism (Aarts 1957a; Fujino and Reid 1983; Marousky 1969, 1971a; VanderMolen et a1. 1983). A reaction to cutting could lead to the deposition of

1.

WATER RELATIONS OF CUT FLOWERS

17

material in the lumen of the xylem conduits, for example, suberin, lignin, tannin, or various gums. It could also result in exudation, at the cut surface, of substances such as latex, mucilage, or resin, which may partially enter the xylem conduits. Similarly, cutting may lead to the formation of tyloses in the conduit lumen. The occlusion may also relate to microbial growth (Aarts 1957a) or may be due to air bubbles. The latter occur as a result of air that is aspired directly after cutting or is due to cavitation in the water-conducting elements (Dixon et al. 1988; Dixon and Peterson 1989; de Stigter and Broekhuysen 1989). A. Deposition of Lignin, Suberin, and Tannin Cutting gives rise to a complex wound reaction that involves ethylene synthesis and/or the synthesis and activation of peroxidases and phenylalanineammonia lyase, enzymes involved in the biosynthesis of lignin and other substances that are deposited in the cell walls (Yang and Pratt 1978) or in the vessel lumen (Rhodes and Wooltorton 1978; Cline and Neely 1983). Durkin (1967) suggested that some active process might lead to accumulation of compounds such as lignins and tannins in the xylem of cut rose flowers. Others, however, found little staining with ferric chloride or with phloroglucinol-HCI, thus excluding the presence of either lignins or tannins (Burdett 1970; Gilman and Steponkus 1972; Parups and Molnar 1972). The hypothesis that phenol polymerization is involved in the vascular blockage of cut roses was investigated using vase solutions of pH 4 to 8, because the activity of the polyphenoloxidase, a key enzyme in phenol polymerization, is zero at pH 4 (Vamos-Vigiazo 1981). After 7 days of vase life vascular occlusion was independent of pH, which is evidence against the involvement of phenol polymerisation in the development of the stem occlusion (van Doorn et al. 1989). B. Deposition of Gum in Conduits by Xylem Cells In several plants the xylem conduits become blocked by gums that are deposited into their lumen. The presence of gums has been investigated in numerous Australian plants by Chattaway (1948), who concluded that their occurrence was generally related to the family level. Plugging of the xylem with gums was found, for example, in the Asteraceae, Malvaceae, Mimosaceae, Proteaceae, and Rutaceae. Gums are deposited by the ray cells of the vascular bundle, and generally not by the parenchyma cells that border the conduits. Ray cells exude gum material through pores in the pit membranes between

18

W. G. VAN DOORN

vessels and ray cells (Chattaway 1948). The gums found in xylem vessels are polysaccharides commonly based on glucuronic acid with associated hexose and pentose sugars, such as galactose, mannose, arabinose, xylose, and rhamnose (Brown et al. 1948; Hough and Pridham 1959; Jones 1939, 1950). Some of the gums consist of pectic arabinogalactans (Smith and Montgomery 1959). The role of gum deposition in the xylem conduits in the development of water stress has been studied in Acacia, Alnus, Amelanchier, Dahlia, Prunus, and Rosa. 1. Acacia. Many plants of the Mimosaceae (for example, Acacia mollissima) , secrete gum into the xylem lumen (Chattaway 1948). Cut flowering branches of Mirandole mimosa (A. dealbata), imported into Holland from southern France, showed almost no water uptake during vase life and desiccated within a few days. When the stems were placed in a preservative solution specially developed for mimosa (whose composition was not revealed), water uptake was much improved, flower buds opened and developed normally, and branches had a good vase life (Bakker and Stephan 1964a,b). Similar results were obtained with Le Gaulois, Super Mirandole, Quatre and Tournaire mimosa, and with a related species, A. moutteana (Sytsema 1968a). Gum deposition in the xylem lumen of Acacia species may be a reason for their precocious wilting, but a relationship between wilting and gum formation has apparently not been reported.

2. Alnus Glutinosa, Amelanchier Spicata,

and Dahlia Variabilis. When bacterial growth in the water was completely suppressed by a mixture of antimicrobial compounds and no bacteria could be isolated from stem segments, an occlusion was sometimes still detected in stems of Alnus glutinosa, Amelanchier spicata, and Dahlia variabilis (Aarts 1957a). Light microscopy revealed gum-like material in the vessel lumen. The plugs were initially almost colorless, then turned brown. In stems placed in the antimicrobial solution, the hydraulic conductivity of the basal segment decreased little, but a steady decrease in hydraulic conductivity was found in the 8to 16-cm and 16- to 24-cm segments until it was very low by day 7. Stems of A. spicata that were placed in water without the antimicrobial compound showed low hydraulic conductivity in the basal 8cm stem segment within 2 days of vase life. The blockage due to the presence of gums, therefore, was normally not the cause of early water stress, but when microbial growth was excluded gums did limit flower life to about a week (Aarts 1957a).

1.

WATER RELATIONS OF CUT FLOWERS

19

3. Prunus. Gums were observed in the xylem vessels in cut shoots of peach, P. persica (Davies et al. 1981; Munoz et al. 1982); cherry, P. avium (Stosser 1978a,b); and sour cherry, P. cerasus (Olien and Bukovac 1982a,b). Hydraulic conductance of the branches was inversely correlated with their gum content and the number of plugged xylem conduits. In some of these experiments, however, microbial growth as a partial cause of plug formation cannot be excluded, because sucrose was included in the vase water. Because gum formation in both cherry species was stimulated by ethephon, a molecule that releases ethylene, it was concluded that gum formation is regulated by ethylene (Munoz et al. 1982). Ethanol included in the vase solution at 1 % reduced the number of plugged vessels and increased the time to flower wilting. At such a low concentration ethanol is probably not an antibacterial agent, but does inhibit both ethylene synthesis and ethylene action (Heins 1980; Wu et al. 1992). Ethanol also considerably reduces the surface tension of water, which can then bypass the blockage (see Section VILF). The 3 day vase life of P. triloba (Bakker and Stephan 1964b) may be related to deposition of gums in the xylem lumina. Gum deposition, in contrast, if it occurs, is apparently not a problem in several other Prunus species. Branches of P. serrulata had a vase life of 9 days (Bakker and Stephan 1964b), while P. lusitanica and P. laurocerasus remained unwilted for 15 and 16 days, respectively (Moll 1880).

4. Rosa. In cut rose flowers, amorphous plugs-plugs that do not contain bacteria-were found in the xylem conduits and were considered to be due to a response of the rose stem to cutting (Parups and Molnar 1972). The material was supposed to be a gum, and chemical analysis revealed that it mainly consisted of polysaccharides, with monomers such as arabinose, rhamnose, mannose, galactose, mannose, and galacturonic acid (Lineberger and Steponkus 1976), lipids, and proteins (Parups and Molnar 1972; Dixon and Peterson 1989). The vascular plugs stained with ruthenium red (Burdett 1970; Parups and Molnar 1972). Antibacterial compounds have been found to prolong vase life and to prevent development of the plugs (Burdett 1970). Bacterial slime also mainly consists of polysaccharides (Sutherland 1977; Wilkinson 1977), and slime from several bacterial strains isolated from the stems of cut rose flowers also stained with ruthenium red. Scanning electron microscopy indicated that the structure of the amorphous plug material in xylem vessels was like that of the slime of isolated bacterial colonies growing on agar (van Doorn et al.

20

W. G. VAN DOORN

1990b, 1991e). Lipids and proteins have also been detected in the slime excreted by bacteria (Pier et a1. 1978). It may be tentatively concluded, therefore, that the amorphous plugs found in cut rose stems are a fraction of the material excreted by bacteria and remnants of dead bacteria, small enough to pass the small pores in the pit membranes. Since the bacteria cannot pass these pores, some xylem conduits are found with amorphous material only. The amorphous vascular plugs were mainly found at about 15 to 30 cm from the cut surface (Burdett 1970; Lineberger and Steponkus 1976). With the exception of Burdett (1970), no author has suggested that the plugs found higher up the stem are related to low hydraulic conductance. The role, if any, of these plugs for the blockage of rose stems is unclear, since other authors have found lowest hydraulic conductance in the basal stem segment (de Stigter and Broekhuysen 1986a; Durkin and Kuc 1966; van Doorn et a1. 1989). The total number of conduits with amorphous plugs also seems too low to account for the blockage. Rasmussen and Carpenter (1974) reported up to 4% of the conduits in transverse stem sections, Burdett (1970) detected plugs in 3-11 %, Lineberger and Steponkus (1976) a maximum of 20%, and Dixon and Peterson (1989) in 8-23%. Only a few conduits contained amorphous plugs in Sonia roses (van Doorn et a1. 1989).

C. Exudation of Latex and Other Substances at the Cut Surface Many plant species show exudation at the cut surface when a cut is made. For example, Tradescantia warscewiczii and Abutilon malvaeflorum exude gum, and Ficus asperata exudes latex. The stems of these species, when cut and placed in water, often take up little or no water (Moll 1880; de Vries 1881). The apparent main function of exuded latex, gum, and resin is to protect the plant by sealing lesions (Sperlich 1939; Ledbetter and Porter 1970; Sen and Chawan 1972). Mucilage, gum, latex, or resin are generally present in specialized cells or in ducts lined with secretory cells; a good anatomical description of these secretory structures is given by Fahn (1974) and Mauseth (1988). The secreted substances can hardly be separated in groups, since many intermediary forms exist. Exuded gum, for example, may be difficult to distinguish from mucilage. Mucilage, an aqueous mixture of polysaccharides, is exuded at the cut surface of plants from many families, including Cactaceae, Lauraceae, Malvaceae, Sterculiaceae, and Tiliaceae, and also in such

1.

WATER RELATIONS OF CUT FLOWERS

21

genera as Aloe, Althaea, and Ulmus (de Bary 1877; Metcalfe and Chalk 1983). Gums are also found in several families, including Aroideae, Convulvulaceae, Magnifoliaceae, and Musaceae. In some of these families the gum contains resin particles (de Bary 1877). Resins mainly consist ofterpenes mixed with volatile oils, which give them fluidity. When exposed to air the oils evaporate and the substance becomes hard. Plants that exude resins are found in several Gymnosperm families (including Araucariaceae, Cupressacea, Pinaceae, Taxaceae, and Taxodiaceae) and in some Angiosperm families, such as Rosaceae and Anacardiaceae (de Bary 1877; Bordeau and Schopmeyer 1958; Kisser 1958). Some Gymnosperm parts, however, do not become blocked rapidly after being cut and placed in water. Cut stems of Taxus baccata and Pinus abies, for example, placed in water shortly after harvest stayed fresh for about 14 days (Moll 1880). Latex contains particles that give it color. Latex-exuding plants are estimated to include more than 12,000 species in about 900 genera (van Die 1955). The main families include the Apocynaceae (e.g., Nerium oleander, Vinca spp.), Asdepiadaceae (Asclepias), Asteraceae (Cichorium, Lactuca, Taraxacum), Caricaceae (Carica papaya), Euphorbiaceae (Euphorbia, Hevea), Liliaceae (Allium), Moraceae (Ficus), and Papaveraceae. Latex is also found in some Campanulaceae, Convulvulaceae (e.g., Ipomoea), Cucurbitaceae, and Papilionaceae (de Bary 1877; Haberlandt 1896; Mauseth 1988). Latex consists of high molecular polyterpenes that are deposited in the vacuole (Esau 1965; Sheldrake 1969; Ledbetter and Porter 1970; Mauseth 1988). Among various plant species the latex is highly variable in composition: It may contain high concentrations of rubber, resins, mucilage, proteins, tannins, starch, sugars, and alkaloids (Archer et al. 1969; Dickenson 1969; Homans et al. 1948; Mahlberg 1973). Latex hardens when exposed to air, and hardens even faster when the cut end is placed in water (de Bary 1877), hence vascular blockage may rapidly ensue. Moll (1880) and de Vries (1881) noted that cut stems of latex-bearing plants are often unsuitable for experimentation, since water uptake is low or absent. An interaction may exist between secretion of material into the lumina of the xylem conduits and exudation of the same material from the cut surface. Anastomosing laticifers have branches adjacent to xylem vessels (de Vries 1881), and the nonanastomosing laticifers are located just outside the vascular bundle (Haberlandt 1896; Mauseth 1988). In conifers the resin ducts are generally present in the xylem rays, that is, they are also adjacent to xylem vessels (de Vries 1881). This means that upon cutting, the material flowing out

22

W. G. VAN DOORN

at the cut surface is close to the conduits opened by cutting and can, therefore, both flow into them and cover them. Four genera of cut flowers, in which exudation may be important, are discussed: Euphorbia, which exudes latex, Heliconia and daffodil (Narcissus), which both exude mucilage, and Prunus, which exudes gum.

Euphorbia. Branches of poinsettia (Euphorbia pulcherrima) placed in water exude latex, which inhibits water uptake and results in premature wilting of the leaves and bracts. Exudation of latex can be stopped by dipping the cut stern ends in hot water (70°C) for at least 1 min or at higher temperatures for a shorter period; in boiling water a I-s dip was adequate. The sterns are then recut in air through the treated stern segment (Sytsema 1967, 1968b). The treatment apparently results in coagulation of the latex within the laticifers, preventing its subsequent flow. The vase life of E. pulcherrima Cardinalis was 4.8 days when this hot-water treatment was not given, and 11.5 days after the treatment (Sytsema 1967). In Euphorbia fulgens a similar hot-water treatment resulted in a small increase in vase life, though not in all tests (Sytsema 1969, 1976; Hermann 1975). Farina and Paterniani (1984) found that the best treatment was placing the sterns in a heated preservative solution (at 95°C for 20 s) followed by holding in the preservative solution for a day, before transferring the sterns to water. The preservative solution contained 40 giL sucrose, 100 mglL silver nitrate, and 200 mglL 8-HQS. Hot-water dipping, however, generally results in death of the treated part of the stern and this often leads to excessive microbial growth later in vase life. Staden and Slootman (1976) observed that placing the sterns of Euphorbia fulgens in citric acid at pH 2.8 for 1 h prevented the flow of latex and increased vase life without stern damage. 1.

2. Heliconia. Cut flowering sterns of Heliconia spp. often show leaf inrolling and early fading of the colored bracts (Tjia and Sheehan 1984; Tjia 1985; Criley and Broschat 1992). In H. psittacorum, early leaf wilting and bract fading is especially notable after a period of dry storage. After harvest and direct placement in water, the sterns take up little water (Broschat and Donselman 1983a,b; Donselman and Broschat 1987; Ka-Ipo et al. 1989). In a test with freshly picked flowers that were transported dry to the laboratory within an hour, the initial rate of water uptake was high but rates dropped to low values within an hour and stayed very low for several days (Reid, Paull, and van Doorn, unpublished). Placing the flowers in standard

1.

WATER RELATIONS OF CUT FLOWERS

23

sugar-containing preservative solutions did not prolong vase life (Broschat and Donselman 1983a,b), but treatments with antitranspirants resulted in a small improvement (Ka-Ipo et al. 1989). De Bary (1877) and de Vries (1881) mentioned gum exudation from the cut surface of stems of several species in the Musaceae family, in which the genus Heliconia is usually classified (Stiles 1979), although it is sometimes considered to be a separate family (Kress 1990). Exudation of slimy material was observed at the cut surface of Heliconia stems. Unlike in narcissus, the inclusion of antibacterial compounds, such as HQC, silver nitrate, or sodium hypochlorite, in the vase water did not increase water uptake or vase life (Reid and van Doorn, unpublished).

3. Narcissus. When daffodil flowers are cut, an aqueous substance is usually exuded from the cut surface. This substance is slightly viscous and therefore called narcissus mucilage. Because narcissi often negatively affect the length of vase life of other flowers in the same vase, it has been suggested that the mucilage results in blockage of water uptake and would especially affect flower s-pecies that are sensitive to vascular occlusion (Carow 1981). Placement of one Carlton narcissus flower in 200 mL of water with one Sonia rose resulted in precocious wilting of the flowers and the leaves of the roses, in failure of flower opening, and in bent neck. No negative effect was observed on the narcissi themselves. Measurements of hydraulic conductance of the rose stems showed that by day 2 of vase life, the stem ends were completely occluded. Bacterial counts of the xylem and the cut surface of the basal 5 cm of the stems showed high numbers by day 2 of vase life. Inclusion of 300 mg/L of HQC in the vase water at the onset of the experiment completely prevented the water stress symptoms in the rose flowers and prevented the increase in bacterial numbers in the basal end of the stems. HQC is an antibacterial compound, but also inhibits ethylene production, and could, therefore, act via a stem-related process. Treatment with the anti-ethylene compound silver thiosulphate (STS; at 2 mM for 3 h) at the onset of the experiments, had no effect on the occlusion in the rose stems induced by the presence of narcissi, and did not affect the number of bacteria in the stems. Inclusion of sodium hypochlorite in the water at 40, 60, or 80 mg/L (active ingredient) prevented the water stress symptoms just as much as inclusion of 300 mg/L HQC, and also prevented the increase in the number of bacteria. Sodium hypochlorite does not inhibit ethylene production. It was concluded that the mucilage by itself does not result in vascular occlusion in stems of cut rose flowers. The mucilage is a

24

W. G. VAN DOORN

rich source of nutrition for bacteria, which are the apparent main cause of the blockage induced by the slime. In tulips, however, the slime results in premature leaf yellowing, which is not due to vascular blockage, and was also observed when slime is placed on the leaf surface (van Doorn, unpublished). 4. Prunus. Gum formation in intact

Prunus species occurs just under the periderm, which results in its rupture, after which the material flows out on the plant surface. Gum production in Prunus is considered to be a common response to stress and wounding and to be mediated by ethylene (Olien and Bukovac 1982a). As outlined in Section IV,B, gum is also deposited in the xylem vessels of Prunus species. The relative role of gum exuded at the cut surface, which is then probably partially taken up by the xylem conduits, and gum deposited into the vessels by the ray cells is as yet unclear. D. Tyloses

Tyloses are outgrowths of cells that form a balloon-like structure in the lumina of the xylem conduits and may completely fill the conduit lumen (Zimmermann 1983). They may function as a means for blocking the entry of microorganisms, and are frequently found just under the cut surface of woody branches, after pruning (Haberlandt 1896). Tyloses generally originate from ray cells, occasionally from paratracheal parenchyma cells. Chattaway (1948) observed that tyloses were general in several families, for example, Magnoliaceae, Oleaceae, Scrophulariaceae, and Urticaceae, while in other families they were found in some genera only, for example Betulaceae, Ericaceae, Euphorbiaceae, Myrtaceae, Papilionaceae, and Proteaceae. Tylose formation in the petiole abscission zone in Phaseolus vulgaris was accelerated by low doses of ethylene and auxin (Scott et a1. 1964), and in the abscission zone of the petioles and stems of cotton (Gossypium hirsutum) it was stimulated by auxin but inhibited by ethylene (Bornman 1967; Bornman et a1. 1967). Although tyloses were found in Eucalyptus (Chattaway 1948), cut branches from several members of this genus have a relatively long vase life (Bazzocchi et a1. 1987; Mayne et a1. 1985; Gotz 1986; Vaughan 1988; Broschat and Donselman 1987), hence they apparently do not seriously inhibit water uptake. Other Eucalyptus species, however, have a short vase life due to wilting (Benny et a1. 1982; Vaughan 1988), which could be related to tylose formation. The presence of tyloses has been investigated in a few cut flowers such as Prunus spp, roses, and lilacs.

1.

WATER RELATIONS OF CUT FLOWERS

25

1. Prunus. Tyloses were found in the xylem of apricots (P armeniaca), and their formation was increased by 2-chloroethyl phosphonic acid (ethephon), a compound that releases ethylene. as well as by 2,4,5-trichlorophenoxyacetic acid, a synthetic auxin. Thus, tylose formation in apricot stern could be under control both of endogenous ethylene and of auxin (Bradley et a1. 1969). Prunus species show gum formation as a result of wounding, both at the cut surface and inside the xylem conduits (Section VI.B), so it may be that vascular blockage in this plant is due to both gum deposition and tylose formation. 2. Rosa. Absence of tyloses from cut flowering sterns of Forever Yours roses was reported by Parups and Molnar (1972). Lineberger and Steponkus (1976) were also unable to demonstrate tyloses in cut sterns of Red American Beauty roses, nor were these structures found in sterns of Sonia, Madelon, Cara Mia, and Frisco roses (van Doorn and Reid 1995).

3. Syringa. A blockage developed in cut flowering sterns of lilacs (Syringa vulgaris) placed in water. Lowest hydraulic conductance was found not at the stern base, but higher up the stern (SytsemaKalkman 1991). Several tyloses have been found in the xylem vessels, and hydraulic conductance of stern segments was inversely correlated with tylose development. Premature wilting and tylose formation was delayed by AVG (aminoethoxyvinylglycine), an antiethylene compound, suggesting that tylose formation in this species is regulated by ethylene (van Doorn et a1. 1991d). However, the number of vessels blocked by tyloses seemed much too small to account for the reduction in hydraulic conductance of the sterns. VanderMolen et a1. (1983) have shown that tylose formation is often accompanied by deposition of mucilage in the xylem lumen. The reduction of hydraulic conductance in lilac sterns may, therefore. be mainly due to blockage of the pit membrane pores, by material released in parallel with tylose formation. E. Microbial Growth

Arnold (1930) probably was the first to attribute low water uptake in cut flowers to blockage by bacteria and their degradation products. Although he did not test this hypothesis, later authors attempted to do so by including antimicrobial compounds or bacteria (alive, dead, or decomposed) in the vase water, or by correlating microorganism growth associated with the sterns with the development of the oc-

26

W. G. VAN DOORN

clusion. Aarts (19S7a) was the first to demonstrate that microorganism growth in the vase water resulted in low hydraulic conductance of the stems, especially in the basal stem segment. 1. Correlation Between Microbial Growth and Vascular Occlusion. The development of occlusions in the stems of cut rose flowers has been correlated with an increase in the number of bacteria in the stems. The main blockage developed in the lowermost stem segment, in which many more bacteria were found than in more distal segments. When vascular blockage developed, the bacterial population in the basal S-cm stem segments was about 10 6 colony forming units (cfu) per gram fresh weight. Whenever the number of bacteria exceeded this number, the blockage was found, be it after 3 days in pure water or after a longer period in the presence of an antimicrobial compound (van Doorn et al. 1989). High bacterial counts in stems were also correlated with vascular occlusion in the petioles of fronds from the fern Adiantum raddianum (van Doorn et al. 1991a). Whereas the numbers of bacteria in the stems are correlated with the blockage, such a correlation does not always exist between numbers in the stems and in the vase water. After a few days of vase life, no bacteria can sometimes be found in the vase water containing cut rose flowers, but a considerable number of bacteria, high enough to result in vascular occlusion, were present in the stems (van Doorn and Perik 1990). During vase life, the numbers of bacteria in the vase water of several cut flower species have been measured. Bacterial counts reach a maximum of about 10 7 cfu/mL of water after a few days of vase life in roses, carnations, tulips, and chrysanthemums. Such bacterial counts corresponded with symptoms of water deficit in roses and some chrysanthemum cultivars, but not in carnations and tulips (0. 1. Staden, unpublished). 2. Microscopical Evidence. Light microscopy of cut dahlia flowers

(Dahlia variabilis) that had been placed in water for some days showed a high number of bacteria at the cut surface (Aarts 19S7a). Ultrastructural investigations of cut roses placed in water also led to the conclusion that the region close to the cut surface contained bacteria; the presence of a population of bacteria at the cut surface and inside the water-conducting elements preceded the onset of measurable occlusion (Lineberger and Steponkus 1976; de Stigter and Broekhuysen 1986a; van Doorn et al. 1991c).

1.

WATER RELATIONS OF CUT FLOWERS

27

3. Role of Yeasts, Filamentous Fungi, and Bacteria. When the vase water is held at neutral pH there is usually no development of a yeast population in the vase solution, and only a few filamentous fungi are found. When, however, the pH is kept at a low level, for example, by using citric acid, bacterial growth is initially suppressed but a population of yeasts rapidly develops and many filamentous fungi are found (0. 1. Staden, unpublished). Both yeasts and filamentous fungi may lead to vascular blockage (Put and Clerkx 1988). Vase water held at pH higher than about 4 may contain a few fungi and yeasts, but no yeasts are observed at the cut surface or inside the xylem of rose stems held in such water. In vase water of pH 4-7, the role of yeasts in vascular occlusion is, therefore, apparently minimal. Within 3-4 days of vase life at such pH, a few fungal hyphae are found at the cut surface. Because the development of fungi occurs after the development of the occlusion, a role of fungi in vascular occlusion of cut roses is also apparently minimal. At pH 4-7 population of bacteria rapidly develops at the cut surface and inside the xylem conduits (van Doorn et al. 1991b). Fungi may have a role in the stem occlusion of some other flowers, since captan, a fungicide (with the active compound Ntrichloromethylthio-3 a,4, 7,7 a-tetrahydrophthalimide), improved water uptake (Aarts 1957a). The inclusion of another fungicide, Cladox (a commercial preparation based on 2,4-dinitrorhodane benzene), in a mixture of antimicrobial compounds also improved flower vase life (Aarts 1957a), and inclusion of thiabendazole (Apelbaum and Katchansky 1977) in a solution that already contained an antibacterial compound and a sugar, had a positive effect on Gypsophila flowers. However, the antibacterial properties of these fungicides in vase water remain to be evaluated. 4. Identification of Bacteria and Fungi. Ford et al. (1961) identi-

fied bacteria in containers at four flower wholesalers in Detroit, Michigan, as belonging to the genera Achromobacter, Alcaligenes, Bacillus, Escherichia, Flavobacterium, Micrococcus, and Pseudomonas. At rose growers, the water used for rehydration shortly after harvest and for storage, held at 4°C, also contained Pseudomonas as the main genus. When aluminum sulfate was routinely included (at 0.8 giL) in this water, another bacterium developed, apparently in monoculture, identified as Klebsiella ozonae (Y. de Witte, unpublished). Identification of bacteria also occurred in hospital vases, because of the risk of infection, especially by Pseudomonas aeruginosa. Vase

28

W. G. VAN DOORN

water at two Miami hospitals contained several Pseudomonas species (P. aeruginosa, P. alcaligenes, and P. cepacia), Aeromonas hydrophila, Acinetobacter spp., Escherichia coli, Flavobacterium spp., Klebsiella ozonae, Proteus mirabilis, and a number of unidentified species (Taplin and Mertz 1973). In a hospital in Memphis, Tennessee, flower vases containing different flowers (carnations, chrysanthemums, daisies, gardenias, gladioli, orchids, and roses) contained a similar bacterial flora. Pseudomonas aeruginosa was present in all vases, and the genera Enterobacter, Serratia, and Escherichia were found in most vases. Genera less frequently found included Acinetobacter, Aeromonas, Erwinia, Flavobacterium, Klebsiella, Proteus, and Staphylococcus (McClary and Layne 1977). The similarity of the flora in the vase water of the various flowers may relate to reuse of the vases. In laboratory investigations Dansereau and Vines (1975) found the genera Pseudomonas and Flavobacterium in the vase water of snapdragons, and Marousky (1976) identified an Erwinia species from chrysanthemum vase water. Put (1990) made an exhaustive survey of both the bacteria and the fungi in the vase water of rose, chrysanthemum, and gerbera flowers, and Nooh et al. (1986) identified the fungi in the vase water of Ruscus foliage. In the vase water of roses the predominant bacteria were Pseudomonads, while Enterobacter was a minor accompanying genus. In repeat experiments, several other genera were incidentally present and may be chance contaminants (de Witte and van Doorn 1988; van Doorn et al. 1991b). In vase water of carnations, about 50% of the bacteria were Pseudomonads, along with about 20% Acinetobacter and 20% Alcaligenes (van Doorn et al. 1994). Some results on the composition of the bacterial flora in vase water are summarized in Table 1.2 and those on the fungal flora are given in Table 1.3. 5. Effects of Antimicrobial Compounds. In many cut flowers the suppression of microbial growth in the vase solution results in a delay of wilting. Ratsek (1935) and Laurie (1936), for example, found that metallic copper resulted in a small (1.0 to 2.7 days) delay of wilting in a number of the flowers tested (Argyranthemum, Aster, Calendula, Dendrantbema, Clarkia, Godetia, Mattbiola, Narcissus, Nemesia, Salpiglossis, and Viola), a response they attributed to partial inhibition of microorganism growth. Most antimicrobial substances, when used at concentrations that adequately control microbe growth, are toxic to cut flowers. Antimi-

WATER RELATIONS OF CUT FLOWERS

1.

29

Table 1.2. Identification of bacteria from flower vase water, after several days of vase life, as related to the flower species.

Bacteria GRAM-NEGATIVE RODS Acinetobacter sp. Achromobacter sp. Alcaligenes sp. Citrobacter C.freundii C. freundii var. amalonaticus Enterobacter R ag~omernns R cloacae E. gergovinae Enterobacter sp. Flavobacterium sp. Pseudomonas P. aeruginosa P.c~acfu

P. P. P. P. P. P.

fluorescens maltophilia mendocina pikettii putida putrefaciens

P.~u~~

P. vesicularis

Pseudomonas sp. GRAM-POSITIVE RODS Bacillus B. cereus (lecithinase-) B. cereus (lecithinase+) B. circulans B. licheniformis B. mycoides B. polymyxa B. subtilis B. subtilis var. niger B. thiaminolyticus Corynebacteria GRAM-POSITIVE COCCI Streptococcus lactis group

Rose

+ +

Chrysanthemum

Gerbera

+

+ +

+ + + +

+ + + +

+

+ + + + + + + + + + + +

+ +

+

+ + +

+ + + + + +

+ +

+

+ +

+

+ +

+ +

+ + +

+ + +

+ +

+ +

+

Note. Results from McClary and Layne (1977), de Witte and van Doorn (1988), Put (1990), and van Doorn et al. (1991e). +, positive identification.

W. G. VAN DOORN

30

Table 1.3. Identification of filamentous fungi and a yeast in the vase water of some cut flowers after 3-12 days and Ruscus cut foliage after 30 days of vase life. Fungus

Rose

Chrysanthemum

Gerbera

Aspergillus niger A. terreus

Aureobasidium pullulans (yeast) Botrytis cinera Botrytis sp. Cladosporium herbicola. Fusarium solani F. oxysporum Mucor hiemalis M. racemosus Penicillium brevicompactum Penicillium sp. Rhizopus stolonifer Rhizopus sp. Trichoderma pseudokoningii Verticillium brevicompactum

+ +

Ruscus

+ +

+ + + +

+ +

+ + + +

+

+ + +

+

+

+ +

Note. Data on Ruscus foliage from Noah et a1. (1986) and on cut flowers from Put (1990) and O. L. Staden (unpublished). All identifications, except those on Ruscus, were made by the Central Bureau for Fungal Cultures in Baarn, Holland.

crobial compounds that delay wilting without being toxic to cut flowers include (1) salts of copper, zinc, cobalt, and nickel; (2) quinoline compounds, such as HQC or HQS, (3) chlorine compounds, such as sodium hypochlorite, slow-release chlorine chemicals, and chloramine-T; (4) quaternary ammonium compounds, such as benzalkone; and (5) chlorinated aromatic compounds, such as dichlorophen and chlorhexidine. Examples of flowers and cut greens in which these compounds positively affected the length of vase life are presented in Table 1.4. Treatment with an antimicrobial compound shortly after harvest is beneficial for several flower species. In the Netherlands, gerbera flowers must be pulse treated with a sodium hypochlorite solution prior to sale at the auctions, and most of the flowers that are grown outdoors in the summer are mandatorily treated by a chloramine-T solution (Table 1.5). Physan-20 increased the vase life of China asters (Callistephus chinensis) , but was not as effective as a lO-min silver nitrate dip, which increased vase life threefold. The effect of silver and Physan-

1.

WATER RELATIONS OF CUT FLOWERS

31

Table 1.4. Examples of flowers and cut greens in which antimicrobial compounds had a positive effect on the length of vase life. Compound METAL SALTS Aluminium sulfate

Species

Concentration (mg/L)

Forsythia 800 Lupinus hartwegii 100-300

Cobaltous acetate

Phalaenopsis Rosa hybrida Rosa hybrida

800 400-800 266

Cobaltous chloride

Rosa hybrida

260

Tagetes patula Adiantum raddianum (fern) Rosa hybrida

13-65 185-290

Cobaltous sulfate

Rosa hybrida

132-310

Nickel chloride

Phalaenopsis

1500 (10 min)

Nickel sulfate

Rosa hybrida

Silver acetate

Rosa hybrida

1548 (10-20 min) 10-100

Silver nitrate

Addiantum raddianum (fern)

Cobaltous nitrate

Antirrhinum majus Argyranthemum frutescens Calendula

275

12.5-25 1000 (20 min) 1000 (10-40 min) 25 1000 (10-40 min) 1000 (10 min)

Callistephus chinensis 30 (24 h) Dendranthema (chrysanthemum) 30 Dendrobium Gerbera jamesonii 20-30

30 (20-24 h, 12°C) 30 Leptospermum Polystichum (fern) 25

Reference A. Ruting, pers. comm. Mohan Ram and Ramanuja Rao 1977 A. Ruting, pers. comm. de Stigter 1980a Venkatarayappa et al. 1981 Venkatarayappa et al. 1981; Reddy 1988 Chandra et al. 1981 van Doorn et al. 1991g Murr et al. 1979; Reddy 1988 Venkatarayappa et al. 1981; Reddy 1988 Aharoni and Mayak 1977 Reddy et al. 1988 Ryan 1957; Scholes and Boodley 1964 van Doorn et al. 1991g Fujino et al. 1983 Awad et al. 1986 Byrne et al. 1979; Accati Garibaldi and Deambrogio 1988 Awad et al. 1986 Kofranek et al. 1978 Nichols 1973a, 1975 Ketsa and Boonrote 1990 Penningsfeld and Forchthammer 1966 Penningsfeld and Forchthammer 1966 Joyce at al. 1993 Carow 1978; 1981

W. G. VAN DOORN

32

Table 1.4. Continued. Compound

Zinc acetate

Species

Reference

Rosa hybrida

30-50

Zinnia

170-340 (30 min) 1000 (10-40 min) 1-100

Awad et al. 1986

250-500

van Doorn et al. 1991g

Gladiolus Gypsophila paniculata Rosa hybrida

450-600 250

Marousky 1968a,b Jones and Hill 1993

250

Ruhmohra adiantiformis (fern) Scilla campanulata Syringa vulgaris Anigozanthos Argyranthemum frutescens Dendranthema

800 (10 min)

Burdett 1970; van Doorn et al. 1990a Stamps and Nell 1983

Rosa hybrida

QUINOLINE COMPOUNDS HQC Adiantum raddianum (fern)

HQS

Concentration (mg/L)

250 400 Not reported 300

Scholes and Boodley 1964; van Doorn et al. 1990a

Ryan 1957

200

Jones and Hill 1993 van Doorn et al. 1991d Faragher 1989 Accati Garibaldi and Deambrogio 1988 Gay and Nichols 1977

Dendrobium Leptospermum

100 200

Ketsa and Boonrote 1990 Joyce at al. 1993

Rosa hybrida Syringa vulgaris

200 300

Burdett 1970 Sytsema-Kalkman 1991

10

Joyce at al. 1993

12 12 12 12

Jones Jones Jones Jones

(chrysanthemum)

CHLORINE COMPOUNDS BCDMH Eucalyptus (cut foliage)

Gerbera jamesonii Ulium parkmannii Rosa hybrida Scilla campanulata Chloramine-T Chlorine bleach (sodium hypochlorite)

Rosa hybrida 20-40 Adiantum rad10-20 dianum (fern) Gerbera jamesonii 7.5 40 (24 h)

Rosa hybrida

20-40

and and and and

Hill Hill Hill Hill

1993 1993 1993 1993

Ryan 1957 van Doorn et al. 1991g Carow 1981 J.N. van der Sprong, pers. comm. van Doorn et al. 1990a

1.

WATER RELATIONS OF CUT FLOWERS

33

Table 1.4. Continued.

Compound DDMH

DICA

Species

Antirrhinum majus Argyran th em um frutescens Gladiolus Gypsophila paniculata Rosa hybrida Antirrhinum majus Argyranthemum frutescens Aster Dianthus caryophyllus Gerbera jamesonii Gladiolus Gypsophila paniculata Ulium parkmannii Rosa hybrida Scilla campanulata Telopea speciosissima Thryptomene ca lycin a

Concentration (mg/L) 200-300

50 50-300 50 50 100-300

Dichlorophen Hexachlorophen

Marousky 1976 Accati Garibaldi and Deambrogio 1988 Marousky 1976 Marousky 1976

200

Marousky 1976 Kofranek et al. 1974; Marousky 1976 Kofranek et al. 1974

200 50

Kofranek et al. 1974 Marousky 1976

50

Barendse 1978; Jones and Hill 1993 Marousky 1976 Marousky 1976; Jones and Hill 1993 Jones and Hill 1993 Marousky 1976; van Doorn et al. 1990a Jones and Hill 1993

50 50 50 50 50 25 avail. chlorine 50

QUATERNARY AMMONIUM COMPOUNDS Physan Callistephus 200 chinensis CHLORINATED HYDROCARBONS Chlorhexidine Aster

Reference

100

Gerbera jamesonii 10-40 Aster 100

Faragher 1986 Jones et al. 1993

Kofranek et al. 1978

Smellie and Brincklow 1963 van Meeteren 1978 Smellie and Brincklow 1963

Note. The compounds were either included in the vase solution at the onset of vase life at about 20°C, or were given as a pulse treatment shortly after harvest, at various periods and temperatures, which are indicated in parentheses. Pulse treatments temperatures were about 20°C unless mentioned otherwise. In these tests no sugar was included in the vase solution. BCDMH = 1-bromo-3-chloro-5,5-dimethylhydantoin; DDMH = 1,3-dichloro-5,5-dimethylhydantoin; DICA = dichlorocyanuric acid = sodium dichloro-s-triazone trione (SDT); HQC = a-hydroxyquinoline citrate; HQS = a-hydroxyquinoline sulfate; Physan = mixture of dimethylbenzylammonium chlorides and dimethylethylbenzylammonium chlorides.

W. G. VAN DOORN

34

Table 1.5. Mandatory pulse treatment with chloramine-T, in produce sold at the flower auctions in Holland.

Achilles Aconitum Agapanthus Alchemilla Allium Amaranthus Ammi majus Anethum (dill) Argyranthemum frutescens (marguerite daisy) Aster [except novi-belgii group] Astrantia Calendula Celosia Centaurea Chelone Cirsium Crocosmia (montbretia) Cynara Dahlia Digitalis Doronicum Echinops Eremurus Erigeron Eryngium (lisianthus) Eustoma Godetia Gomphrena Helianthus

Helichrysum Helipterum Hesperus Hypericum Ixia Kniphofia Leonotis Liatris Lunaria Lysimachia Matricaria Matthiola (stocks) Molucella Myosotis Nigella Ornithogalum Paeonia Papaver Phlox Primula Rudbeckia Saponaria Sedum Limonium (statice) Trachelium Triteleia (brodiaea) Tritonia Trollius Veronica Zinnia

Note. Some common plant names are given in parentheses.

20 was correlated with the absence of stem plugging with microorganisms (Kofranek et al. 1978). Physan-20 was also used by Farnham et al. (1978a) to facilitate water uptake in Gypsophila paniculata, and it was similarly assumed that the antibacterial properties of Physan were the reason for its positive effect. Physan-20 consists of a mixture of quaternary ammonium salts that also have surfactant properties, which may explain part of their effect (see Section VIILD). The inclusion of the surfactant Agral-LN (active ingredient: alkylphenoxypolyethoxy ethanol with an average ethoxy chain length of 8.5) in the vase water increased the longevity of Vuylstekeara (van Mourik and van as 1991), Thryptomene calycina (Jones et al. 1993a) and Bouvardia (van Doorn et al. 1993a), when they are placed in water directly after harvest. Agral-LN has no antibacterial proper-

1.

WATER RELATIONS OF CUT FLOWERS

35

ties and even tends to increase the number of bacteria in vase water and stems (van Doorn et al. 1993a), hence its mode of action is apparently in bypassing the (bacterial) blockage and circumventing or repairing the emboli caused by air entry and cavitation, and possibly also improving water conduction in the unobstructed vessels (see Sections VLF and VILC). The bacterial count in the vase solution was generally reduced by the antimicrobial treatments mentioned in Table 1.4 (Scholes and Boodley 1964; Larsen and Cromarty 1967; Nichols 1968b; McClary and Layne 1977; Marousky 1976,1977), and the number of bacteria associated with the cut surface and the xylem interior of rose stems was also shown to be reduced by several of these compounds (van Doorn et al. 1990a). Some substances not shown in Table 1.4 are also reported to delay flower wilting but it remains unclear to what degree this effect is due to their antibacterial activity. Sodium benzoate at 100 mg/L, for example, somewhat inhibited bacterial growth in Gypsophila vase water (Marousky and Nanney 1972), but also inhibits ethylene production (Baker et al. 1977; Wang and Baker 1979), a more likely reason for its effect since Gypsophila petal wilting is regulated by ethylene (van Doorn and Reid 1995). Thiabendazole [2-(4-thiazolyl)1H-benzimidazole], is a fungicide that has also been included in pulse treatment formulations for flowers (Apelbaum and Katchansky 1977), although its effect on the fungal or bacterial count in unknown. After a 24-h pulse with 300 mg/L thiabendazole glycolate and 10% sucrose, the fresh weight of carnation flowers during the first 6 days of vase life was much higher than in controls. The number of open flowers on Gypsophila panicles was increased by a 72-h pulse with these mixtures (Apelbaum and Katchansky 1977). Antibiotics have generally not been found useful for cut flowers. Penicillin, streptomycin, terramycin, actinobolin, viridogrisein, and grisioviridin had no effect on the vase life of Indianapolis chrysanthemum, and penicillin, streptomycin, terramycin, neomycin, and hygromycin had no effect in Rockwood snapdragons. The compounds were not toxic to flowers (Wiggans and Payne 1963). Streptomycin was ineffective in controlling bacterial growth in flower vases (McClary and Layne 1977). On the other hand, in Gladiolus natalensis the inclusion of 25 mg/L streptomycin, together with sucrose and gibberellic acid, promoted sucrose uptake and resulted in a small increase in longevity (Ramanuja Roa and Mohan Ram 1982), which may partially be due to inhibition of bacterial growth. Acid solutions have also been found to facilitate water uptake (Weinstein and Laurencot 1963; Marousky 1969, 1971a; Durkin 1980).

36

W. G. VAN DOORN

When the acid is effectively buffered around pH 3 it results in inhibition of bacterial growth in rose stems (van Doorn and Perik 1990). Aarts (1957a) reported that inclusion of acids in the water will prevent bacterial growth, but addition of acids to a solution that was already preventing bacterial growth further increased the length of vase life of roses, Dahlia, and Can vallaria. The type of acids used was not critical; good results were obtained with citric, malic, malonic, oxalic, phosphoric, sulfuric, and tartaric acid, as long as the pH did not fall below 3. Citric acid also had a positive effect on the vase life of several other flowers (Fourten and Ducomet 1906; Arnold 1931; Durkin 1980). A solution of citric acid at pH 3-3.5 has been suggested for rehydration of cut flowers (Durkin 1979b, 1980; Sacalis 1993). Because of its acidity, this solution may initially inhibit bacterial growth, though when included in the vase water for 2 days or longer it usually results in a higher number of bacteria than in controls. The vase water of White Horim chrysanthemum flowers, for example, contained 33 bacteria/mL after 2 days at 20°C. Following the inclusion of 100 mg/L of citric acid (pH 3.1 at the onset of vase life), the number of bacteria in the water was 1.3 x 106 /mL on day 2. On days 4 and 6 of vase life the number of bacteria in the citric acid treatment was 100 times higher than in the control treatment (Staden et a1. 1980). The buds of several cut flowers (e.g., Gypsophila and Gladiolus) that are placed in water tend not to open without an exogenous source of carbohydrates. Sugars also delay the autocatalytic rise in ethylene production and the concomitant petal wilting in carnations. When sugar is given without an antimicrobial compound, the stem xylem rapidly becomes occluded by microorganisms, preventing further entry of water and the dissolved sugar (Larsen and Frolich 1969). An antimicrobial compound is therefore also added. Table 1.6 gives examples of the compounds used, the flowers and cut greens in which this had a positive effect on the length of vase life, and the concentrations of the antimicrobial compounds used in combination with the effective concentration of sugar. The toxicity to the flowers of several of the antimicrobial compounds tended to be reduced by the sugar (Marousky 1976). 6. Inclusion of Bacteria and Their Products in Vase Water. Adding vase-water bacteria to the vase solution of cut Sonia roses, at a final inoculum of 10 9 cfu/mL, resulted in immediate wilting and bending of the stem just under the flower head. A reduction of water uptake was found with inocula of 10 7-10 8 cfu/mL, but these concentrations resulted in only a small decrease of vase life (van Doorn et a1. 1986;

1.

WATER RELATIONS OF CUT FLOWERS

37

Table 1.6. Examples of flowers and cut greens in which antimicrobial compounds, combined with a sugar, had a positive effect on the length of vase life.

Compound

Concen- Cone. tration sucrose (mg/L) (w/w)

Species

METAL SALTS Aluminium Antirrhinum majus sulfate Aster Cobaltous chloride

Nickel chloride Silver nitrate

800 800 800 75

1.5% 1.5% 2% 3.4%

30-300

3%

Nichols 1968b

150

2%

30

4%

E. Accati-Garibaldi, pers. comm. Aarts 1957b

Rosa hybrida Dendranthema (chrysanthemum)

Copper nitrate Dianthus

caryophyllus Gerbera jamesonii Amelanchier canadensis Antirrhinum majus Antirrhinum majus Argyran th em um frutescens Calendula

Reference

30 4% 1000 5-10% (10-40 min) 25 1%

1000 5-10% (10-40 min) Convallaria majalis 30 7% Cosmos bipinnatus 30 7% Cyclamen 25 5% 5-6% Dahlia variabilis 30 3-4% Dendranthema 30 (chrysanthemum) 4% Dianthus caryophyllus 30 Dianthus plumarius 30 6% 4% Dendrobium 30 GLU Freesia 30 6% 3-6% 20-30 Gerbera jamesonii

Iberis sempervirens Iris germanica Lathyrus odoratus Malus purpurea Matthiola incana Muscari armenia cum Ribes sanguineum Rosa hybrida Scabiosa atropurpurea Scabiosa caucasica Strelitzia reginae

30 1-1.5% 5% 30 6-8% 30 4% 30 30 2% 30 6% 2.5% 30 30 4% 30 1.5-2% 30 4% 10% 50 (48-h)

Miigge 1983 Miigge 1983 0.1. Staden, unpublished Chandra et al. 1981; Pardha Saradhi 1989

Aarts 1957b Awad et al. 1986 Accati Garibaldi and Deambrogio 1988 Awad et al. 1986 Aarts 1957b Aarts 1957b Kohl 1972 Aarts 1957b Aarts 1957b Aarts 1957b Aarts 1957b Ketsa and Boonrote 1990 Aarts 1957b Steinitz 1982; AbdelKader and Rogers 1986 Aarts 1957b Aarts 1957b Aarts 1957b Aarts 1957b Aarts 1957b Aarts 1957b Aarts 1957b Aarts 1957b Aarts 1957b Aarts 1957b Halevy et al. 1978

W. G. VAN DOORN

38

Table 1.6. Continued.

Compound Silver nitrate (continued)

Species

Syringa vulgaris Tulipa stellata Zinnia

QUINOLINE COMPOUNDS HQC An thirrhin um

Concen- Cone. tration sucrose (mg/L) (w/w) 30 3% 30 4% 1000 5-10% (10-40 min)

Reference Aarts 1957b Aarts 1957b Awad et a1. 1986

200-400

1-2%

200 (chrysanthemum) 300-400 Dianthus

2%

Marousky 1971b

4%

Larsen and Frolich 1969; Nichols 1973b Woodson 1987

majus Den dran thema

caryophyllus Freesia

200 (24-48 h) Gladiolus 600 Gloriosa virescens 100 Gypsophila 200

paniculata Matthiola incana 300-400 Nephrolepis (fern) 300 Rosa hybrida 200 Ruscus hypoglossum 300

20% 4% 1.5% 2% 1-2% 22.5% 3% 22.5%

Larsen and Scholes 1966

Marousky 1968a,b Slootman 1981 Marousky and Nanney 1972 Larsen and Scholes 1967 Nooh et a1. 1986 Marousky 1969 Nooh et a1. 1986

(foliage)

Strelitzia reginae HQS

Anigozanthos Banksia Boronia Chamelaucium uncinatum Dendrobium Dendrobium Eremurus Gerbera jamesonii Gypsophila paniculata Nerine bowdenii

CHLORINE COMPOUNDS Chlorine bleach (sodium Banksia prionotes hypochlorite) Verticordia

250 10% (48 h) Not reported 2% Not reported 1% Not reported 2% 1-3% 200 200 225 400 (24-48 h) 200

8% 4% GLU 20% 3%

Halevy et a1. 1978 Faragher 1989 Faragher 1989 Faragher 1989 Joyce 1988 Ketsa 1989 Ketsa and Boonrote 1990 Carow 1981 Abdel-Kader and Rogers 1986 Downs et a1. 1988

200 (24 h) 200

15% 5%

Downs and Reihana 1987

100 50

2% 2%

Joyce et a1. 1993 Joyce et a1. 1993

1.

WATER RELATIONS OF CUT FLOWERS

39

Table 1.6. Continued.

Compound mCA

Species

Anigozanthos Antirrhinum majus Argyranthemum frutescens Aster

Concen- Cone. tration sucrose (w/w) (mg/L) 200 100-300

3% 2%

100

2%

50

2%

50-250

2-3%

50

2-4%

50

2%

Gypsophila paniculata 50 Matthiola incana 100 50 Rosa hybrida 500 Strelitzia reginae (48 h) Swainsonia formosa 100 50 Aster Dianthus caryophy11us 50 50 Gladiolus 50 Gerbera jamesonii

2% 2% 2% 10%

50 50

2% 2%

Dianthus caryophyllus Gladiolus Gerbera jamesonii

DDMH

Gypsophila paniculata Rosa hybrida

4% 2% 2% 4% 2%

QUATERNARY AMMONIUM COMPOUNDS 4% Vantoc AL Dianthus 50-100 caryophyllus 4% 25-50 Dianthus Vantoc CL caryophyllus 200 5-10% Dianthus Physan-20 (24-48 h) caryophyllus 200 5% Den dran thema (16 h) (chrysanthemum) 10% 200 Gypsophila paniculata 2-4% 200 Grevi11ea 100-200 2% Triteleia laxa 10% 500 Dianthus Benzalkone caryophyllus

Reference Faragher 1989 Kofranek et a1. 1974; Marousky 1976 Kofranek et a1. 1974 Kofranek et a1. 1974; Marousky 1976 Kofranek et a1. 1974; Marousky 1976 Kofranek et a1. 1974; Marousky 1976 E. Accati Garibaldi, pers. comm. Marousky 1976 Kofranek et a1. 1974 Marousky 1976 Halevy et a1. 1978 Barth 1990 Marousky 1976 Marousky 1976 Marousky 1976 E. Accati Garibaldi, pers. comm. Marousky 1976 Marousky 1976

Nichols 1978 Nichols 1978 Farnham et a1. 1978b Kofranek and Halevy 1981 Farnham et a1. 1978a Lacey 1983 Kofranek 1986 Casp et a1. 1980

W. G. VAN DOORN

40

Table 1.6. Continued.

Compound

Species

Concen- Cone. tration sucrose (mg/L) (w/w)

CHLORINATED HYDROCARBONS Dichlorophen Dianthus caryophyllus

10

4%

Reference Nichols 1968a, 1973b

Note. The treatment was at 20°C and continuous, unless otherwise indicated in parentheses. When a pulse treatment was given, both the antimicrobial compound and the sugar were given only during the pulse. The sugar is sucrose; in some experiments glucose (GLU) was used. BCDMH = 1-bromo-3-chloro-5,5dimethylhydantoin; DDMH = 1,3-dichloro-5,5-dimethylhydantoin; DICA = dichlorocyanuric acid sodium dichloro-s-triazone trione (SDT); HQC = 8-hydroxyquinoline citrate; HQS = 8-hydroxyquinoline sulfate; Physan = mixture of dimethylbenzylammonium chlorides and dimethylethylbenzylammonium chlorides; Vantoc AL = 10% mixture of alkyltrimethyl bromide; Vantoc CL 50% lauryldimethylbenzylammonium chloride.

de Witte and van Doorn 1988). In experiments with Royalty roses the length of vase life was not reduced by final inocula of up to 10 8 cfu/mL of bacteria in the vase water (Reid et al. 1990). Put and Jansen (1989) found a reduction of vase life of Sonia roses with bacterial inocula of only 106 cfu/mL. In these experiments the roses had been stored dry prior to use. The interaction between the effect of bacteria and dry storage on the water relations and the vase life of cut rose flowers is further discussed in Section VII. Inclusion of bacteria in the vase water of gerbera flowers resulted in premature bending of the scapes. The numbers of bacteria that induced a curvature of more than 90° were 10 6 and 10 8 cfu/mL in the cultivars Liesbeth and Mickey, respectively (van Doorn et al. 1994). The longevity of White Sim carnation flowers was also reduced when the vase water inoculum was about 10 8 cfu/mL or higher (van Doorn et al. 1991f, 1995). No effect of such bacterial inoculation was found on the vase life of Apeldoorn tulips and White Horim chrysanthemum (0. 1. Staden, unpublished). Zagory and Reid (1986) isolated numerous bacterial strains from carnation vase water, of which two inhibited water uptake at 10 5 cfu/mL, a considerably lower titer than that of the other strains. One of these strains (Bl) produced much more slime than the other strains isolated. In subsequent experiments with carnation flowers it was concluded that these two strains, if at all spontaneously present in the vase water, did not reach concentrations high enough to limit

1.

WATER RELATIONS OF CUT FLOWERS

41

vase life (van Doorn et a1. 1991f, 1995). In rose and gerbera the effects on water uptake and vase life were independent of the bacterial strain (de Witte and van Doorn 1988; van Doorn and de Witte 1994). The method of growing bacteria in vitro and then adding them in the vase water will introduce living and usually also dead bacteria. Only when colonies had been grown on Plate Count Agar for 24 h were all the bacteria alive. When incubated for 48 h, the number of living bacteria had not increased with respect to the 24-h incubation, but the total mass had considerably increased, so that about 80% of the total number were dead (van Doorn and de Witte 1991a). Dead bacteria also resulted in vascular blockage (de Witte and van Doorn 1992). Extracellular polysaccharides (EPS) produced by bacteria and fungi may also lead to blockage. Put and Klop (1990) included EPS from three bacterial species and two species of fungi in the vase water of cut rose flowers and found vascular occlusion, but the number of bacteria in the vase water or the stems was not determined. De Witte and van Doorn (1992) isolated the EPS from Pseudomonas cepacia and included this in a sterile vase solution and found rapid blockage in the basal end of the stems of cut roses. The number of bacteria associated with the xylem was also very high, indicating that bacteria introduced by the nonsterile stems were able to use the EPS as a source of energy, and that the blockage was at least partially due to these bacteria. When EPS was included in vase water, control of bacterial growth in the xylem was found to be difficult. Keeping the pH at a low level by either adding a phosphate-citrate buffer (pH 3.1) or including HQC in the vase solution (up to 500 mg/ L) was incapable of stopping bacterial growth, but the combination of these two treatments was effective. When bacterial growth was eliminated, vascular occlusion was still found, apparently due to the EPS added. Dextrans are the EPS from the bacterium Leuconostoc mesenteroides, and are commercially available in fractions of different molecular weight. Inclusion of the 750-kD dextran in vase water, together with 24 mM phosphate-citrate buffer at pH 3.1 and 350 mg/ L HQC to control bacterial growth, had little effect on hydraulic conductance of the stems of Sonia roses, whereas a dextran of 2000 kD, at a concentration of about 150 mg/L, decreased hydraulic conductance to low levels (R. M. J. Krutzen, unpublished). Similarly, de Stigter and Broekhuysen (1986b, and unpublished results) perfused rose stems with solutions of dextrans of different molecular weight.

42

W. G. VAN DOORN

Small molecules readily passed through the stems, but passage decreased with increasing molecular weight and became minimal, though detectable, at 2000 kD. Only blue dextran, which has a molecular weight somewhat over 2000 kD, failed to pass. This agreed with the rapid wilting observed when Sonia roses were placed in blue dextran solutions (de Stigter and Broekhuysen 1986b). 7. Mode of Action of Bacteria. Burdett (1970) suggested that bacteria partially degrade cell walls or pit membranes by cellulolytic or pectolytic activity. Fragments would then accumulate and result in an occlusion. The inner walls of the xylem conduits are cellulosic in nature and the pit membrane is a remnant of the primary wall in which the matrix material is hydrolyzed during differentiation, leaving a cellulose microfibrillar web (O'Brien and Thimann 1969; Butterfield and Meylan 1982). Mayak et al. (1974) found vascular blockage in cut roses placed in a solution of cellulase, but only when using a relatively high concentration (1 giL). When comparing the effect of cellulase (about 50 kD) with ovalbumin, an inert protein of 50 kD, both at 1 giL, low hydraulic conductance was found shortly after placing rose stems in the solutions (de Witte and van Doorn 1992), indicating that the pores in the pit membranes can be blocked by any globular protein with a molecular weight of 50 kD. Put and Rombouts (1989) found an occlusion after feeding rose stems with low concentrations of pectolytic enzymes, and reiterated the hypothesis of Burdett (1970) that these enzymes are involved in the blockage. This paper, however, can be criticized on several counts. First, from the above discussion it follows that no pectolytic material is available for bacterial degradation unless most of the secondary wall, consisting mainly of cellulose, is first degraded. Second, according to F. W. van Went (who holds the chair of plant ultrastructure at Wageningen), the loose material observed by Put and Rombouts (1989) was actually ice crystals, not wall fragments (personal communication). Third, Put and Rombouts noted vascular occlusion when placing rose stems in a solution of pectic enzymes, but they did not include a control for blockage by the protein itself. Fourth, bacteria that are able to degrade the walls, such as the cellulolytic bacteria from the bovine rumen, leave depressions in the walls of the xylem conduits (Akin 1976; Engels 1989). Although numerous bacteria are present in the xylem conduits of rose stems placed in water for some days, no depressions were present in the wall, indicating the absence of degradation (van Doorn et al. 1991e). Fifth, it was found that the bacteria isolated from vase water of cut rose

1.

WATER RELATIONS OF CUT FLOWERS

43

flowers did not show pectolytic activity (de Witte and van Doorn 1988; Put and Klop 1990). Burdett (1970) also suggested that bacteria may be responsible for the occlusion in stems of cut rose flowers by influencing the paratracheal parenchyma cells or the ray cells, which then would produce plugs in the xylem conduits. This has not been substantiated, since only a few amorphous plugs are present in cut rose stems (van Doorn et al. 1989). Because some of the bacteria isolated from vase water induced leakage in the beetroot test, Put and Klop (1990) hypothesized that the wilting symptoms of cut rose flowers placed in water are due not to an occlusion in the stems, but rather to a direct effect on the plasma membrane. This, however, is unlikely. First, the observed symptoms of wilting in cut roses are always correlated with low hydraulic conductance in the stem. Second, recutting of the basal stem segment of rose flowers under water results in recovery of the wilting symptoms and this procedure can be repeated, showing that the plasma membrane of the petal cells remains fully functional. Several lines of evidence seem to support the idea that the effect of bacteria is purely physical. Hydraulic conductivity of rose stems placed in either 5 x 10 9 or 2 x 107 cfu/mL of living or dead bacteria declined rapidly, just as rapidly at 1°C as at 20°C (de Witte and van Doorn 1992). This indicates that the occlusion by bacteria does not depend on physiological activity of the bacteria (or the plant). A suspension of lysed bacteria placed in the water of cut roses also rapidly resulted in low hydraulic conductance of the basal 5-cm stem segment. As discussed above, bacterial extracellular polysaccharides and globular proteins also result in vascular blockage. Thus, living and dead bacteria, bound together by their EPS, bacterial EPS itself, and the degradation products from bacteria, all apparently block the pores in the pit membranes in a physical manner. After a few days in the vase, a thick layer of material, consisting of bacteria with their EPS is often found to cover the xylem at the cut surface, which may also impede water entry. F. Cavitation

The theory of water cohesion to explain the ascent of sap in plants implies the existence of negative pressures in the xylem (Dixon 1914). Negative pressures in cells were measured by early workers to be up to about -20 MPa (Renner 1911). A low pressure may cause the conduits to cavitate, after which the lumen of a conduit becomes

44

W. G. VAN DOORN

nonfunctional for water conduction. In intact plants, cavitation may occur spontaneously: The water column is thought to break because of a nucleus that suddenly gives rise to water vapor. The vapor will immediately fill the lumen of the conduit, but will subsequently be diluted by air diffusing into the lumen from the aqueous solution in the walls. Cavitation may also occur in xylem conduits that are adjacent to a conduit that already contains an embolus. Air in embolized conduits will be at atmospheric pressure, whereas adjacent conduits are filled with water, at subatmospheric pressure. The pressure difference at which the gas-water interface moves into the adjacent conduits depends on the pressure difference across the pit membranes and the diameter of the pores in these membranes (Zimmermann 1983). Although Stocking (1948) suggested that cavitation would not occur before the water potential reached -3.0 MPa, the measurement of cavitations by monitoring acoustic emissions at the plant surface reveals that they can occur at higher values, -0.5 MPa in some plant species, determined using the Scholander et al. (1965) pressure-chamber technique (Milburn and Johnson 1966; Tyree and Sperry 1989; Tyree and Ewers 1991). Early authors noted that the xylem vessels of intact plants are often devoid of water. Sachs (1887, p. 215-225) found that water uptake in woody branches was high even when most or all of the xylem conduits contained air; he hence questioned the role of air emboli for water uptake. Strasburger (1891, p. 678-688) resolved this by showing that most of the xylem elements produced during the current year of growth were filled with sap, even during the summer, when most of the older elements contained air. Embolization in the elements formed during the current year, he hypothesized, can become limiting for water transport. De Stigter and Broekhuysen (1989) placed Sonia roses in water and at intervals divided the stems into 2.5-cm segments, that were connected to air at a pressure of 0.01 kPa. In freshly cut stems the conduits were filled with xylem sap, and the pressure applied was insufficient to move this sap. After 1 or 2 days, the basal 2.5-cm segment started to allow passage of air. Later on, the 2.5-cm segments further up the stem progressively started to allow air passage, until even the peduncle became permeable. It was concluded that the stems become filled with gas, starting at the base and eventually affecting the whole stem. H. C. M. de Stigter (personal communication 1992) found that the increase in air conductivity in the basal

1.

WATER RELATIONS OF CUT FLOWERS

45

segment started after hydraulic conductance in this segment had become close to zero, apparently due to bacterial blockage. In most studies acoustic emissions, either in the auditory (Milburn and Johnson 1966; Milburn and MCLaughlin 1974) or ultrasonic (Tyree and Sperry 1989) ranges, measured at the plant surface, are used to detect cavitation. The work of Tyree and Sperry (1989) shows that hydraulic conductance in stems is inversely correlated with the number of cavitated xylem conduits. From their pioneering study with cut air-dehydrated Samantha roses, Dixon et al. (1988) concluded that cavitation occurred at water potentials that were high relative to that in other plants studied, and that hydraulic conductance had become reduced to 30% of initial conductance after only a few cavitations. A further drop in hydraulic conductance occurred concomitant with an increase in the rate of cavitation, starting at about -2.0 MPa. Using this experience, Dixon and Peterson (1989) showed the presence of cavitated xylem elements in Samantha roses that had not been stored dry but were transported to the laboratory packed on ice and placed in water after recutting the basal 5 cm under water. A qualitative method was used, based on staining with berberine hemisulfate, a fluorescent tracer. This dye stains intact xylem conduits, but does not enter cavitated ones. Plugs were observed in a few vessels at the basal stem end and it was concluded that these plugs may relate to the initial development of the occlusion. Cavitation, also occurring further up the stem, was thought to be a main cause of water stress in the flowers. Since only a few plugs were observed it was concluded that cavitation occurred after insignificant degrees of physical blockage at the cut end. Data on hydraulic conductance at the cut end, however, were not given. Pores in the pit membranes can become blocked by material of microbial origin, in the absence of visible plugs. Using ultrasonic acoustic emissions, numerous cavitations were detected at the surface of Thryptomene saxicola stems that were placed in water within a few seconds after harvest. These emissions were not found when the stem was recut under water, at least when removing the longest opened vessel. This indicates that the aspired air in the conduits opened by cutting was a prerequisite for cavitation in this species (van Doorn and Jones 1994). In Samantha roses Dixon and Peterson (1989) removed 5 cm under water, which contained about 95 % of the conduits opened by cutting. This technique will leave a few long, opened vessels, that have a relatively large diameter, which may explain why cavitation occurred. We also found ultrasonic acoustic emissions in cut roses that were placed in water

46

W. G. VAN DOORN

within minutes of harvest. In stems that were not recut under water, cavitation frequency depended on the cultivar, the light intensity, and the bacterial count in the vase water (unpublished). The difference between cultivars and species in their sensitivity to bacteria in the vase solution may relate to what happens after the bacterial blockage. Bacteria are found in all stems placed in water, but this may not lead to serious problems in the conduction of water unless it is followed by a large number of cavitations in the xylem. After a good number of cavitations, water uptake becomes reduced, the water potential drops further, and more conduits will cavitate, which further decreases water flow. From the above discussion it follows that cavitation may definitely playa role in the decrease in water uptake in flowers placed in water directly after harvest. Because cavitation apparently also plays an important role in the vascular blockage that develops during dry storage, details such as the relationship between cavitation and conduit morphology and the repair of cavitated conduits are further discussed in Section VII.C. VII. VASCULAR OCCLUSION IN DRY-STORED FLOWERS

Early workers noted that shoots of some plants, when cut in air and then placed in water, wilted within hours. For example, Sachs (quoted by de Vries 1873) found this with Tithonia tagetifolia, Nicotiana latissima, Cucurbita pepo, and Helianthus annuus. Strasburger (1891, p. 679-681) noted that shoots of Bryonia were still able to take up substantial amounts of water when left dry for 30 min, but absorbed little water when left dry for more than 60 min. Renner (1911) and Stocking (1948) observed that shoots of some plants that were cut in air, then allowed to wilt in air, and subsequently placed in water, rapidly regained turgor, whereas shoots of other species did not. Such differences also clearly exist in cut flowers: species that are sensitive to a period of storage include Astilbe, Bouvardia, Gypsophila, and many species grown in the open in Western Europe, such as Alchemilla, Celosia, Gaillardia, Godetia, Hesperis, Limonium, Molucella, Nigella, Ornithogalum, and Salvia (Carow 1981). Other species, such as Lilium, Tulipa, Iris, and Dianthus, can be stored dry for a relatively long period of time without a significant reduction in subsequent water uptake. In some species the effect is clearly dependent on the cultivar; in Dendranthema (chrysanthemum), for example, several cultivars can be stored dry without subsequent inhibition of water uptake, but other cultivars show a

1.

WATER RELATIONS OF CUT FLOWERS

47

low uptake rate and rapid leaf wilting. Among rose cultivars a wide range of sensitivity to dry storage has also been found. The relative sensitivity of cut flowers to dry storage, called DRYFAC, has been used in modeling the effect of dry storage on vase life (van Doorn and Tijskens 1991): Rd

=

f DRYFAC time

X

Presd

X

dtime

o

where Rd is the decrease of vase life (in days) and Presd is the vapor pressure deficit, that is, the difference between the vapor pressure around the product and that inside the product (which is close to saturated). Presd is dependent on relative humidity (RH) and temperature: Presd

=

100 - RH

RH

X

10[2.7857 + (7.5'

Temp /

237.3

+ Temp)]

where Temp is the temperature in ac. Values of DRYFAC were experimentally obtained and varied from 0.0017 for Carlton daffodil, which is relatively insensitive to dry storage, to 0.6670 for Sonia roses, which among roses show intermediate sensitivity. A decrease of vase life by previous dry storage may be due to low water uptake, but in some species also relates to other factors. In Iris, for example, flower opening is often inhibited after dry storage, a process that is apparently not due to low water uptake. Water uptake into stems that have been held dry can reportedly be facilitated by a number of treatments.

Recutting Under Water. When shoots of Helianthus tuberosus were cut in air and placed in water they soon wilted, but recutting a part of the stem under water resulted in recovery, and it was concluded that the short period between cutting and placement in water resulted in a blockage in the stem (de Vries 1873). Holle (1916) showed that recutting under water removed the blockage that developed during dry storage of Sinapis alba, and this has also been shown with numerous commercial cut flowers. Increase ofPressure. Shoots of Helianthus annuus rapidly wilt when cut in air and placed in water; but increasing the pressure above the water resulted in recovery (de Vries 1873).

48

W. G. VAN DOORN

Decrease of Pressure. Wilted stems that were placed in water rapidly recovered when the water was placed under subatmospheric pressure, which probably facilitates removal of air from the stems, and then returned to atmospheric pressure (Neger 1912; Hamner et al. 1945; Stocking 1948). Degassing of Water. Water that has been boiled, and thus made free of dissolved gas, and then cooled to ambient temperature can rapidly refill air-filled tracheids in Gymnosperm wood (Strasburger 1891, p. 717). Tests with cut roses also showed more rapid rehydration in water that had been degassed (Durkin 1979a). High Water Temperature. Wilting in flowers of Helianthus tuberosus was rapidly reversed when the flowers were held briefly in water of 35-40°C (de Vries 1873). Holle (1916) held cut flowering stems of Sinapis alba dry at 18°C for some hours, then placed them in water of 15-17°C or 35-40°C, without recutting the stems. Flowers placed in warm water regained turgidity more quickly. The use of warm or tepid water after recutting of the stems is often recommended to consumers. Sacalis (1993) advised the use of warm water for rehydration of many flowers, including Dahlia, Delphinium, Eustoma, Forsythia, Freesia, Gladiolus, Gypsophila, Hippeastrum (amaryllis), Lathyrus, Liatris, Lilium, Limonium, Matthiola, Narcissus, Nerine, Paeonia, Protea, Strelitzia, Syringa, and Tulipa. In Holland the use of warm water (about 50°C) is also advised for the rehydration of Phalaenopsis flowers. The mechanism of action of warm water is not understood. Temperature has little effect on surface tension (Neumann 1978), but viscosity decreases with temperature. Low Water Temperature. Cut roses placed in water at 2°C rehydrated much more rapidly than roses placed in water at 23°C (Durkin 1979b). Placing fronds of Pteris ferns in water at 4°C shortly after harvest was also beneficial (Carow 1981). The water uptake of fronds from the leatherleaf fern (Rumohra adiantiformis) was similarly improved by placing them in cold water (about 3°C [Stamps 1986]), and a similar effect was found in dry-stored chrysanthemum flowers (van Meeteren 1989). The mechanism of the effect of cold water is as yet unknown. Although water is more viscous at low temperatures (at O°C viscosity is twice as high as at 20°C), which could impede flow velocity, the solubility of gases in water is higher at lower temperatures. Freshly cooled water can, therefore, absorb some gas.

1.

WATER RELATIONS OF CUT FLOWERS

49

Decrease of Solution pH. Low pH has been shown to be favorable for rehydration of dry-stored roses and chrysanthemums (Durkin 1979a,b, 1980). Rehydration of flowers in retail premises with a citric acid solution at pH 3.5 is recommended by Sacalis (1993) for flowers such as Acacia, Alstroemeria, Antirrhinum, Argyranthemum frutescens (marguerite daisy), Bouvardia, Callistephus chinensis (china aster), Delphinium, Dendranthema (chrysanthemum), Eremurus, Freesia, Gladiolus, Gypsophila, Heliconia, Iris, Lilium, Paeonia, Protea, and Syringa, while for roses a pH of 3.0 is recommended. The acid treatment is usually advised to be combined with a warm water treatment. Low pH is known to increase the rate of flow in isolated 5-cm stem segments of rose flowers (Durkin 1979a). This may relate to the dissociation constant of carboxyl groups. Cellulose contains numerous carboxyl groups, which are the main reason why the xylem wall is negatively charged (Veen and van de Geijn 1978). In aqueous solution above pH 3 the carboxyl groups are dissociated and, therefore, negatively charged. Water is a partial dipole and forms a mantle around each of the carboxyl groups, and especially in the narrow pores in the pit membranes these mantles may impede water flow. At pH 3 the carboxyl groups become protonated and uncharged, and the water mantles are thus lost. Decrease of Surface Tension. Mertens (1944) held flowering stems of Hydrangea macrophylla dry until they wilted, then placed them in a vase with cut stems of lily-of-the-valley (Convallaria majalis), which exude saponins into the vase solution. The Hydrangea stems recovered much quicker than those placed in water without lily-ofthe-valley flowers. Although the saponins may have several effects, the foaming capacity of these compounds was explicitly mentioned and they probably improved water uptake through their surfactant action. The addition of surfactants to the vase solution is very effective in overcoming the occlusion that develops during dry storage. The use of Tween-20 in the vase water was reported by Durkin (1980) to overcome vascular blockage in dry-stored chrysanthemum flowers. Pulsing with Tween-20 or Tween-80 alleviated the blockage in drystored rose stems, but also resulted in severe leaf abscission (van Doorn et a1. 1993a). A pulse treatment of roses with a solution of Agral-LN prior to dry storage was effective in promoting water uptake after dry storage and had no toxic effect at the concentrations used to promote water uptake (Perik and van Doorn 1988). The flower auctions in Holland further tested the surfactant Agral-LN and after

50

W. G. VAN DOORN

finding that it improved vase life, its use was made mandatory for roses, Astilbe, and Bouvardia. The water-treatment authorities, however, have objected to the discharge of considerable amounts of this surfactant in the sewage, because of the relatively slow breakdown of the phenoxy ring by microorganisms. Linear alkylethoxy surfactants have subsequently been identified that are both not toxic to the flowers and easily biodegradable (Pak and van Doorn 1992; van Doorn et al. 1993a,b), and these have now replaced Agral-LN. A pulse treatment with Triton X-l00, a phenoxy type of surfactant, prior to dry storage also increased the length of vase life of roses, Bouvardia, and Astilbe (van Doorn et al. 1993a), as well as sunflowers (Heliantbus annuus) (Jones et al. 1993b). Similarly, pulse treatment of some chrysanthemum cultivars with quaternary surfactants, prior to dry storage, delayed leaf wilting from about 2 days (controls) to 7-10 days for treated flowers (D'hont and van der Sprong 1989). Experiments at the Aalsmeer flower auction also showed a positive effect of a pulse treatment with surfactants on the vase life of Aster novi-belgii, Cartbamus, Gentiana, Pbysostegia, Rudbeckia, and Solidago (K. D'hont, personal communication 1990). Pulse treatment with a surfactant solution has been made mandatory by the Dutch flower auctions for Aster (only the novi-belgii group of cultivars) Astilbe, Bouvardia, Cartbamus, Dendrantbema, Gentiana, Gypsopbila (combined with sugars), and Rosa; for Solidago a surfactant pulse is advised. The mechanism of action of surfactants on cut flowers is probably based on a decrease in surface tension (Myers 1991). In dry-stored stems the decrease in surface tension facilitates entry of water into the air-filled lumen of xylem conduits (van Doorn et al. 1993b). In practice, when surfactant solutions are used for several days, the bacterial population may increase. Agral-LN, for example, increased bacterial growth. To prevent excessive growth, aluminum sulfate at 0.8 giL was included in the solution. Chlorine compounds were ineffective in combination with this surfactant (K. D'hont, personal communication 1990). The literature indicates that the presence of air in the xylem conduits is at least partially a cause of the low water uptake after a period of dry storage. Air may be aspired, immediately after cutting, into the xylem conduits that are opened. Later, xylem conduits not opened by cutting may cavitate (suddenly fill with gas). Factors other than air bubbles may also contribute to the blockage. It has been suggested that tylose formation occurs after the xylem conduits become devoid of water (Klein 1923). Deposition of gums into the xylem conduits could also be a result of holding the stems dry

1.

WATER RELATIONS OF CUT FLOWERS

51

(Chattaway 1948). When stems have been placed in water prior to dry storage they have become contaminated with microorganisms. If the microbes are able to multiply inside the stems during dry storage, they may also be partially responsible for the occlusion (van Doorn and de Witte 1991b). A. Aspired Air: The Lumen Pathway and the Cell Wall Pathway for Water

Hales (1748, cited by Strasburger 1891, p. 689) showed that air was taken up at the cut end of apple branches, and Strasburger (1891, p. 688-690) found considerable air uptake after cutting branches of several other Angiosperm trees, such as oak, beech, and linden. As compared with these Angiosperm trees much less air was absorbed by branches of Gymnosperm wood. He found gas bubbles in the lumina of the conduits. These bubbles are named emboli (singular, embolus), from the Greek C:/-lP01to<;, or stopper, because they were thought to block the flow of water in the conduits. Scholander et a1. (1957) exposed liana stems (Tetracera sp.) to air for 15 min and found that 80 mL of air was aspired at the cut surface. When the stems were subsequently placed in water, the rate of uptake was as high as in controls, hence no blockage was apparent. Experiments with Sonia roses also showed that air was taken up after cutting. The highest rate of uptake occurred immediately after cutting and in leafy stems the initial rate of air uptake correlated with the rate of transpiration, indicating that air was aspired into the xylem conduits. When only the leaf closest to the cut surface was left attached to the stem, the amount of air taken up correlated with the size of that leaf and air uptake ceased only when the leaf had completely desiccated. When such leaves close to the cut surface were removed prior to the experiment and the leaf scar was covered with laboratory grease, only about 0.04 mL of air was taken up, which corresponded with the lumen of the conducting elements opened by cutting. In Sonia roses, the absorption of air was complete within half an hour. Because water uptake is clearly inhibited only after 24-36 h of exposure to air, the mere uptake of air into the xylem conduits opened by cutting, therefore, is not responsible for the occlusion upon extended exposure to air (van Doorn 1990). The uptake of water into stems that were exposed to air could involve two pathways. Normally, in stems unexposed to air, the bulk of water transport will be through the lumina of the conducting elements. However, when emboli are present in these elements, water transport may also occur in the xylem conduit walls, although the

52

W. G. VAN DOORN

resistance for water transport in the walls is much higher than that in water-filled lumina (Lauchli 1976). The walls of Sonia roses have been found to be a good conductor for water-soluble dye, indicating that they can be used as a pathway for long-distance water transport. Experiments with a girdled-stem system, in which a ring of bark was removed at the basal end of the stems and the cut surface was sealed with laboratory grease, showed that water uptake through a small wall area could maintain turgidity when stems were either not held dry or only briefly stored dry. When the stems were desiccated for a prolonged period, the uptake of water trough the walls was no longer adequate. These results could be interpreted to show that the blockage occurring during prolonged periods of air dehydration of cut Sonia stems is related to interruption of the water pathway in the xylem conduit walls. The effect of surfactants, however, was not consistent with this hypothesis. Surfactants included in the vase solution promoted water uptake into normal cut stems exposed to air, but had no effect on the dry-stored girdled stems with a grease-covered cut surface (van Doorn 1993). The results of experiments in which dry-stored Sonia roses were placed in a dilute suspension of India ink demonstrated that in stems that were exposed to air for a short period the suspension was still able to enter a relatively large number of xylem conduits opened by cutting, although not to the maximum length of the longest conduits. Prolonged exposure to air resulted in a decrease of both the number of conduits in which water was able to enter and in the height of water penetration. Adding surfactants to the vase solution promoted entry of water into the xylem conduits that were opened by cutting. These results show that, at least in Sonia roses, a restriction of the lumen pathway for water in the xylem elements opened by cutting may be a cause of the occlusion that develops upon exposure to air (van Doorn and Otma 1994). Dehydration of the walls may be the cause of the inability of water to enter these lumina, since a dry wall reduces the contact angle of wetting (Siau 1984). Following reduced penetration into the lumina of the opened conduits, the water column continuity is disrupted, since the water can no longer make contact with the conduits still filled with sap. A remarkable difference was found between rose cultivars in their sensitivity to air exposure. Among a number of cultivars, Cara Mia was the most sensitive, followed by Madelon, Sonia, and Frisco. In dry-stored Frisco roses the stomata often closed more rapidly than in Sonia, but the main difference was its low cuticular transpiration. The differences among Cara Mia, Madelon, and Sonia roses were

1.

WATER RELATIONS OF CUT FLOWERS

53

not related to their water loss or water potential. (van Doorn and Reid 1995). A difference among species or cultivars in the height of water entry in the xylem conduits opened by cutting could explain their differences in rehydration after dry storage. In Cara Mia and Sonia roses, however, no difference was observed in the height of water entry into the opened conduits as measured by the distribution of India ink after various periods of dry storage (van Doorn and Otma 1994). This indicates that the blockage in various rose cultivars cannot fully be explained by inhibition of water entry into the lumina of the xylem elements opened by cutting.

B. Cavitation The mechanisms by which water-conducting elements can become cavitated have been discussed in Section VI.F. As mentioned in that section, Dixon et al. (1988) studied the ultrasonic acoustic emissions (DAEs) in cut Samantha roses that were fully hydrated and then allowed to dehydrate in air. The flowers and leaves were removed in these measurements. A few DAEs were observed to start at a water potential of -0.2 to -0.4 MPa. About 70% of the hydraulic conductance had been lost when the rate of DAEs started to increase, at about -2.0 MPa. Hydraulic conductance is measured by passing water through stem segments, hence the loss of a few wide xylem vessels, given the Hagen-Poiseuille law that flow is proportional to the fourth power of the conduit radius, could account for its relatively large decrease. A 70% reduction in hydraulic conductance, however, is of little consequence for the rate of water uptake. The water potential of the flowering stems has become low, hence a high rate of water uptake will still be possible even when a relatively low number of xylem elements are still conducting water. In Sonia roses we showed that blocking about 66% of all vessels and tracheids (with a razor blade) did not impair water uptake. The rate of water flow in the remaining one-third of the vessels and tracheids, measured with the heat-conductance method, was considerably higher than before the insertion of the blade into the stem. Water uptake rates by these stems was the same, irrespective of blocking two-thirds of their water-conducting elements (van Doorn et al. 1989). Also, the experiments of Dixon's group may have been complicated by the presence of bacteria, since the stems had been placed in water before dry storage (see Section VILD).

54

W. G. VAN DOORN

In cut roses that were not placed in water prior to dry storage, and hence did not contain bacteria, a clear reduction of water uptake was found after a short or longer dehydration period, depending on the cultivar. In Cara Mia stems it was observed after about 3 h of dry storage at 20°C, in Madelon after 9-14 h, and in Sonia after 24-36 h. In all three cultivars the onset of cavitation occurred after about 30 to 60 min, and hence was not correlated with the development of inhibition of water uptake. In contrast, the onset of a high frequency of UAEs occurred prior to the development of the reduction in water uptake capacity. Cavitation may, therefore, be a cause of the vascular blockage that develops during dry storage (van Doorn and Suiro 1996), which corroborates the findings of Dixon et a1. (1988). A high percentage of vessels and tracheids must apparently become inoperative before the rate of water uptake becomes reduced. Differences between species and cultivars of cut flowers, with respect to the onset of cavitation, may relate to xylem anatomy. During dehydration wider xylem conduits tend to cavitate earlier, at less negative water potentials than narrower ones (Salleo and Lo Gullo 1986; 1989a,b; Lo Gullo and Salleo 1993). Neither these reports, nor recent reviews that discuss the effect of xylem conduit diameter on cavitation (Zimmermann 1983; Grace 1993) refer to Strasburger (1891, p. 678-679 and 682-688), who made detailed microscopical observations on embolization as related to xylem anatomy. In Gymnosperm wood, the small-diameter tracheids were more frequently filled with gas than tracheids that were wider. In Robinia branches the xylem fibers and the widest vessels were often embolized, leaving only the narrow vessels and the tracheids filled with sap; in Wisteria all conduits were filled with gas, except those formed the current year, but even among these a number of vessels, especially the widest ones, contained gas. Similar observations were made with Quercus branches. The size distribution of xylem vessels and tracheids has also been studied in Cara Mia, Madelon, and Sonia roses, which clearly differ in their sensitivity to cavitation and dry storage, but no clear differences were observed (van Doorn and Reid 1995). Refilling of cavitated elements may determine the rate of water uptake after a period of dry storage and may also partially account for the differences in recovery of dry-stored shoots. Disappearance of emboli is apparently due to dissolution of the gases in water (Milburn and McLaughlin 1974; Sperry et a1. 1987). Strasburger (1891, p. 700-710) investigated the refilling of cavitated tracheids in small blocks of pine wood and showed that it usually started at the tapered end of a conduit, from the direction where the water was sup-

1.

WATER RELATIONS OF CUT FLOWERS

55

plied. Refilling occurred faster in relatively wide tracheids grown early in the season. Water flow initially occurred between the wall of the tracheids and the air bubble in their lumen. Refilling was impeded when the wood was taken from the older parts of the tree, which were more impregnated with resin and suberin. Similar findings were obtained with dicotyledonous wood (e.g., Acacia, Castania, Fagus, Robinia, Quercus, and Salix). De Stigter and Broekhuysen (1989) devised a method (described in Section VLF) to quantify the presence of embolized conduits in stem segments. Preliminary observations with Sonia roses using this method showed that the cavitated conduits in dry-stored stems were slowly refilling when the stems were subsequently placed in water and also showed that surfactants aided in refilling (V. Suiro, unpublished).

c.

Deposition of Material and Tylose Formation

Water uptake in cut stems of grapevine was inhibited after prolonged exposure to air, and a layer of exuded material was observed on the cut surface, hence the blockage was suggested to be due to this material (Scholander et al. 1955). Klein (1923) found that tylose formation often occurred after a xylem vessel became air-filled. In drystored cut flowers these mechanisms of blockage may also occur, but little is known about their role. Dry storage of Sonia, Madelon, Cara Mia, and Frisco roses resulted in vascular occlusion, but did not result in any visible material in the conduit lumen (van Doorn and Reid 1995). No data seem available for other cut flowers. D. Microbial Growth When flowers are held in water prior to dry storage, the xylem interior and the cut surface of the stems always become contaminated with bacteria. During dry storage these bacteria multiply, even at a rate similar to that found in stems that remain in water (van Doorn and de Witte 1991b). This result was unexpected since bacteria are found only in the xylem conduits that have been opened by cutting and these will contain air when the stems are held dry. The result may be explained by the polysaccharide capsules which envelop most of the bacteria found in the xylem, and by the presence of extracellular (loose) bacterial polysaccharides. These materials have a high water-holding capacity and absorbed water may enable multiplication. When rose stems were held dry immediately after

56

W. G. VAN DOORN

cutting, no bacteria were present in the stem interior, but this exclusion of bacterial growth did not prevent blockage after dry storage. The introduction of a low number of bacteria in the xylem alone does not lead to low water uptake, nor does a short period of dry storage necessarily lead to a low uptake rate. When the two are combined, however, a threshold is apparently crossed and water uptake is very low (van Doorn and D'hont 1994). In the flower trade this combination of an introduction of bacteria directly after harvest and dry storage for some time prior to placement in the vase by the consumer almost always occurs. For cut roses this is apparently the reason why bent neck and lack of flower opening is commonplace. Recutting of the stems may remove most of the bacterial blockage but does not eliminate the emboli above the site of recutting. When stems are placed in water prior to dry storage, the bacteria adhering to the outside of the stem and the cut surface, and those that have entered the stem will add to the population that will develop in vase water. Even if the stems are recut after dry storage the remaining bacteria may still inoculate the vase water. VIII. EVALUATION OF THE CAUSES OF VASCULAR BLOCKAGE

As yet, only in roses is there enough evidence for a preliminary assessment of the contribution made to the inhibition of water uptake by various causes such as reactions of the stem, microorganisms, and emboli. In a few other flowers, such as Astilbe, Bouvardia, Dendranthema (chrysanthemum), Gerbera, Gypsophila, and Syringa, the available information is less conclusive. A. Astilbe and Bouvardia

Both Bouvardia and Astilbe flowers show wilting shortly after they are placed in the vase water, especially when the flowers have been stored dry. The period of dry storage (at 20°C and 60% RH) after which the rate of water uptake is too low to meet the transpirational demand is about 1 day in Bouvardia and less than a day in Astilbe (van Doorn et al. 1993a). A blockage to water uptake develops in the lowermost part of the stems in both species. The low rate of water uptake after dry storage can be overcome by placing the stems in a surfactant solution, or by pulse treatment with a surfactant solution prior to dry storage (van Doorn et al. 1993a,b).

1.

WATER RELATIONS OF CUT FLOWERS

57

B. Dendranthema (chrysanthemum)

Several cultivars of chrysanthemum show leaf wilting shortly after being placed in the vase solution, even when the stems are not stored dry prior to vase life. The flowers of these cultivars may last for several weeks, just as in the cultivars that do not show this typical leaf wilting, an effect that may relate to the lower osmotic potential in the petals (Halevy and Mayak 1974). Leaf wilting depends on the place where the stems have been cut at harvest. When the cut is made higher up the stems, the rate of water uptake is higher and symptoms of water stress are observed later, an effect attributed to the woody nature of the basal stem end. A lower rate of water uptake has been related to thicker and more lignified walls (Laurie et al. 1958), and could possibly relate to deposition of suberin or other water-repellent materials. In Florida, leaves on flowers harvested in April or May tended to wilt earlier and the stem tissue did appear more woody (Marousky 1973). The degree of stem "hardiness" was quantified using an Instron pressure device. Hardiness of stem segments taken from similar positions above the root-shoot junction, was, among chrysanthemum cultivars, inversely related to the rate of water uptake (Harkema 1980). Silver ions promoted water uptake and maintained leaf turgidity. Silver pretreatment also reduced the blockage developing in the stems during dry storage (Nichols 1973a), which may indicate that the blockage either relates to bacteria or to a process controlled by ethylene. The blockage is apparently not mediated by ethylene, since feeding with ACC had no effect (Singh and Moore 199Zb). Placing the stems of dry-stored chrysanthemums in hot water for a few minutes prior to vase life largely prevented premature leaf wilting. This hot-water treatment was also effective when it occurred prior to dry storage, though less so than when applied immediately before placing the flowers in vase water (Boer and Harkema 1979; Harkema 1979). Treatment with surfactants (Durkin 1980) or placement of the stems in cold water (van Meeteren 1989) similarly overcame the blockage. Nichols (l973a) found visible occlusions in the xylem vessels of chrysanthemum stems after more than 7 days of vase life, and Singh and Moore (199Za) observed such plugs after 3 days of vase life. Visible plugs in the xylem lumina thus did not appear to be responsible for the rapid fall in water conductivity of the stems that occurs shortly after cutting. Using berberine hemi-sulfate staining, the percentage of functional vessels was inversely correlated with the decrease in water conductivity (Singh and Moore 199Za), which also indicates

58

W. G. VAN DOORN

an occlus ion other than those related to the visible plugs. The loss of functio nal vessel s may be due to rapid cavita tion, but this has not yet been report ed. In additio n, Chatta way (1948) mentio ned that the condu it lumen in Astera ceae can becom e occlud ed with gums secreted by the ray cells, but a detaile d micros copica l invest igation of the blocka ge in chrysa nthem um stems is lackin g, hence no judgm ent can be made about gums, tyloses , or other materi al that may becom e deposi ted in the condu its. The exact role of microb ial growth has also not been evalua ted. In experi ments by van Meete ren (1989) the weigh t of freshly cut stems placed in water increa sed. When the stems were held dry on a labora tory bench for 1 h they lost about 5% of their origin al weigh t, and when subseq uently placed in water they increa sed in weigh t again, indica ting no seriou s inhibi tion of water uptake . When stems were sealed in plastic and held dry for 24 h they lost less than 2% of their weigh t, but when placed in water their fresh weigh t did not recove r, indica tive of a seriou s occlus ion. When air was drawn from the cut end the flower , fresh weigh t rapidl y increa sed. These results indica te that the presen ce of embol i in the condu its is a cause of the blocka ge develo ping during dry storage , althou gh it may not completely explai n the blocka ge. An additio nal reactio n appare ntly occurs when the stems are held in air of high humid ity for 24 h, as compa red with stems left on the labora tory bench for an hour. C. Gerbe ra

The scapes of cut Gerbera flower s often bend during vase life, which may relate both to a blocka ge of water uptake and to proper ties inherent in the scapes . Scape bendin g during vase life was allevia ted by the inclus ion of antimi crobia l compo unds such as silver nitrate and dichlo rophen in the vase solutio n (Penni ngsfel d and Forcht hamm er 1966; van Meete ren 1978). Silver may also act as an antieth ylene agent, but such action is not known for dichlo rophen . Inclus ion of bacter ia in the vase solutio n did result in scape bending, with the averag e scape angle reflect ing the numbe r of bacter ia in the vase water (van Doorn and de Witte 1994). Excess ive bacter ial growth , therefo re, may be one of the reason s of the bendin g. The xylem of Astera ceae can becom e occlud ed by gums (Chatt away 1948). Wheth er this also applie s to cut Gerbera flower s remain s to be established . Gerbera flower s are usuall y transp orted dry. After dry storag e the scapes are usuall y wilted and bent; when placed in water most cul-

1.

WATER RELATIONS OF CUT FLOWERS

59

tivars rapidly regain turgor, but others do not. The degree of permanent bending was clearly increased after a simulated dry transport (van Doorn et a1. 1994). Cultivars widely differ in their tendency to scape bending during vase life, which may be due to anatomical features. Marousky (1976) found that the scapes of Appleblossom gerberas were less vulnerable to bending than those of Tropic Gold gerberas. The former had more vascular material per unit circumference, which is consistent with the idea that more cell wall material prevents bending. Out of nine gerbera cultivars, the two that were most likely to bend also had a lower percentage dry weight in the 12 cm of the scape just under the flower head, but the differences between the other cultivars did not correlate with scape dry weight percentage (van Doorn et a1. 1994). D. Gypsophila Senescence in Gypsophila flowers (and in those of many other Caryophyllaceae) is regulated by ethylene. Freshly cut branches show a water deficit early in vase life, which results in a rise in ethylene production and premature flower wilting (van Doorn and Reid 1992). The water stress is apparently due to vascular occlusion by bacteria because chlorine compounds or BQS controlled bacterial growth in the vase water and considerably extended longevity (Marousky 1977; Marousky and Nanney 1972). Whether the bacterial blockage is followed by cavitations is as yet unknown. Gypsophila is very sensitive to dry storage, during which a blockage also develops, but the nature of this blockage is as yet not known. E. Rosa Unsevered rose flowers had a longevity of 8 to 23 days, depending on the cultivar, whereas cut flowers placed in water had a longevity of only 3.5 to 7 days. In cut sterns an occlusion developed in the xylem prior to the development of the water deficit (Durkin and Kuc 1966; Mayak et a1. 1974; Zieslin et a1. 1978). In roses placed in water directly after cutting, the microscopical evidence showed no reaction by the plant itself. Neither suberin nor tannin was found inside the xylem conduits, nor tyloses, and all the gum-like material was apparently related to microorganism growth. Cutting the sterns of roses results in a high rate of ethylene production, but the development of occlusion in the sterns was not affected by either inhibition

60

W. G. VAN DOORN

or stimulation of ethylene production. These observations indicate that ethylene production as a response to wounding was not the cause of occlusion (Burdett 1970; Parups and Molnar 1972; van Doorn et al. 1989). Marousky (1969, 1971a) held the stems of cut Better Times roses in a hypochlorite solution for some minutes and then transferred them to sterilized bottles with sterile water, containing either a buffer at pH 6 or pH 3 or 8-hydroxyquinoline citrate (HQC). After 2 days of vase life, most of the bottles contained no bacteria in the vase water, yet hydraulic conductance was lower in water controls than in the HQC treatment and lower at pH 6 than at pH 3. From this it was inferred that pH 3 and HQC, apart from acting as antibacterial agents, reduced some stem-induced blockage. The experiments of Marousky (1969, 1971a) were repeated with Sonia, Ilona, Polka, and Frisco roses and it was found that a bacterial population grew inside the stem even though no bacteria were found in the water for several days. In all cases a reduction of hydraulic conductance was correlated with the presence of a high number of bacteria in the stems (van Doorn and Perik 1990). When the growth of microorganisms was prevented in the stems, no decrease in stem hydraulic conductance was observed, indicating that there was no contribution by the stem cells to vascular occlusion (van Doorn et al. 1989). When roses are stored dry, the growth of bacteria at the cut surface and in the xylem interior goes on, and this may lead to blockage following reimmersion in water. However, when bacteria were excluded, an occlusion was still found. This occlusion could not be accounted for by the aspiration of air at the cut surface (van Doorn 1990), nor was it due solely to a blockage in the xylem wall pathway for water (van Doorn 1993). In some rose cultivars it is related to the inability of water to enter the lumina of the conduits opened by cutting, which is probably related to the size of the contact angle between the water meniscus and the dry walls of the conduits. In three rose cultivars tested, a high frequency of cavitations preceded the maximum blockage, which occurred from 3 to 36 h of dry storage, depending on the cultivar. The water stress that is frequently observed in cut rose flowers is apparently due to the combined effect of the occlusion developing during dry storage, for which bacteria are only partially responsible, and the blockage developing during vase life, which is mainly or entirely bacterial in origin. Although some insight has been gained, the exact nature of the blockage developing during dry storage remains to be further evaluated.

1.

WATER RELATIONS OF CUT FLOWERS

61

F. Syringa

Cut stems of lilacs placed in water wilt within about 4.5 days, but silver nitrate in the vase water delayed wilting until about day 7 (Sytsema 1962). Silver nitrate reduced the number of bacteria in the lowermost 10 cm of lilac stems (van Doorn et al. 1991d). The use of the antifungal preparation Cladox (2,4-dinitrorhodanebenzene) reduced the number of hyphae in the water and on the cut surface and also delayed wilting, so it was concluded that the development of a bacterial and fungal population may limit the vase life of lilacs (Sytsema 1962). Flower wilting was also delayed by HQS or HQC (Kalkman 1987; Sytsema-Kalkman 1991). HQC reduced the number of bacteria in the stem and prevented the decrease in hydraulic conductance. As discussed in Section VLD, the 10- to 20-cm segment was limiting the flow of water in the stems. Tyloses were observed in the xylem vessels, especially in the 10- to 20-cm stem segment. Aminoethoxyvinylglycine (AVG), an effective inhibitor of ethylene synthesis, did not prevent bacterial growth or the decrease in hydraulic conductance in the lowermost 10 cm of the stems, but it reduced tylose formation and the blockage in the 10- to 20-cm segment. The results indicate that the blockage is mainly related to tylose formation, which is regulated by ethylene. Although the number of tyloses was not adequate to explain the blockage, the secretion of material that usually accompanies tylose formation could be the main cause of occlusion. HQS and HQC are antimicrobial, but also inhibit ethylene synthesis (Parups and Peterson 1973, van Doorn et al. 1989). Silver nitrate (Sytsema 1962), although not very mobile in the xylem, may reach the 10- to 20-cm segment and there inhibit ethylene action. Although they are not conclusive, the data indicate that the processes related to tylose formation constitute the factor that limits water uptake. G. Ferns

In Adiantum raddianum (maidenhair fern), the pinnae often rapidly curl and desiccate in fronds that are placed in water shortly after harvest. The desiccation starts in a part of the frond only, comprising several pinnae. Recutting under water temporarily reduces the symptoms, indicative of a blockage in the lower part of the petiole. Because of their vulnerability to desiccation, fronds of A. raddianum are not widely used as vase foliage. The fronds are usually also not stored dry, because this results in an even more premature desicca-

62

W. G. VAN DOORN

tion. Initial investigations indicated a role of ethylene in the blockage (Fujino and Reid 1983), but it was later found that the blockage is mainly bacterial in origin (van Doorn et al. 1991a,g). Leatherleaf fern (Rumohra adiantiformis) is one of the most important species used as cut foliage in the United States. Its vase life is 1-3 weeks, usually terminated by the partial or complete desiccation-induced infolding of the pinnae (Mathur et al. 1982). When the fronds were placed in water, the rate of water uptake decreased exponentially and the water potential of the fronds decreased rapidly (Stamps and Nell 1983; Nell et al. 1983). Recutting of the petiole base reduced premature curling, but did not completely prevent it (Renny 1982; Marousky 1983). A comparison between material from several growers, who used different levels of shade, showed that the incidence of premature curling was positively correlated with stomatal density (Marousky 1983). Water loss shortly after harvest may be a cause of the problem: After desiccation of freshly harvested fronds to a range of water potentials, the number of fronds with curled pinnae, determined after 1 week of vase life, was correlated with water loss during the desiccation treatment (Nell et al. 1983). An effect of the rate of transpiration after harvest may also explain the finding that in Florida a reduction in postharvest longevity occurred most frequently during the summer months (Conover et al. 1979; Stamps et al. 1989). Interestingly, fronds cut in the summer and immediately placed in ice water (3°C) in the field showed a higher rate of water uptake than the controls placed in water at ambient temperature (Stamps 1986). Since no visible blockages were observed in the tracheids (Conover et al. 1979; Nell et al. 1983), the results may indicate that tracheids become cavitated both before harvest and when the fronds are held dry shortly after harvest. The number of cavitated tracheids may be higher due to the higher evaporative demand during growth and harvesting. Ice-water treatment may facilitate the repair of cavitated tracheids. The giant holly fern (Polystichum munitum) also often shows premature inrolling of the pinnae, which can be prevented by daily recutting of the petiole or placement in 25 mg/L silver nitrate, suggesting that the folding is due to vascular occlusion (Carow 1981). The vase life of Pteris spp. ferns varied among 12 tested cultivars, from 4 to 22 days. The 22-day vase life can be achieved by placing the fronds of some species in water at 4°C for 10-20 h, and only if the total period of dry storage does not exceed 2 days (Carow 1981). The vase life of the fern Nephrolepis exaltata was 2-4 weeks. Treatments with HQC, with or without sucrose, had no effect on the length

1.

WATER RELATIONS OF CUT FLOWERS

63

of vase life of this long-lived foliage green (Nooh et al. 1986). Dry storage of Nephrolepis exaltata (as well as N. cordifolia) may, however, result in early fall of the pinnae during vase life (Carow 1981).

H. Other Cut Foliage Vaughan (1988) reported that the length of vase life of some species of cut foliage is usually less than 2 weeks. Species with a relatively short vase life include Asparagus asparagoides (7-10 days), Camellia japonica (7 days), Cordyline sp. (7-14 days), Codiaeum variegatum (7 days), Hedera helix «6 days), Galax aphylla (10 days), Magnolia spp. (5-7 days), Myrtus communis (10 days), and Podocarpus macrophyllus (10 days). The vase life of branches of several Eucalyptus species is also less than 10 days (Henny et al. 1982; Vaughan 1988). Although adverse water relations may be involved in the short vase life of these species, details have as yet not been reported. The vase life of Cotinus foliage is usually short, especially during summer, and longevity cannot be increased by inclusion of HQC in the vase solution. Cotinus was found to excrete an oily substance, that reduces the length of vase life of flowers or foliage placed in the same vase, and this substance may also be involved in the short vase life of the Cotinus foliage (A. Ruting, personal communication 1993).

IX. RELATIONSHIPS BETWEEN WATER STRESS, HORMONAL CONTROL OF FLOWER OPENING, AND SENESCENCE Water stress in plants may lead to increased ethylene production (Apelbaum and Yang 1981). A rise in ethylene production during flower development has been detected in noncut and cut roses (Mayak et al. 1972; Halevy and Mayak 1975; Faragher and Mayak 1984; Faragher et al. 1986, 1987a,b). In Golden Wave roses little difference was found between ethylene production in noncut and cut flowers (Halevy and Mayak 1975), but the effect of water stress during vase life on ethylene production has not been studied in detail. In intact roses petal life is usually limited by abscission, which is regulated by ethylene (Woltering and van Doorn 1988). Ethylene may inhibit or stimulate flower opening in cut roses or result in abnormal flower form, depending on the cultivar (Reid et a1. 1989), but the rise in ethylene production in cut roses placed in water directly following harvest (Halevy and Mayak 1975; Faragher and Mayak 1984) occurred after flower opening and therefore has no

64

W. G. VAN DOORN

consequence for opening. Opening could be affected, however, by ethylene production during and shortly after dry storage, when rose buds produce considerable amounts of ACC and ethylene (Faragher et al. 1987b). In roses, moderate water stress during dry storage may have little effect on the time to wilting and increased ethylene production during vase life, if a considerable part of the stem is recut in air or a few centimeters are recut under water. In contrast, after inadequate recutting of the stems the blockage that develops during dry storage is not removed and water stress occurs earlier than in nonstored stems. Indeed, after such a treatment, a rise in ACC levels and ethylene production during the vase life of roses occurred earlier than in control stems (Faragher et al. 1987b). Some flowers in which petals wilt when they senesce show regulation of senescence by ethylene, whereas in others petal wilting is apparently not regulated by ethylene (Woltering and van Doorn 1988). In species where petal wilting is ethylene-regulated, water stress may thus lead to advanced senescence. In carnations, for example, petal inrolling and subsequent wilting are closely related to a rise in ethylene production, the onset of which is determined by factors such as the rate of base level ethylene production (Nichols 1968a) and ethylene sensitivity (Barden and Hanan 1972). Water loss resulted in a transient increase in the ethylene production of White Sim carnation flowers and in a shorter vase life. The effect of desiccation was abolished when the stems were treated with ADA, an inhibitor of ethylene production, prior to a period of dry storage (Mayak et al. 1985). The reduction of White Sim carnation vase life, however, is found only after extreme desiccation. When carnation flowers are left dry on the laboratory bench for up to 2 days at 25°C, no subsequent effect on vase life is found, except for the days lost by the period of desiccation (C. H. Whitehead and W. G. van Doorn, unpublished). In the flower trade, carnations are also transported dry for many days without (in most cultivars) an apparent effect on their subsequent vase life, except for the time lost in the period of transport. As discussed (in Section VIII.F), an early water deficit during vase life and dehydration during dry storage prior to vase life, resulted in increased ethylene production and early flower wilting in Gypsophila paniculata, which shows petal wilting that is sensitive to ethylene. When panicles are cut and directly placed in water the flowers wilt rapidly, an effect counteracted by a pulse treatment with STS (van Doorn and Reid 1992). In dry-stored panicles placed in

1.

WATER RELATIONS OF CUT FLOWERS

65

water, bud opening was inhibited, an effect also alleviated by STS. Bud opening in this species, therefore, is not primarily inhibited by a low water potential itself but by increased ethylene production as a result of the water deficit. In flowers in which perianth wilting is not regulated by endogenous ethylene, there is apparently little effect of dry storage on the time to petal wilting during vase life. Examples are gerbera, daffodil, and several cultivars of chrysanthemum. Although ethylene production during desiccation has not been evaluated in these flowers, they are transported dry for extended periods and although stem bending in gerbera, flower opening in daffodil, and loss of leaf turgor of chrysanthemum may be hastened by this treatment, petal wilting is generally not. In contrast, the wilting of another ethylene-insensitive flower, Iris germanica, was hastened by a temporary water deficit (Paulin 1972). Low water potential may also lead to increased concentrations of abscisic acid (ABA) in plants (Aspinall 1980). Borochov et al. (1976) reported an increase in petal ABA levels after 4 h of dry storage of cut Superstar roses. ABA levels returned to normal during the next 18 h when the roses were placed in water, but increased more than threefold when the stems were held dry during this period. As discussed in Section IV.A.4, the inclusion of ABA in the vase water has been found to accelerate petal wilting of cut roses, at least when the stems were held in darkness, whereas exogenous ABA may delay early petal wilting in stems placed in the light. Cytokinin levels in rose petals have been found to decrease during vase life (Mayak et al. 1972). Inclusion of kinetin in the vase solution of leafless rose stems delayed flower wilting; leafless stems were used to avoid increased transpiration due to stomatal opening in the leaves by kinetin. Control flower buds did not open and wilted, but kinetin-treated flowers fully opened prior to wilting. A water deficit occurred somewhat later in the kinetin-treated flowers (Mayak and Halevy 1974). An effect of water stress, prior to vase life, on endogenous cytokinin levels has apparently not been reported. X. CONCLUSIONS

Many cut flowers placed in water show an early water deficit due to an occlusion in the xylem. Two types of occlusion can be distinguished. One is related to dry storage, to which almost all commercially sold cut flowers are subjected, and one develops during vase

66

W. G. VAN DOORN

life. Water stress will develop because of a blockage, but also as a function of transpiration rate. Detailed information on stomatal opening and closure in cut flowers is as yet lacking. The reduction of stomatal and cuticular transpiration, by physiological means or by breeding, may be one of the strategies to prevent early water stress in cut flowers. Upon cutting, several plants exude mucilage, gums, resin, or latex at the cut surface. Mucilage, gums and resin can also be deposited into the xylem conduits by the living cells around them. Furthermore, cells may protrude into the xylem lumina to form tyloses. The presence of these materials and tyloses in the xylem conduits has been observed in several species used as cut flowers, but their exact role in the vascular occlusion is as yet largely unknown. When cut flowers are placed in water directly after harvest, their xylem becomes blocked by bacteria growing at the cut surface and inside the xylem conduits. This bacterial blockage apparently is a physical phenomenon, due to the presence of living bacteria and the extracellular polysaccharides they produce and to dead bacteria and their degradation products. The bacterial blockage may be followed by cavitations in the xylem, that is, the filling of the conduits with gas. The bacterial blockage probably develops in the basal stem end of all cut flowers, but some are much more sensitive to this blockage than others. Bacterial counts in vase water of various cut flower species do usually not differ substantially. The same is true for bacterial counts in stems of cut flowers. The reason for the differences in sensitivity to bacteria is as yet unknown, but may relate to differences in leaf area, in initial differences in the rates of transpiration per unit leaf area, the reaction of the stomata to moderate water stress, the rate and area of cuticular transpiration, the xylem anatomy, the number of xylem conduits per unit leaf area, and the sensitivity of the conduits to cavitation. Cavitation may playa role in the blockage observed in flowers placed in water directly after harvest, but it is as yet unclear whether cavitation occurs after the main blockage has already been established, and hence only exacerbates it, or whether it starts after even minor blockage and thus plays an essential role. This is an important topic for further research. When cut flowers are placed in water prior to dry storage the xylem conduits become contaminated with bacteria that keep growing during dry storage, which may explain why the rate of water uptake after dry storage is often low. However, when bacterial growth dur-

1.

WATER RELATIONS OF CUT FLOWERS

67

ing dry storage is excluded, a blockage nonetheless develops. The nature of this blockage is still not fully elucidated. In cut rose flowers it was not due solely to the presence of air in the lumen of the conduits opened by cutting. Nor was it solely related to the drying of the xylem walls, which inhibits the flow of water in the xylem walls and prevents the entry of water into the lumen of the conduits opened by cutting. When cut flowers are held dry and reach a critical water potential, the xylem conduits start to cavitate. Although a correlation has been found between the presence of a high number of cavitated conduits and occlusion, the role of cavitation in the blockage occurring during dry storage and the rate of refilling of the cavitated conduits following reimmersion in water need further evaluation. Increased resistance for flow of water occurs in two parallel pathways in the xylem conduits opened by cutting: the lumina and the walls. In addition, the flow of water may become impeded in the lumen of the conduits that remain unopened, by cavitation and/or deposition of material. Inhibition of water uptake by cut flowers may be measurable only when several or all of these resistances are high. In practice, early water deficit of cut flowers during vase life may be due to a combination of the blockages developing during dry transport and the blockages developing during subsequent vase life. In cut rose flowers, for example, the bacterial blockage by itself does normally not explain the observed symptoms of water stress, nor does the vascular occlusion developing during dry storage. The water deficit, however, can be fully explained by the combination of bacterial occlusion during vase life and nonbacterial blockage that occurs during dry storage. In many cut flowers the occlusion developing during vase life can be prevented by inclusion of an antibacterial compound in the vase solution, or, in some woody stems such as lilacs, by adding a compound that apparently inhibits tylose formation. The vascular occlusion related to dry storage (when excluding bacterial contamination) can usually be overcome by a surfactant treatment. However, the rapid occlusion in many other flowers, such as Canna and Heliconia, can as yet not be prevented. The role of exudation of materials at the cut surface and that of deposition of materials in the xylem lumen of cut flowers have rarely been studied in detail, and may provide ways to prevent early wilting in cut parts of many species, including those whose short vase life has as yet precluded their use as a commercial commodity.

68

W. G. VAN DOORN

LITERATURE CITED Aarts, ]. F. T. 1957a. On the keepability of cut flowers (in Dutch, with English summary). Meded. Landbouwhogesch. 57(9):1-62. Aarts, ]. F. T. 1957b. The development and keepability of cut flowers (in Dutch). Meded. Directeur van de Tuinbouw 20:690-702. Abdel-Kader, H., and M. N. Rogers. 1986. Postharvest treatment of Gerbera jamesonii. Acta Hort. 181:169-176. Abeles, F. B., P. W. Morgan, and M. E. Saltveit. 1992. Ethylene in plant biology, 2d ed. Academic Press, San Diego. Accati Garibaldi, E., and F. Deambrogio. 1988. Conservazione del fiore reciso di margherita. L'Informatore Agrario 44(19):97-104. Acock, B., and R. Nichols. 1979. Effects of sucrose on water relations of cut, senescing, carnation flowers. Ann. Bot. 44:221-230. Aharoni, M., and S. Mayak. 1977. Water conductance in stems of Phalaenopsis cut flowers (in Hebrew). p. 76-77. In: Dept. Ornamental Hort., Hebrew Univ., Annu. Rep. 1975-1977. (cited by Halevy and Mayak 1981). Akin, D. E. 1976. Ultrastructure of rumen bacterial attachment to forage cell walls. Appl. Environ. Microbiol. 31:562-568. Apelbaum, A., and M. Katchansky. 1977. Improving quality and prolonging vase life of bud cut flowers by pretreatment with thiabendazole. ]. Am. Soc. Hort. Sci. 102:623-625. Apelbaum, A., and S. F. Yang. 1981. Biosynthesis of stress ethylene induced by water deficit. Plant Physiol. 68:594-596. Archer, B. L., B. G. Audley, G. P. McSweeney, and T. C. Hong. 1969. Studies on composition of latex serum and 'bottom fraction' particles. ]. Rubber Res. Inst. Malaya 21:560-569. Arnold, Z. 1930. Einige orientierende Versuche zur Frage der kunstlichen Frischerhaltung der Schnittblumen. Gartenbauwissenschaft 3:47-58. Arnold, Z. 1931. Weitere Versuche zur Frage der kunstlichen Frischerhaltung der Schnittblumen. Gartenbauwissenschaft 5:255-266. Aspinall, D. 1980. Role of abscisic acid and other hormones in adaptation to water stress. p. 155-172. In: N. C. Turner and P. ]. Kramer (eds.), Adaptation of plants to water and high temperature stress. Wiley, New York. Auge, R. M., A. ]. W. Stodola, and B. D. Pennell. 1990. Osmotic and turgor adjustment in Rosa foliage drought-stressed under varying irradiance. ]. Am. Soc. Hort. Sci. 115:661-667. Awad, R. E., A. Meawad, A. Kamel Dawh, and M. El-Saha. 1986. Cut flower longevity as affected by chemical pre-treatment. Acta Hort. 181:177-182. Baker,]. E., C. Y. Wang, M. Lieberman, and R. E. Hardenburg. 1977. Delay of senescence in carnation by a rhizobitoxine analog and sodium benzoate. HortScience 12:38-39. Bakker, ]., and H. Stephan. 1964a. Mimosa (Acacia dealbata 'Mirandole') (in Dutch). p. 139-140. In: Annu. Rep. 1963. Res. Sta. for Floriculture, Aalsmeer. Bakker, ]., and H. Stephan. 1964b. Prunus serrulata and Prunus triloba (in Dutch). p. 138. In: Annu. Rep. 1963. Res. Sta. for Floriculture, Aalsmeer. Barden, L. E., and ]. ]. Hanan. 1972. Effects of ethylene on carnation keeping life. ]. Am. Soc. Hort. Sci. 97:785-788. Barendse,L. 1978. Keepability research on cut flowers (in Dutch). Annu. Rep. 1977. Res. Sta. for Floriculture, Aalsmeer.

1.

WATER RELATIONS OF CUT FLOWERS

69

Barth, G. 1990. Cut flower potential of Sturt's Desert Pea. Austral. Hort. 88:48-53. Bazzocchi, R., M. Giorgioni, and Calzolari, S. 1987. Eucalyptus per la produzione di fronde recise. Colture Protette 6:27-32. Bieleski, R. L., and M. S. Reid. 1992. Physiological changes accompanying senescence in the ephemeral daylily flower. Plant Physiol. 98:1042-1049. Blanke, M. M. 1991. Wie sieht die Erdbeerbliite unter dem Mikroskop aus? Erwerbsobstbau 33:108-110. Blanke, M. M., and C. J. Lovatt. 1993. Anatomy and transpiration of the avocado inflorescence. Ann. Bot. 71:543-547. Boer, W. c., and H. Harkema. 1978. Storage experiments with daffodil cv. Carlton (in Dutch). Rep. 2023. Sprenger Inst., Wageningen. Boer, W. C., and H. Harkema. 1979. Effect of postharvest treatments on vase life of chrysanthemum Horim (in Dutch). Vakblad voor de Bloemisterij 34(6):44-46. Boer, W. C., and R. A. Hilhorst. 1978. Storage experiments with cut flowers (in Dutch). Rep. 2000. Sprenger Inst., Wageningen. Bordeau, P. F., and C. S. Schopmeyer. 1958. Oleoresin exudation pressure in slash pine: its measurement, heritability and relation to oleoresin yield. p. 313-319. In: K. V. Thimann (ed.), The physiology of forest trees. Ronald, New York. Bornman, C. H. 1967. The relationship between tylosis and abscission in cotton (Gossypium hirsutum L.) explants. S. Afr. J. Agr. Sci. 10:143-154. Bornman, C. H., A. R. Spurr, and F. T. Addicott. 1967. Abscisin, auxin, and gibberellin effects on the developmental aspects of abscission in cotton (Gossypium hirsutum). Am. J. Bot. 54:125-135. Borochov, A., and W. R. Woodson. 1989. Physiology and biochemistry of flower petal senescence. Hort. Rev. 11:15-43. Borochov, A., T. Tirosh, and A. H. Halevy. 1976. Abscisic acid content of senescing petals on cut rose flowers as affected by sucrose and water stress. Plant Physiol. 58:175-178. Bradley, M. v., N. Marei., and J. C. Crane. 1969. Morphological and histological effects of ethrel on the apricot, Prunus armeniaca L., as compared with those of 2,4,5-trichlorophenoxyacetic acid. J. Am. Soc. Hort. Sci. 94:316-318. Broschat, T. K., and H. Donselman. 1983a. Heliconias: a promising new flower crop. HortScience 18:2. Broschat, T. K., and H. Donselman. 1983b. Production and postharvest culture of Heliconia psittacorum flowers in Southern Florida. Proc. Florida State Hort. Soc. 96:272-273. Broschat, T. K., and H. Donselman. 1987. Potential of 57 species of tropical ornamental plants for cut foliage use. HortScience 22:911-913. Brown, F., E. L. Hirst, and J. K. N. Jones. 1948. The structure of almond-tree gum, I: the constituents of the aldobionic acid derived from the gum. J. Chern. Soc. 1948: 1677-1679. Burdett, A. N. 1970. The cause of bent neck in cut roses. J. Am. Soc. Hort. Sci. 95:427-431. Butterfield, B. G., and B. A. Meylan. 1982. Cell wall hydrolysis in the tracheary elements of the secondary xylem. p. 71-84. In: P. Baas P. (ed.), New perspectives in wood anatomy. Nijhoff, The Hague. Byrne, T. G., D. S. Farnham, and L. S. Pyett. 1979. Postharvest studies with marguerite daisies. p.1-4. In: Florist Nurs. Rep. (Univ. of Calif.). Carow, B. 1978. Zur Haltbarkeit der Schwertfarns. Gartnerb6rse Gartenwelt 78:12171218.

70

W. G. VAN DOORN

Carow, B. 1981. Frischhalten von Schnittblumen, 2d ed. Eugen Ulmer, Stuttgart. Casp, A. M., P. J. Salvador, and M. J. Ibanez. 1980. A new solution for carnation bud opening with promising improvements due to a quaternary ammonium compound. Acta Hort. 113:183-189. Chandra, G., K. S. Reddy, and H. Y. Mohan Ram. 1981. Extension of vase life of cut marigold and chrysanthemum flowers by the use of cobalt chloride. Ind. J. Exp. BioI. 19:150-154. Chattaway, M. M. 1948. The development of tyloses and secretion of gum in heartwood formation. Austral. J. Sci. Ser. B BioI. Sci. 2:227-240. Cline, M. N. and D. Neely. 1983. The histology and histochemistry of the woundhealing process in Geranium cuttings. J. Am. Soc. Hort. Sci. 108:779-780. Conover, C. A., R. T. Poole, and 1. 1. Loadholz. 1979. Update on leatherleaf fern wilt. Univ. Florida, Apopka Agr. Res. Center, Rep. RH-79-1. Conrado, 1. 1., R. Shanahan, and W. Eisinger. 1980. Effect of pH, osmolarity, and oxygen on solution uptake by cut rose flowers. J. Am. Soc. Hort. Sci. 105:680683. Criley, R. A., and T. K. Broschat. 1992. Heliconia: botany and horticulture of a new floral crop. Hort. Rev. 14:1-55. Crombie, D. S., M. F. Hipkins, and J. A. Milburn. 1985. Gas penetration of pit membranes in the xylem of Rhododendron as a cause of acoustically detectable sap cavitation. Austral. J. Plant Physiol. 12:445-453. Cr6ssmann, G. 1968. Uber den Einfluss eines Frischhaltemittels auf die Haltbarkeit und Aussehen verschiedener Schnittblumenarten und -sarten. Taspo 102(31):1011. Dansereau, B. and H. M. Vines. 1975. In-stem movement, isolation, and identification of two bacteria and their antibiotic sensitivity. Acta Hort. 41:183-197. Davies, F. S., C. E. Munoz, and W. B. Sherman. 1981. Opening and vase life extension of peach flowers on detached shoots with sucrose and ethanol. J. Am. Soc. Hort. Sci. 106:809-813. de Bary, A. 1877. Vergleichende Anatomie der Vegetationsorgane der Phanerogamen und Fame. Wilhelm Engelmann, Leipzig. de Stigter, H. C. M. 1980a. Effects of glucose with 8-hydroxyquinoline sulfate or aluminium sulfate on the water balance of cut Sonia roses. Z. Pflanzenphysiol. 101:95-105. de Stigter, H. C. M. 1980b. Water balance aspects of cut and intact 'Sonia' rose plants and effects of glucose, 8-hydroxyquinoline sulfate and aluminium sulfate. Acta Hort. 113:97-107. de Stigter, H. C. M., and A. G. M. Broekhuysen. 1986a. Role of stem cut surface in cut rose performance. Acta Hort. 181:359-363. de Stigter, H. C. M., and A. G. M. Broekhuysen. 1986b. Experimentally induced plugging of cut-rose xylem by particulate or macromolecular matter. Acta Hart. 181:365-370. de Stigter, H. C. M., and A. G. M. Broekhuysen. 1989. Secondary gas embolism as an effect of disturbed water balance in of cut roses. Acta Hort. 261:17-25. de Vries, H. 1873. Uber das Welken abgeschnittener Sprosse. Arbeiten an der botanischen Institut in Wiirzburg 1:287-301. de Vries, H. 1881. Uber einige Nebenprodukte des pflanzlichen Stoffwechsels. Landwirtsch. Jahrbucher 10:687-730. de Witte, Y., and W. G. van Doorn. 1988. Identification of bacteria in the vase water of roses, and the effect of the isolated strains on water uptake. Sci. Hort. 35:285291.

1.

WATER RELATIONS OF CUT FLOWERS

71

de Witte, Y., and W. G. van Doorn. 1992. The mode of action of bacteria in the vascular occlusion of cut rose flowers. Acta Hort. 298:165-170. D'hont, K., and J. van der Sprong. 1989. Postharvest treatment of chrysanthemum. Acta Hort. 261:305-307 Dickenson, P. B. 1969. Electron microscopical studies of latex vessels of Hevea brasiliensis J. Rubber Res. Inst. Malaya 21:543-558. Dixon, H. H. 1914. Transpiration and the ascent of sap in plants. MacMillan, London. Dixon, H. H., J. A. Butt, D. P. Murr, and M. J. Tsujita. 1988. Water relations of cut greenhouse roses: the relationships between stem water potential, hydraulic conductance and cavitation. Sci. Hart. 36:109-118. Dixon, M. A., and C. A. Peterson. 1989. A re-examination of stem blockage in cut roses. Sci. Hort. 38:277-285. Donnelly, D. J., and F. E. Skelton. 1989. Comparison of hydathode structure in micropropagated plantlets and greenhouse-grown 'Queen Elisabeth' rose plants. J. Am. Soc. Hort. Sci. 114:841-846. Donselman, H. M., and T. K. Broschat. 1986. Production of Heliconia psittacorum for cut flowers in South Florida. Bull. Heliconia Soc. Int. 1(4):4-6. Downs, e.G., and M. Reihana. 1987. Extending vase life and improving quality of Nerine cut flowers with preservatives. HortScience 22:670-671. Downs, C. G., M. Reihana, and H. Dick. 1988. Bud-opening treatments to improve Gypsophila quality after transport. Sci. Hort. 34:301-310. Durkin, D. 1967. Role of tannins in senescence of the cut rose flower. Abstracts, Am. Soc. Hart. Sci. 185:78. Durkin, D. J. 1979a. Some characteristics of water flow through isolated rose segments. J. Am. Soc. Hart. Sci. 104:777-783. Durkin, D. J. 1979b. Effect of millipore filtration, citric acid, and sucrose on peduncle water potential of cut rose flower. J. Am. Soc. Hart. Sci. 104:860-863. Durkin, D. J. 1980. Factors affecting hydration of cut flowers. Acta Hart. 113:109117. Durkin, D. J., and R. H. Kuc. 1966. Vascular blockage and senescence of the cut rose flower. Proc. Am. Soc. Hart. Sci. 89:683-688. Engels, F. M. 1989. Some properties of cell wall layers determining ruminant digestion. p. 80-87. In: A. Chesson and E. R. Orskov (eds.), Physicochemical characterisation of plant residues for industrial and feed use. Elsevier, New York. Esau, K. 1965. Plant anatomy, 2d ed. Wiley, New York. Evans, R.Y., and M. S. Reid. 1986. Control of petal expansion during diurnal opening of roses. Acta Hort. 181:55-63. Evans, R. Y., and M. S. Reid. 1988. Changes in carbohydrates and osmotic potential during rhythmic expansion of rose petals. J. Am. Soc. Hart. Sci. 113:884-888. Eveling, D. W. 1984. Examination of the cuticular surfaces of fresh delicate leaves and petals with a scanning electron microscope: a control for artifacts. New Phytol. 96:229-237. Fahn, A. 1974. Plant anatomy, 2d ed. Pergamon, Oxford, UK. Faragher, J. D. 1986. Postharvest physiology of waratah inflorescences (Telopea speciosissima, Proteaceae). Sci. Hart. 28:271-279. Faragher, J. D. 1989. A review of research on postharvest physiology and horticulture of Australian native flowers. Acta Hart. 261:249-256. Faragher, J. D., and S. Mayak. 1984. Physiological responses of cut rose flowers to exposure to low temperature: changes in membrane permeability and ethylene production. J. Exp. Bot. 35:965-974.

72

W. G. VAN DOORN

Faragher, J. D., S. Mayak, and T. Tirosh. 1986. Physiological responses of cut rose flowers to cold storage. Physiol. Plant. 67:205-210. Faragher, J. D., E. Wachtel, and S. Mayak. 1987a. Changes in the physical state of membrane lipids during senescence of rose petals. Plant Physiol. 83:1037-1042. Faragher, J. D., Y. Mor, and F. Johnson. 1987b. Role of aminocyclopropane-1-carboxylic acid (ACC) in control of ethylene production in fresh and cold-stored rose flowers. J. Exp. Bot. 38:1839-1847. Farina, E., and T. Paterniani. 1984. Trattamenti termici ad elevata temperatura a steli recisi di Euphorbia fulgens all'atto della raccolta per la conservatione dei caratteri di commerciabilita: prove sperimentali per la definizione delle migliori condizioni di esecuzione. Ann. 1st. Spero Flor. 15:105-112. Farnham, D. S., A. M. Kofranek, and J. Kubota. 1978a. Bud opening of Gypsophila paniculata 1. cv. Perfecta with Physan-20. J. Am. Soc. Hort. Sci. 103:382-384. Farnham, D. S., T. Ueda, A. M. Kofranek, A. H. Halevy, A. H. McCain, and J.Kubota. 1978b. Physan-20, an effective biocide for conditioning and bud opening of carnations. Florists' Rev. 162:24-26, 58-60. Figueiredo, A. C. S., and M. S. Pais. 1992. Ultrastructural aspects of the nectary spur of Limodorum abortivum (1.) Sw. (Orchidaceae). Ann. Bot. 70:325-331. Ford, H. E., D. T. Clark, and R. F. Stinson. 1961. Bacteria associated with cut flower containers. Proc. Am. Soc. Hort. Sci. 77:635-636. Fourten, L., and V. Ducomet. 1906. Sur la conservation des fleurs coupees. Rev. Hort. 78:260-262. Fujino, D. W., and M. S. Reid. 1983. Factors affecting the vase life of fronds of maidenhair fern. Sci. Hort. 21:181-188. Fujino, D. W., M. S. Reid, and H. C. Kohl. 1983. The water relations of maidenhair fronds treated with silver nitrate. Sci. Hort. 19:349-355. Gay, A. P., and R. Nichols. 1977. The effects of some chemical treatments on leaf water conductance of cut flowering stems of Chrysanthemum morifolium. Sci. Hort. 6:167-177. Gilman, K. F., and P. L. Steponkus. 1972. Vascular blockage in cut roses. J. Am. Soc. Hort. Sci. 97:662-667. Goldsmith, G. W., and A. 1. Hafenrichter. 1932. Anthokinetics: the physiology and ecology of floral movements. Carnegie Inst., Washington DC, Publ. 420. G6tz, W. 1986. Eucalyptus-Arten im Test. Zierpflanzenbau 26:973-978. Grace, J. 1993. Consequences of xylem cavitation for plant water deficits. p. 109128. In: J. A. C. Smith and H. Griffiths (eds.), Water deficits: plant responses from cell to community. Bios, Oxford, UK. Haberlandt, G. 1896. Physiologische Pflanzenanatomie, 2d ed. Wilhelm Engelmann, Leipzig. Halevy, A. H. 1960. Diurnal fluctuations in water balance factors of gladiolus leaves. Bull. Res. Counc. Israel 8D:239-246. Halevy, A. H. 1976. Treatments to improve water balance of cut flowers. Acta Hort. 64:223-230. Halevy, A. H., and S. Mayak. 1974. Improvement of cut flower quality, opening and longevity by pre-shipment treatments. Acta Hort. 43:335-347. Halevy, A. H., and S. Mayak. 1975. Interrelationships of several phytohormones in the regulation of rose petal senescence. Acta Hort. 41:103-116. Halevy, A. H., and S. Mayak. 1981. Senescence and postharvest physiology of cut flowers, 2. Hort. Rev. 3:59-143.

1.

WATER RELATIONS OF CUT FLOWERS

73

Halevy, A. H., S. Mayak, T. Tirosh, H. Spiegelstein, and A. M. Kofranek. 1974. Opposing effects of abscisic acid on senescence of cut rose flowers. Plant Cell Physiol 15:813-821. Halevy, A. H., A. M. Kofranek, and S. T. Besemer. 1978. Postharvest handling methods for bird-of-paradise flowers (Strelitzia reginae Ait.). J. Am. Soc. Hort. Sci. 103:165-169. Hammond, J.B.W. 1982. Changes in amylase activity during rose bud opening. Sci. Hort. 16:283-289. Hamner, C. L., R. F. Carlson, and H. B. Tukey. 1945. Improvement in keeping quality of succulent plants and cut flowers by treatment under water in partial vacuum. Science 102:332-333. Hanson, A. D., and W. D. Hitz. 1982. Metabolic responses of mesophytes to plant water deficits. Annu. Rev. Plant Physiol. 33:163-203. Hanson, A. D., and H. Kende. 1975. Ethylene-enhanced ion and sucrose efflux in morning glory flower tissue. Plant Physiol. 55:663-669. Harkema, H. 1979. Hot water treatments of chrysanthemums (in Dutch). Intern. Rep. 365. Sprenger Inst., Wageningen. Harkema, H. 1980. Effect of stem lignification on the keepability of chrysanthemum (in Dutch). Rep. 2097. Sprenger Inst., Wageningen. Harkema, H., and P. C. Boom. 1983. Effect of pulse treatment duration and sugar concentration on the keepability of Sonia and Ilona roses (in Dutch). Interim Rep. 31. Sprenger Inst., Wageningen. Harkema, H., and W. G. van Doorn. 1985. Water uptake of cut flowers during pulse treatments (in Dutch). Vakblad voor de Bloemisterij 40(6):106-107. Heins, R. D. 1980. Inhibition of ethylene synthesis and senescence in carnation by ethanol. J. Am. Soc. Hort. Sci. 105:141-144. Henny, R. J. 1982. Reversing postharvest wilt of leatherleaf fern. Univ. Florida, Apopka Agr. Res. Center, Rep. RH-82-23. Henny, R. J., R. H. Stamps, and E. M. Rasmussen. 1982. Preliminary evaluation of different Eucalyptus species for use as a cut-foliage crop. Foliage Dig. 5:3-4. Hermann P. 1975. Euphorbia fulgens als Schnittblume-frisch vom Erzeuger in die Vase. Gartenwelt 74:158-159. Hew, C. S., F. Y. Lee, and K. H. Wee. 1987. Factors affecting the longevity of Aranda flowers. Acta Hort. 205:195-202. Holle, H. 1916. Untersuchungen tiber Welken, Vertrocknen und Wiederstraffwerden. Flora 108:73-126. Homans, L. N. S., J. W. van Dalfsen, and G.E. van Gils. 1948. Complexity of fresh Hevea latex. Nature 161:177-178 Hough, 1., and J. B. Pridham. 1959. The composition of plum gums. Biochem. J. 73:550-559. Jones, J. K. N. 1939. The constitution of cherry gum, I: composition. J. Chern. Soc. 1939:558-561. Jones, J. K. N. 1950. The structure of peach gum, I: the sugars produced on hydrolysis of the gum. J. Chern. Soc. 1950:534-537. Jones, R. B., and M. Hill. 1993. The effect of germicides on the longevity of cut flowers. J. Am. Soc. Hart. Sci. 118:350-354. Jones, R. B., J. D. Faragher, and W. G. van Doorn. 1993a. Water relations of cut flowering branches of Thryptomene calycina (Lindl.) Stapf. (Myrtaceae). Postharvest BioI. Technol. 3:57-67.

74

W. G. VAN DOORN

Jones, R B., M. Serek, and M. S. Reid. 1993b. Pulsing with Triton X-l00 improves hydration and vase life of cut sunflowers (Helianthus annuus L.). HortScience 28:1178-1179. Joyce, D. 1988. Postharvest characteristics of Geraldton wax flowers. J. Am. Soc. Hort. Sci. 13:738-742. Joyce, D., R Jones, and J. Faragher. 1993. Postharvest characteristics of native Australian flowers. Postharvest News Inform. 4:61-67. Ka-Ipo, R, W. S. Sakai, S. C. Furutani, and M. Collins. 1989. Effect of postharvest treatment with antitransparants on the shelf life of H. psittacorum cv. parakeet cut flowers. Bull. Heliconia Soc. Int. 4(3):13-14. Kalkman, E. C. 1987. Postharvest treatment of lilacs (in Dutch). Rep. 45. Res. Stat. Floriculture, Aalsmeer. Kamp, H. 1930. Untersuchungen tiber Kutikularbau und kutikuHire Transpiration von BliHtern. Jahrb. f. Wiss. Bot. 72:403-465. Kende H., and A. D. Hanson. 1977. On the role of ethylene in aging. p. 172-180. In: P. E. Pilet (ed.), Plant growth regulation. Springer, Berlin. Ketsa, S. 1989. Vase-life characteristics of inflorescences of Dendrobium 'Pompadour.' J. Hort. Sci. 64:611-615. Ketsa, S., and A. Boonrote. 1990. Holding solutions for maximizing bud opening and vase-life of Dendrobium 'Youppadeewan' flowers. J. Hort. Sci. 65:41-47. Kisser, J. 1958. Die Ausscheidung von atherischen Olen und Harzen. p. 91-131. In: W. Ruhland (ed.), Handbuch der Pflanzenphysiologie, Part X. Springer, Berlin. Klein, G. 1923. Zur Atiologie der Thyllen. Zeitschr. f. Botanik 15:417-439. Kofranek, A. M. 1986. Studies on the postharvest handling of Triteleia laxa. Acta Hort. 181:231-236. Kofranek, A. M., and A. H. Halevy. 1981. Chemical pretreatment of chrysanthemum before shipment. Acta Hort. 113:89-95. Kofranek, A. M., H. C. Kohl, and J. Kubota. 1974. A slow release chlorine compound as a vase water additive. Florists' Rev. 154 (4000):63-65. Kofranek, A. M., E. Evans, J. Kubota, and D. S. Farnham. 1978. Chemical pretreatments for China asters to increase flower longevity. Florists' Rev. 162 (4206):26, 70-72. Kohl, H. C. 1972. Cyclamens as cut flowers. Florists' Rev. 150 (3884):17. Kohl, H. c., and D.L. Rundle. 1972. Decreasing water loss in cut roses with abscisic acid. HortScience 7:249. Koning, R E. 1984. The roles of plant hormones in the growth of the corolla of Gaillardia grandiflora (Asteraceae) ray flowers. Am. J. Bot. 71:1-8. Koning, R. E. 1986. The role of ethylene in corolla unfolding in Ipomoea nil (Convulvulaceae). Am. J. Bot. 71:152-155. Kramer, P. J. and J. S. Boyer. 1995. Water relations of plants and soils. Academic Press, New York. Kress, W. J. 1990. The phylogeny and classification of the Zingiberales. Ann. Missouri Bot. Garden 77:698-721. Lacey, S. A. 1983. Vase life of Grevillea Sandra Gordon. p. 111-113. In: P. Watkins and R Collins (eds.), Production and marketing of Australian wildflowers for export. Proc. National Technical Workshop. Univ. Western Australia, Perth. Larsen, F. E., and R W. Cromarty. 1967. Micro-organism inhibition by 8-hydroxyquinoline citrate as related to cut flower senescence. Proc. Am. Soc. Hort. Sci. 90:546-549.

1.

WATER RELATIONS OF CUT FLOWERS

75

Larsen, F. E., and M. Frolich. 1969. The influence of 8-hydroxyquinoline citrate, Ndimethylamino-succinamic acid, and sucrose on respiration and water flow in 'Red Sim' cut carnations in relation to flower senescence J. Am. Soc. Hort. Sci. 94:289-292. Larsen, F. E., and J. F. Scholes. 1966. Effects of 8-hydroxyquinoline citrate, Ndimethylamino succinamic acid and sucrose on vase life and spike characteristics of cut snapdragon. Proc. Am. Soc. Hort. Sci. 89:694-701. Larsen, F. E., and J. F. Scholes. 1967. Effect of 8-hydroxyquinoline citrate, sucrose and Alar on vase life and quality of cut stocks. Florists' Rev. 139 (3608) 46-47, 117.

Uiuchli, A. 1976. Apoplasmic transport in tissues. p. 3-34. In: U. Liittge and M.E. Pitman (eds.), Encyclopedia of plant physiology, new series, Vol. 2: Transport in plants IIB. Springer, Berlin. Laurie, A. 1936. Studies on the keeping qualities of cut flowers. Proc. Am. Soc. Hart. Sci. 34:595-597. Laurie, A., D. C. Kiplinger, and K. S. Nelson. 1958. Commercial flower forcing, 6th ed. McGraw-Hill, New York. Ledbetter, M. c., and K. R. Porter. 1970. Introduction to the fine structure of plant cells. Springer, Berlin. Lineberger, R. D., and P. L. Steponkus. 1976. Identification and localization of vascular occlusions in cut roses. J. Am. Soc. Hort. Sci. 101:246-250. Livne, A. and Y. Vaadia. 1972. Water deficits and hormone relations. p. 255-276. In: T. T. Kozlowski (ed.), Water deficits and plant growth. Vol. 3. Academic Press, New York. Lo Gullo, M. A., and S. Salleo. 1993. Different vulnerabilities of Quercus ilex L. to freeze and summer drought-induced xylem embolism: an ecological interpretation. Plant Cell Environ. 16:511-519. Losch, R., and J. D. Tenhunen. 1981. Stomatal responses to humidity: phenomenon and mechanism. p. 136-161. In: P.G. Jarvis and T.A. Mansfield (eds.), Stomatal physiology. Cambridge Univ. Press, Cambridge, UK. Loveys, B. R., and P. E. Kriedemann. 1973. Rapid changes in abscisic acid-like inhibitors following alterations in vine leaf water potential. Physiol. Plant. 28:476479. Mahlberg, P. G. 1973. Scanning electron microscopy of starch grains from latex of Euphorbia terracina and E. tirucalli. Planta 110:77-80. Marousky, F. J. 1968a. Physiological role of 8-hydroxyquinoline citrate and sucrose in extending vase-life and improving quality of cut gladiolus. Proc. Florida State Hort. Soc. 81:409-414. Marousky, F. J. 1968b. Influence of 8-hydroxyquinoline citrate and sucrose on vaselife and quality of cut gladiolus. Proc. Florida State Hart. Soc. 81:415-419. Marousky, F. J. 1969. Vascular blockage, water absorption, stomatal opening and respiration of cut Better Times roses treated with 8-hydroxyquinoline citrate and sucrose. J. Am. Soc. Hort. Sci. 94:223-226. Marousky, F. J. 1971a. Inhibition of vascular blockage and increased moisture retention in cut roses induced by pH, 8-hydroxyquinoline citrate and sucrose. J. Am. Soc. Hort. Sci. 96:38-41. Marousky, F. J. 1971b. Handling and opening bud-cut chrysanthemum flowers with 8-hydroxyquinoline citrate and sucrose. USDA, Agr. Res. Service. Marketing Rep. 905. Washington, DC.

76

W. G. VAN DOORN

Marousky, F. J. 1972. Water relations, effects of floral preservatives on bud opening and keeping quality of cut flowers. HortScience 7:114-116. Marousky, F. J. 1973. Recent advances in opening bud-cut chrysanthemum flowers. HortScience 8:13-16. Marousky, F. J. 1976. Control of bacteria in vase water and quality of cut flowers as influenced by sodium dichloroisocyanurate, 1,3-dichloro-5,5-dimethylhydantoin and sucrose. USDA, Agr. Res. Service S-115. Marousky, F. J. 1977. Control of bacteria in Gypsophila vase water. Proc. Florida State Hart. Soc. 90:297-299. Marousky, F. J. 1983. Premature wilt of leatherleaf fern with different pinnae maturities from various growing environments. Proc. Florida State Hort. Soc. 96:270272. Marousky, F. J., and J. Nanney. 1972. Influence of storage temperatures, handling and floral preservatives on postharvest quality of gypsophila. Proc. Florida State Hart. Soc. 85:419-422. Martin, J. T., and B. E. Juniper. 1970. The cuticles of plants. Edward Arnold, London. Mathur, D. D., R. H. Stamps, and C. A. Conover. 1982. Postharvest wilt and yellowing of leatherleaf fern. Proc. Florida State Hart. Soc. 95:142-143. Mauseth, J. D. 1988. Plant anatomy. Benjamin/Cummings, Menlo Park, CA. Mayak, S., and A. H. Halevy. 1974. The action of kinetin in improving the water balance and delaying senescence processes of cut rose flowers. Physiol. Plant. 32:330-336. Mayak, S., A. H. Halevy, and M. Katz. 1972. Correlative changes in phytohormones in relation to senescence processes in rose petals. Physiol. Plant. 27:1-4. Mayak, S., Y. Vaadia, and D. R. Dilley. 1977. Regulation of senescence in carnation (Dianthus caryophyllus) by ethylene: mode of action. Plant Physiol. 59:591-593. Mayak, S., A. H. Halevy, S. Sagie, A. Bar-Yosef, and R. Bravdo. 1974. The water balance of cut rose flowers. Physiol. Plant. 32:15-22. Mayak, S., A. Borochov, and T. Tirosh. 1985. Transient water stress in carnation flowers: effect of amino-oxyacetic acid. J. Exp. Bot. 36:800-806. Mayne, J., U. Harm, and H. Mittag. 1985. Eucalyptus fur Schnitt und Container. Deutscher Gartenbau 39:2336-2339. McClary, C. 1., and J. S. Layne. 1977. Flower vase water and ornamental potted plants as reservoirs for Gram-negative pathogenic bacteria. Dev. Indust. Microbial. 18:731-739. Meidner, H., and T. A. Mansfield. 1968. Physiology of stomata. McGraw-Hill, London. Mertens, F. 1944. Ein Beitrag zur Frage der VerHingerung der Haltbarkeit von Schnittblumen durch Zusatze. Gartenbauwissenschaft 18:32-39. Metcalfe, C. R., and L. Chalk. 1983. Anatomy of the Dicotyledons, Vol. 2: Wood structure. Clarendon, Oxford, UK. Milburn, J. A., and R. P. C. Johnson. 1966. The conduction of sap, II: Detection of vibrations produced by sap cavitation in Ricinus xylem. Planta 69:43-52. Milburn, J. A., and M. E. McLaughlin. 1974. Studies of cavitation in isolated vascular bundles and whole leaves of Plantago major L. New Phytol. 73:861-871. Mohan Ram, H. Y., and 1. V. Rao. 1977. Prolongation of vase life of Lupinus hartwegii Lindl. by chemical treatments. Sci. Hart. 7:377-382. Moll, J. W. 1880. Untersuchungen uber Tropfenausscheidung und Injection bei Blattern. Verslagen en Mededeelingen der Kon. Akad. van Wetenschappen (Amsterdam), Natuurkunde 14:237-247.

1.

WATER RELATIONS OF CUT FLOWERS

77

Morgan, J. M. 1984. Osmoregulation and water stress in higher plants. Annu. Rev. Plant Physiol. 35:299-319. Miigge, A. 1983. Haltbarkeit und Lagerung von Sommerblumen. Gartenwelt 30:218219. Munoz, C. E., F. S. Davies, and W. B. Sherman. 1982. Hydraulic conductivity and ethylene production in detached flowering peach shoots. HartScience 17:226228. Murr, D. P., T. Venkatarayappa, and M. J. Tsujita. 1979. Counteraction of bent-neck of cut roses with cobalt nitrate. Can. J. Plant Sci. 59:1169-1171. Myers, D. 1991. Surfaces, interfaces, and colloids: principles and applications. VCH, New York. Naidu, S., and M. S. Reid. 1989. Postharvest handling oftuberose (Polianthes tuberosa 1.). Acta Hart. 41:207-215. Neger, F. W. 1912. Spaltoffnungsschluss und kiinstliche Turgorsteigerung. Ber. Deutsch. Bot. Gesellsch. 30:179-194. Nell, T. A., J. E. Barrett, and R. H. Stamps. 1983. Water relations and frond curl of cut leatherleaf fern. J. Am. Soc. Hart. Sci. 108:516-519. Nelson, S. D., and J. M. Mayo. 1975. The occurrence of functional non-chlorophyllous guard cells in Paphiopedilum spp. Can. J. Bot. 53:1-7. Neumann, A.W. 1978. Contact angles. p. 3-35. In: J.F. Padday (ed.), Wetting, spreading and adhesion. Academic Press, London. Nichols, R. 1968a. The response of carnations (Dianthus caryophyllus) to ethylene. J. Hart. Sci. 43:335-349. Nichols, R. 1968b. Micro-organisms, germicides and senescence. p. 38-39. In: Annu. Rep. Ditton Lab, 1967-1968. East MaIling, UK. Nichols, R. 1973a. Water uptake of cut chrysanthemum Chrysanthemum morifolium Ramat. p. 48. In: Annu. Rep. 1972, Glasshouse Crops Res. Inst. Littlehampton, UK. Nichols, R. 1973b. Senescence of the cut carnation flower: respiration and sugar status. J. Hart. Sci. 48:111-121. Nichols, R. 1975. Chrysanthemum (Chrysanthemum morifolium Ramat.): solution uptake and flower quality. p. 50-51. In: Annu. Rep. 1974, Glasshouse Crops Res. Instit. Littlehampton, UK. Nichols, R. 1976. Senescence and sugar status of the cut flower. Acta Hart. 41:2129. Nichols, R. 1978. Quaternary ammonium compounds and flower preservatives. p. 62. In: Annu. Rep. 1977, Glasshouse Crops Res. Inst. Littlehampton, UK. Nobel, P. S. 1991. Physicochemical and environmental plant physiology. Academic, New York. Noah, A. E., T. EI-Kiey, and M. Khattab. 1986. Studies on the keeping quality of cut green Ruscus hypoglossum 1. and Nephrolepis exaltata Schott. Acta Hart. 181:223229. O'Brien, T. P., and K. V. Thimann. 1969. Observations on the fine structure of the oat coleoptile III. Protoplasma 63:443-478. Olien, W. C., and M. J. Bukovac (1982a). Ethephon-induced gummosis of sour cherry (Prunus cerasus L.), I: Effect on xylem function and shoot water status. Plant Physiol. 70:547-555. Olien, W. c., and M. J. Bukovac (1982b). Ethephon-induced gummosis of sour cherry (Prunus cerasus L.), II: Flow characteristics of gum solutions. Plant Physiol. 70:556-559.

78

W. G. VAN DOORN

Pak, C., and W. G. van Doorn. 1992. The relationship between structure and function of surfactants used for rehydration of cut Astilbe, Bouvardia and roses. Acta Hort. 298:171-176. Pardha Saradhi, P. 1989. Prolongation of vase-life of chrysanthemum blooms by cobalt chloride and its reversal by IAA. Acta Hort. 261:309-312. Parups, E. V., and J. M. Molnar. 1972. Histochemical study of xylem blockage in cut roses. J. Am. Soc. Hart. Sci. 97:532-534. Parups, E. v., and E. A. Peterson. 1973. Inhibition of ethylene production in plant tissues by 8-hydroxyquinoline. Can. J. Plant Sci. 53:351-353. Paulin, A. 1972. Influence d'un deficit temporaire en eau sur Ie metabolisme azote des fleurs coupees d'Iris germanica. C. R. Acad. Sci. Paris 275:209-212. Paulin, A. 1980. Influence d'une alimentation continu avec une solution glucosee sur l'evolution des glucides et des proteines dans les divers organes de la rose coupee. Physiol. Plant. 49:55-61. Penningsfeld, F., and L. Forchthammer. 1966. Silbernitrat verbessert die Haltbarkeit geschnittener Gerbera. Gartenwelt 66: 226-228. Perik, R. J. J., and W. G. van Doorn. 1988. Pulse treatment with Agral-LN alleviates vascular blockage in cut roses (in Dutch). Vakblad voor de Bloemisterij 43 (5):4859. Phillips, H. L., and H. Kende. 1980. Structural changes in flowers of Ipomoea tricolor during flower opening and closing. Protoplasma 102:199-215. Pier, G. B., H. F. Sidberry, S. Zolyomi, and J. C. Sadoff. 1978. Isolation and characterization of a high-molecular-weight polyssacharide from the slime of Pseudomonas aeruginosa. Infect. Immun. 22:908-918. Put, H. M. C. 1990. Micro-organisms from freshly harvested cut flower stems and developing during the vase life of chrysanthemum, gerbera and rose cultivars. Sci. Hart. 43:129-144. Put, H. M. c., and A. C. M. Clerkx. 1988. The infiltration ability of micro-organisms Bacillus, Fusarium, Kluyveromyces and Pseudomonas spp. into xylem vessels of Gerbera cv. 'Fleur' and Rosa cv. 'Sonia' cut flowers: a scanning electron microscope study. J. Appl. Bacteriol. 64:515-530. Put, H. M. C., and L. Jansen. 1989. The effects on the vase life of cut Rosa cultivar 'Sonia' of bacteria added to the vase water. Sci. Hart. 39:167-179. Put, H. M. c., and W. Klop. 1990. The effects of microbial exopolysaccharides (EPS) in vase water on the water relations and the vase life of Rosa cv. Sonia. J. Appl. Bacteriol. 68:367-384. Put, H. M. C., and F. M. Rombouts. 1989. The influence of purified microbial pectic enzymes on the xylem anatomy, water uptake and vase life of Rosa cultivar 'Sonia'. Sci. Hart. 38:147-160. Raab, M. M., and R. E. Koning. 1987a. Interacting roles of gibberellin and ethylene in corolla expansion of Ipomoea nil (Convulvulaceae). Am. J. Bot. 74:921-927. Raab, M. M., and R. E. Koning. 1987b. Changes in reponsiveness to ethylene and gibberellin during corolla expansion of Ipomoea nil. J. Plant Growth Regul. 6:121131. Raab, M. M., and R. E. Koning. 1988. How is flaral expansion regulated? BioScience 38:670-674. Ramanuja Rao, I. V., and H. Y. Mohan Ram. 1982. Prevention of compensatory withering and stem-break in cut spikes of gladiolus by overcoming vascular blockage. Proc. Indian National Sci. Acad. Ser. B 48:505-512.

1..

WATER RELATIONS OF CUT FLOWERS

79

Raschke, K. 1989. Movements of stomata. p. 383-441. In: W. Haupt and M. E. Feinleib (eds.), Physiology of movements. Encyclopedia of plant physiology, new series, Vol. 7. Springer, Berlin. Rasmussen, H. P., and W. J. Carpenter. 1974. Changes in the vascular morphology of cut rose stems: a scanning electron microscope study. J. Am. Soc. Hort. Sci. 99:454459. Ratsek, J. C. 1935. Tests of metal containers in an attempt to increase life of cut flowers. Florists' Rev., 9 May. Reddy, T. V. 1988. Mode of action of cobalt extending the vase life of cut roses. Sci. Hort. 36:303-313. Reddy, T. V., C. Nagarajaiah, and B. Raju. 1988. Impregnating cut rose stems with nickel increases vase life. Curro Res. Univ. Agr. Sci., Bangalore 17:108-109. Reid, D. M., and R. 1. Wample. 1985. Water relations and plant hormones. p. 513578. In: R. P. Pharis and D. M. Reid (eds.), Encyclopedia of plant physiology, new series, Vol II. Springer, Berlin. Reid, M. S., and R. Y. Evans. 1986. Control of cut flower opening. Acta Hort. 181:4554. Reid, M. S., R. Y. Evans, 1. L. Dodge, and Y. Mor. 1989. Ethylene and silver thiosulphate influence opening of cut rose flowers. J. Am. Soc. Hort. Sci. 114:436440. Reid, M. S., J. Muisers, D. Zagory, L. L. Dodge, and R. Y. Evans. 1990. Improved methods for rehydrating cut roses and controlling vase solution microbes. p. 2839. In: Roses Inc. Bulletin, October. Renner, O. 1911. Experimentelle Beitriige zur Kenntnis der Wasserbewegung. Flora 103:173-247. Rhodes, J. M., and 1. S. C. Wooltorton. 1978. The biosynthesis of phenolic compounds in wounded plant storage tissues. p. 243-286. In: G. Kahl (ed.), Biochemistry of wounded plant tissues. De Gruyter, Berlin. Robards, A. W. 1970. Electron microscopy and plant ultrastructure. McGraw-Hill, London. Ruting, A. 1991. Effects of wetting agents and cut flower food on the vase life of roses. Acta Hort. 298:69-74. Ryan, W. 1. 1957. Flower preservatives: using silver and zinc ions as disinfectants. Florists' Rev. 121(3129):59-60. Sacalis, J. N. 1974. Inhibition of vascular blockage and extension of vase life in cut roses with an ion exchange column. HortScience 9:149-151. Sacalis, J. N. 1993. Cut flowers: postproduction care and handling, 2d ed. Bael, Batavia, IL. Sachs, J. 1870. Lehrbuch der Botanik, 2d ed. Wilhelm Engelmann, Leipzig. Sachs, J. 1887. Vorlesungen tiber pflanzenphysiologie, 2d ed. Wilhelm Engelmann, Leipzig. Salleo, S., and M. A. Lo Gullo. 1986. Xylem cavitation in nodes and internodes of whole Chorisia insignis H.B. et K. plants subjected to water stress: relations between xylem conduit size and cavitation. Ann. Bot. 58:431-441. Salleo, S., and M. A. Lo Gullo. 1989a. Xylem cavitation in nodes and internodes of Vitis vinifera 1. plants subjected to water stress. p. 33-42. In: K. H. Kreeb, H. Richter, and T. M. Hinckley (eds.), Structural and functional responses to environmental stresses: water storage. SPB, The Hague. Salleo, S., and M. A. Lo Gullo. 1989b. Different aspects of cavitation resistance in Ceratonia siliqua, a drought avoiding Mediterranean tree. Ann. Bot. 64:325-336.

80

W. G. VAN DOORN

Schnabl, H. 1976. Der Einfluss von Aluminium-ionen auf den SUirkemetabolismus von Vicia faba Epidermen. Z. pflanzenphysiol. 77:167-173. Schnabl, H., and H. Ziegler. 1974. Der Einfluss des Aluminiums auf den Gasaustausch und das Welken von Schnittpflanzen. Ber. Deutsch. Bot. Ges. 87:13-20. Schnabl, H., and H. Ziegler. 1975. Uber die Wirkung von Aluminium-ionen auf die Stomatabewegung von Vicia faba-Epidermen. Z. Pflanzenphysiol. 74:394-403. Scholander, P. F., W. E. Love, and J. W. Kanwisher. 1955. The rise of sap in tall grapevines. Plant Physiol. 30:93-104. Scholander, P. F., B. Ruud, and H. Leivestad. 1957. The rise of sap in a tropical liana. Plant Physiol. 32:1-6. Scholander, P. F., H. T. Hammel, E. D. Bradstreet, and E. A. Hemmingsen. 1965. Sap pressures in vascular plants. Science 148:339-346. Scholes, J., and J. W. Boodley. 1964, Improved lasting life of Velvet Times roses with chemicals. New York State Flower Growers Bull. 224:1-4. Schonherr, J. 1982. Resistance of plant surfaces to water loss: transport properties of cutin, suberin and associated lipids. p. 153-179. In: O. 1. Lange, P. S. Nobel, C. B. Osmond, and H. Ziegler (eds.), Physiological plant ecology, II: Water relations and carbon assimilation. Encyclopedia of plant physiology, new series. Vol. 12B. Springer, Berlin. Schrempf, M. 1977. Studies of the circadian rhythm of petal movement in Kalanchoe blossfeldiana. J. Interdiscipl. Cycle Res. 8:396-400. Scott, P. C., B. D. Webster, and A. C. Leopold. 1964. Formation of tyloses during bean leaf abscission. Plant Physiol. 39 (Suppl.):xiv Sen, D. N., and D. D. Chawan. 1972. Leafless Euphorbias on Rajasthan rocks, IV: Water relations of seedlings and adult plants. Vegetatio 24:193-214. Sheldrake, A. R. 1969. Cellulase in latex and its possible significance in cell differentiation. Planta 89:82-84. Siau, J. F. 1984. Transport processes in wood. Springer Series in Wood Science, Springer, Berlin. Singh, K., and K. G. Moore. 1992a. Water relations of cut chrysanthemum flowers. Adv. Hart. Sci. 3:121-124. Singh, K., and K. G. Moore. 1992b. Role of ethylene in keeping quality of chrysanthemum flowers. Adv. Hart. Sci. 6:177-178. Slatyer, R. O. 1977. Plant water relationships, 6th ed. Academic, London. Slootman, J. E. A. 1981. A preliminary study on the vase life of Gloriosa virescens 'Rothschildiana' (in Dutch). Intern. Rep. 481. Sprenger Inst., Wageningen. Slootweg, G., and U. van Meeteren. 1991. Transpiration and stomatal conductance of roses cv Sonia grown with supplemental lighting. Acta Hart. 298:119-125. Smellie, H., and P. Brincklow. 1963. The use of antiseptics for delaying decomposition of cut flowers in hospital wards. Lancet 7841:777-778 Smith, F., and R. Montgomery. 1959. The chemistry of plant gums and mucilages. Reinhold, New York. Smith, J. A. c., and H. Griffiths. 1993. Water deficits: plant responses from cell to community. Bios, Oxford, UK. Sperlich, A. 1939. Das tropische Parenchym. B: Exkretionsgewebe. Handbuch der Pflanzenanatomie. K. Linsbauer, ed. Abteilung 1, Teil 2: Histologie, Band IV. Gebriider Borntraeger, Berlin. Sperry, J. S., N. M. Holbrook, M. H. Zimmermann, and M. T. Tyree. 1987. Spring filling of xylem vessels in wild grapevine. Plant Physiol. 83:414-417.

1.

WATER RELATIONS OF CUT FLOWERS

81

Staden, O. L., and J. A. E. Slootman. 1976. Cause and prevention of rapid wilting of Euphorbia branches, during vase life (in Dutch). Rep. 1974. Sprenger Inst., Wageningen. Staden, O. L., E. C. Kalkman, and J. E. A. Slootman. 1980. Micro-organisms and vase life of cut flowers (in Dutch). Rep. 2112. Sprenger Inst., Wageningen. Stamm, A. J. 1935. The effect of changes in equilibrium relative water vapor pressure upon the capillary structure of wood. Physics 6:334-342. Stamm, A. J. 1952. Surface properties of cellulosic materials. p. 769-792. In: L. E. Wise and E. C. Jahn (eds.), Wood chemistry, Vol. 2. Reinhold, New York. Stamm, A. J., and E. Wagner. 1961. Determining the distribution of interstructural openings in wood. Forest Prod. J. 11:141-144. Stamps, R. H. 1986. Time of harvest and postharvest water dips may affect vase life of leatherleaf fern. Nurseryman's Dig. 20(4):36. Stamps, R. H., and T. A. Nell. 1983. Storage, pulsing, holding solutions and holding solution pH affect solution uptake, weight change and vase life of cut leatherleaf fern fronds. Proc. Florida State Hort. Soc. 96:304-306. Stamps, R. H., T. A. Nell, and D. J. Cantcliffe. 1979. Production temperature affects leatherleaf fern postharvest desiccation. HortScience 24:325-327. Stead, A. D., and K. G. Moore. 1983. Studies on flower longevity in Digitalis: the role of ethylene in corolla abscission. Planta 157:15-21. Stead, A. D., and W. G. van Doorn. 1994. Strategies of flower senescence: a review. p. 215-237. In: R. J. Scott and A. D. Stead (eds.), Molecular and cellular aspects of plant reproduction. Soc. Exp. Biol., Seminar Series 55. Cambridge Univ. Press, Cambridge, UK. Steinitz, B. 1982. The role of sucrose in stabilization of cut Gerbera flower stalks. Gartenbauwissenschaft 47:77-81. Stiles, F. G. 1979. Notes on some natural history of Heliconia (Musaceae) in Costa Rica. Brenesia 15(Suppl.):151-180. Stocking, C. R. 1948. Recovery of turgor by cut shoots after wilting. Plant Physiol. 23:152-155. Stoddard, E. M., and P. M. Miller. 1962. Chemical control of water loss in growing plants. Science 137:224-225. Stasser, R. 1978a. Der histochemische Nachweis einiger Enzyme im Zusammenhang mit der Gummibildung bei Slisskirschen. Angew. Bot. 52:363-369. Stasser, R. 1978b. Untersuchungen liber die Entstehung der Lakunen bei der Gummibildung des Steinobst. Mitt. Klostereneuburg 28:119-125. Strasburger, E. 1891. Uber den Bau und die Verrichtingen der Leitungsbahnen in den Pflanzen. Gustav Fischer, Jena. Stubbs, J. M., and M. J. O. Francis. 1971. Electron microscopical studies of rose petal cells during flower maturation. Planta Medica 20:211-218. Sutherland, I. W. 1977. Bacterial exopolysaccharides: their nature and production. p. 27-96. In: I.W. Sutherland (ed.), Surface carbohydrates of the prokaryotic cell. Academic Press, New York. Suttle, J. C., and H. Kende. 1980. Ethylene action and loss of membrane integrity during petal senescence in Tradescantia. Plant Physiol. 65:1067-1072. Sytsema, W. 1962. Flowering of cut lilacs branches (in Dutch). Meded. Landbouwhogeschool Wageningen 62(2):1-57. Sytsema, W. 1967. The keepability of poinsettia (in Dutch). p. 116-117. In: Annu. Rep. 1966. Res. Stat. Floriculture, Aalsmeer.

82

W. G. VAN DOORN

Sytsema, W. 1968a. The keepability of mimosa (Acacia moutteana and A. dealbata) (in Dutch). p. 122-123. In: Annu. Rep. 1967. Res. Stat. Floriculture, Aalsmeer. Sytsema, W. 1968b. The keepability of poinsettia (in Dutch). p. 122. In: Annu. Rep. 1967. Res. Stat. Floriculture, Aalsmeer. Sytsema, W. 1969. The keepability of Euphorbia fulgens (in Dutch). p. 136-137. In: Annu. Rep. 1968. Res. Stat. Floriculture, Aalsmeer. Sytsema, W. 1976. The keepability of Euphorbia fulgens (in Dutch). Rep. 5. Res. Stat. Floriculture, Aalsmeer. Sytsema-Kalkman, K C. 1991. Postharvest studies on Syringa vulgaris. Acta Hort. 298:127-133. Takimoto, A., and S. Kaihara. 1984. Photocontrol of flower opening in Pharbitis nil. p. 241-253. In: D. Vince-Prue, B. Thomas, and K. K Cockshull (eds.), Light and the flowering process. Academic Press, London. Taplin, D., and P. M. Mertz. 1973. Flower vases in hospitals as reservoirs of pathogens. Lancet 7841:1279-1281. Tenhunen, J. D., R. W. Pearcy, and O. 1. Lange. 1987. Diurnal variations in leaf conductance and gas exchange in natural environments. p. 323-351. In: K Zeiger, G. D. Farquhar, and 1. R. Cowan (eds.), Stomatal function. Stanford Univ. Press, Stanford, CA. Tjia, B. 1985. Longevity and postharvest studies of various Heliconia psittacorum bracts. Bull. Heliconia Soc. Int. 1(1):6. Tjia, B., and K. A. Funnell. 1986. Postharvest studies of Zantedeschia inflorescences. Acta Hort. 181:451-458. Tjia, B., and T. J. Sheehan. 1984. Preserving profits and beauty. Longevity, quality studies help prolong life of Heliconia. Greenhouse Manager 2(11):94-100. Tonecki, J., A. J. Lukaszewska, and N. Gorin. 1989. Effect of L-proline, L-hydroxyproline and gamma-amino butyric acid on the vase life of cut 'Sonia' roses. Gartenbauwissenschaft 54:82-85. Troll, W. 1959. Allgemeine Botanik. Ferdinand Enke, Stuttgart. Troughton, J. H., and 1. A. Donaldson. 1972. Probing plant structure. Chapman & Hall, London. Troughton, J. H., and F. B. Sampson. 1973. Plants: a scanning electron microscope survey. Wiley, Sydney. Turner, N. c., and M. M. Jones. 1980. Turgor maintenance by osmotic adjustment: a review and evaluation. p. 87-103. In: N. C. Turner and P. J. Kramer (eds.), Adaptation of plants to water and high temperature stress. Wiley, New York. Tyree, M. T., and F. W. Ewers. 1991. The hydraulic architecture of trees and other woody plants. New Phytologist 119:345-360. Tyree, M. T., and J. S. Sperry. 1989. Vulnerability of xylem to cavitation and embolism. Annu. Rev. Plant Physiol. Mol. BioI. 40:19-38. Vamos-Vigiyaz6, L. 1981. Polyphenol oxidase and peroxidase in fruits and vegetables. CRC Crit. Rev. Food Sci. Nutr. 15:49-127. Van Alfen, N. K., B. D. McMillan, V. Turner, and W. M. Hess. 1983. Role of pit membranes in macromolecule-induced wilt of plants. Plant Physiol. 73:10201023. VanderMolen, G. K, J. H. Labavitch, L. 1. Strand, and J. K Devay. 1983. Pathogeninduced vascular gels: ethylene as a host intermediate. Physiol. Plant. 59:573580.

1.

WATER RELATIONS OF CUT FLOWERS

83

VanderMolen, G. E., C. H. Beckman, and E. Rodehorst. 1987. The ultrastructure of tylose formation in resistant banana following inoculation with Fusarium oxysporum f.sp. cubense. Physiol. Molec. Plant Pathol. 31:185-200. van Die, J. 1955. A comparative study of the particle fractions from Apocynaceae latices. Ann. Bogor. 2:131-132. van Doorn, W. G. 1990. Aspiration of air at the cut surface of rose stems and its effect on the uptake of water. J. Plant Physiol. 137:160-164. van Doorn, W. G. 1993. Vascular occlusion in cut flowering rose stems exposed to air: role of the xylem wall pathway for water. Physiol. Plant. 90:45-50. van Doorn, W. G., and Y. de Witte. 1991a. Effect of bacterial suspensions on vascular blockage in stems of cut rose flowers. J. Appl. Bacteriol. 71:119-123. van Doorn, W. G., and Y. de Witte. 1991b. Effect of dry storage on bacterial counts in stems of cut rose flowers. HortScience 26:1521-1522. van Doorn, W. G., and Y. de Witte. 1994. Effect of bacteria on scape bending in cut Gerbera jamesonii flowers. J. Am. Soc. Hort. Sci. 119:568-571. van Doorn, W. G., and K. D'hont. 1994. Interaction between the effects of bacteria and dry storage on the opening and water relations in cut rose flowers. J. Appl. Bacteriol. 77:644-649. van Doorn, W. G., and R. B. Jones. 1994. Ultrasonic acoustic emissions from excised stems of two Thryptomene species. Physiol. Plant. 92: 431-436. van Doorn, W. G., and E. Otma. 1994. Vascular occlusion in cut flowering rose stems exposed to air: role of water entry into the xylem conduits opened by cutting. J. Plant Physiol. 145:78-82. van Doorn, W. G., and R. J. J. Perik. 1990. Hydroxyquinoline citrate and low pH prevent vascular blockage in stems of cut rose flowers by reducing the number of bacteria. J. Am. Soc. Hort. Sci. 115:979-981. van Doorn, W. G., and M. S. Reid. 1992. Role of ethylene in flower senescence of Gypsophila paniculata 1. Postharvest BioI. Technol. 1:265-272. van Doorn, W. G., and M. S. Reid. 1995. Vascular occlusion in stems of cut rose flowers exposed to air: role of xylem anatomy and rates of transpiration. Physiol. Plant. 93:624-629. van Doorn, W. G., and V. Suiro. 1996. Relationship between cavitation and water uptake in rose stems. Physiol. Plant. 96:305-311. van Doorn, W. G., and L. M. M. Tijskens. 1991. FLORES: a model on the keeping quality of cut flowers. Agr. Systems 35:111-127. van Doorn, W. G., H. C. E. M. Buis, and Y. de Witte. 1986. Effects of exogenous bacterial concentrations on water relations of cut rose flowers, II: Bacteria in the vase solution. Acta Hort. 181 :463-465. van Doorn, W. G., K. Schurer, and Y. de Witte. 1989. Role of endogenous bacteria in vascular blockage of cut rose flowers. J. Plant Physiol. 134:375-381. van Doorn, W. G., Y. de Witte, and R. J. J. Perik. 1990a. Effect of anti-microbial compounds on the number of bacteria in stems of cut rose flowers. J. Appl. Bacteriol. 68:117-122. van Doorn, W. G., F. Thiel, and A. Boekestein. 1990b. Cryoscanning electron microscopy of a layer of extracellular polysaccharides produced by bacterial colonies. Scanning 12:297-299. van Doorn, W. G., A. Clerkx, and A. Boekestein. 1991a. Bacteria as a cause of vascular occlusion in cut fronds of Adiantum raddianum: a scanning electron microscope study. Sci. Hort. 48:299-309.

84

W. G. VAN DOORN

van Doorn, W. G., H. C. M. de Stigter, Y. de Witte, and A. Boekestein. 1991b. Microorganisms at the cut surface and in xylem vessels of rose stems: a scanning electron microscope study. J. Appl. Bacteriol. 70:34-39. van Doorn, W. G., G. Groenewegen, P. A. van de Pol, and C. E. M. Berkholst. 1991c. Effects of carbohydrate and water status of flower opening in cut Madelon roses. Postharvest BioI. Technol. 1:37-57. van Doorn, W. G., H. Harkema, and E. Otma. 1991d. Is vascular blockage in stems of cut lilac flowers mediated by ethylene? Acta Hort. 298:177-181. van Doorn, W. G., F. Thiel, and A. Boekestein. 1991e. Examination of occlusions in xylem vessels of cut rose flowers, using cryoscanning electron microscopy and cryoultramilling cross-sectioning. Scanning 13:37-40. van Doorn, W. G., D. Zagory, Y. de Witte and H. Harkema. 1991f. Effects of vasewater bacteria on the senescence of cut carnation flowers. Postharvest BioI. Technoi. 1:161-168. van Doorn, W. G., D. Zagory, and M. S. Reid. 1991g. Role of ethylene in vascular blockage in cut fronds from the fern Adiantum raddianum. Sci. Hort. 46:161169. van Doorn, W. G., R. R. J. Perik, and P. J. M. Belde. 1993a. Effects of surfactants on the longevity of dry-stored cut flowering stems of rose, Bouvardia, and Astilbe. Postharvest BioI. Technol. 3:69-76. van Doorn, W. G., C. Pak, and C. J. J. Buddendorf. 1993b. Effects of surfactants on the vascular occlusion induced by exposure to air in cut flowering stems of Astil be, Bouvardia, and rose. J. Plant Physiol. 141:251-253. van Doorn, W. G., L. Bakker, and M. Veken. 1994. Effect of dry storage on scape bending in cut Gerbera jamesonii flowers. Postharvest BioI. Technol. 4:261-269. van Doorn, W. G., Y. de Witte, and H. Harkema. 1995. Effects of high numbers of exogenous bacteria on the water relations and longevity of cut carnation flowers. Postharvest BioI. Technol. 6:111-119. van Meeteren, U. 1978. Water relations and keeping quality of cut Gerbera flowers, I: The cause of stem break. Sci. Hart. 8:65-74. van Meeteren, U. 1979. Water relations and keeping quality of cut gerbera flowers, III: Water content, permeability, and dry weight of the petals. Sci. Hart. 10:261269. van Meeteren, U. 1989. Role of air embolism and low water temperature in water balance of cut chrysanthemum flowers. Sci. Hort. 51:275-284. van Mourik, N. M., and P. C. van Os. 1991. Pulse treatment with a surfactant: vase life of Vuylstekeara Cambria 'Plush' prolonged (in Dutch). Vakblad voor de Bloemisterij 46(8):58-59. Vaughan, M. J. 1988. The complete book of cut flower care. Timber, Portland, OR. Veen, H., and S. C. van der Geijn. 1978. Mobility and ionic form of silver as related to longevity of cut carnations. Planta 140:93-96. Venkatarayappa, T., D. P. Murr, and M. J. Tsujita. 1981. Effect of C0 2+ and sucrose on the physiology of cut Samantha roses. J. Hort. Sci. 56:21-25. Wang, C. Y., and J. E. Baker. 1979. Vase life of cut flowers treated with rhizobitoxine analogs, sodium benzoate, and isopentenyl adenosine. HortScience 14:59-60. Weinstein, L. H. and H. J. Laurencot. 1963. Studies on the preservation of cut flowers. Contr. Boyce Thomp. Inst. 22:81-90. Wiggans, S. C., and R. N. Payne. 1963. Keeping quality of cut flowers as influenced by antibiotics and various other agents. Exp. Stat. Bull B-607. Oklahoma State Univ., Stillwater.

1.

WATER RELATIONS OF CUT FLOWERS

85

Wilkinson, S. G. 1977. Composition and structure of bacteriallipopolysaccharides. p. 97-174. In: I. W. Sutherland (ed.), Surface carbohydrates of the prokaryotic cell. Academic, New York. Winkenbach, F. 1970. Zum Stoffwechsel der aufblUhenden und welkenden Karolle de Prunkwinde Ipomoea purpurea. Ber. Schweiz. Bot. Ges. 80:374-390. Woltering, E. J., and W. G. van Doorn 1988. Role of ethylene in senescence of petals: morphological and taxonomical relationships. J. Exp. Bot. 39:1605-1616. Woodson, W. R. 1987. Postharvest handling of bud-cut Freesia flowers. HortScience 22:456-458.

WU, M-J., L. Zacarias, M. E. Saltveit, and M. S. Reid. 1992. Alcohols and carnation senescence. HortScience 27:136-138. Yang, S. F., and H. K. Pratt. 1978. The physiology of ethylene in wounded plant tissues. p. 596-622. In: G. Kahl (ed.), Biochemistry of wounded plant tissues. De Gruyter & Co., Berlin. Zagory, D. and M. S. Reid. 1986. Role of vase solution microorganisms in the life of cut flowers. J. Am. Soc. Hart. Sci. 111:154-158. Zelitch, I. 1963. The control and mechanisms of stomatal movement. p. 18-42. In: I. Zelitch (ed.), Stomata and water relations in plants. Bull. 664. Connecticut Agr. Exp. Stat. (New Haven). Zer, H., and A. Fahn. 1992. Floral nectaries of Rosmarinus officinalis L. Structure, ultrastructure and nectar secretion. Ann. Bot. 70:391-397. Zieslin, N., H. C. Kohl, A. M. Kofranek, and A. H. Halevy. 1978. Changes in the water status of cut roses and its relationship to bent-neck phenomenon. J. Am. Soc. Hort. Sci. 103:176-179. Zimmermann, M. H. 1983. Xylem structure and the ascent of sap. Springer, Berlin.

2

Tissue Culture of Ornamental Flowering Bulbs (Geophytes) Kiu-Wean Kim*

Department of Horticultural Science Yeungnam University, Kyongsan 712-749, Korea A. A. De Hertagh Department of Horticultural Science North Carolina State University Raleigh, North Carolina 27695-7609, USA

I. II.

III.

Introduction Micropropagation A. Production Stages 1. Stage 0: Selection and Preparation of Mother Plants 2. Stage I: Establishment of the Aseptic Culture 3. Stage II: Multiplication of Propagules 4. Stage III: Obtaining, Hardening, and Bulbing of Plantlets 5. Stage IV: Dormancy Breaking of Bulblets 6. Stage V: Transfer to Natural Environments B. Multiplication Systems 1. Enhancement of Axillary Bud Growth 2. Proliferation of Adventitious Buds/Shoots/Bulblets 3. Induction of Adventitious Buds From Callus 4. Plantlet Regeneration by Somatic Embryogenesis C. Somaclonal Variation Virus Elimination A. Meristem Tip Culture B. Organ Culture C. Callus Culture

* I want to express my appreciation to Yeungnam University for providing financial support for my study leave at North Carolina State University from October 1994 to March 1995 and to Dr. K.J. Kim for his assistance with the preparation of manuscript.

Horticultural Reviews, Volume 18, Edited by Jules Janick ISBN 0-471-57334-5 © 1997 John Wiley & Sons, Inc.

87

88 IV.

V. VI.

K. W. KIM AND A. A. DE HERTOGH Breeding and Genetic Improvement A. In Vitro Pollination and Fertilization B. Embryo Rescue and Endosperm Culture C. Ovary. Ovule, Anther, and Pollen Culture D. Protoplast Culture and Fusion E. Callus and Cell Culture F. Gene Transformation G. Cryopreservation Genera Reviewed A. Dicotyledonae B. Monocotyledonae Conclusions Literature Cited

I. INTRODUCTION

Ornamental flowering bulbs are "geophytes" (Raunkiaer 1934; Rees 1989; Halevy 1990; De Hertogh and Le Nard, 1993a), that is, land plants with buds or shoot apices borne on subterranean storage organs. Generally, they are classified into five groups: bulbs, corms, tubers, tuberous roots, and rhizomes (Hartmann et al. 1990; De Hertogh and Le Nard 1993a). Ornamental geophytes are important floricultural crops, with more than 60 genera being grown commercially (De Hertogh and Le Nard 1993b). They are used as cut flowers, potted flowering plants, and garden plants and are grown throughout the world. The area under cultivation has increased significantly in the past 30 years, and the most important bulb production country is The Netherlands. This review focuses only on ornamental geophytes, but extensive tissue culture studies have been conducted on many edible geophytes, such as asparagus, onion, and potato. Ornamental geophytes are heterozygous and are primarily propagated vegetatively. Conventional methods are natural division, scaling (removal of individual scales to form bulblets), twin scaling (two scales attached to a piece of the basal plate), chipping (a mechanical variation of twin scaling), scooping (removal of the basal plate from scales), scoring (cutting of the basal plate), and stem and leaf cuttings (Hartmann et al. 1990; De Hertogh and Le Nard 1993c). These methods have two serious disadvantages. First, it is often difficult to produce a large number of true-to-type plants in a reasonable period of time. Second, diseases can be easily spread from the plants infected with bacteria, fungi, mycoplasma, viroids, or viruses (Byther and Chastagner 1993). Tissue culture techniques can alleviate some of the problems encountered. Advantages for geophytes are that (1) recalcitrant species and new cultivars can be rapidly multiplied, (2)

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

89

virus-free plants may be obtained from virus-infected plants, and (3) the technique can be used for crop improvement. In vitro techniques can also be used for the production of secondary metabolites. Examples include saffron from Crocus sativus (Sano and Himeno 1987; Koyama et al. 1988; Fakhrai and Evans 1990; Sarma et al. 1990,1991); protoanemonin, an acrid compound, from Anemone and Ranunculus (Bonora et al. 1987); colchicine alkaloids from Colchicum (Hayashi et al. 1988); bufadienolide, a cardiac drug, in Urginea (Jha et al. 1991); irones, a fragrant compound, from Iris (Para and Baratti 1992; Gozu et al. 1993; Jehan et al. 1994); and anthocyanin from Oxalis (Crouch et al. 1993). In this review, three major aspects of tissue culture techniques for ornamental geophytes are discussed: (1) general methods for rapid multiplication; (2) the progress made in the elimination of viruses; and (3) examples in which tissue culture techniques are used for breeding and genetic improvement. The literature was searched for over 60 genera (De Hertogh and Le Nard 1993a,b) and research is reported on 52 of them (Table 2.1). II. MICROPROPAGATION

With conventional propagation methods, approximately 10 to 20 years are required to introduce a new tulip or Narcissus cultivar to the market in reasonable quantities (De Hertogh and Le Nard 1993c). In contrast, about 2500 bulblets of Tulipa can be formed from axillary buds (Paterson and Harper 1986), and 1000 bulblets of Narcissus from meristem tip culture (Brunt 1980) can be produced in a year with tissue culture techniques. Tissue culture techniques for floricultural crops have been summarized by Kim (1986), Debergh (1987), Debergh et al. (1990), Sagawa and Kunisaki (1990), and Pierik (1991). There have also been reviews on ornamental geophytes by Hussey (1975a,1980b), Kromer (1985), Krikorian and Kann (1986), Van Aartrijk and van der Linde (1986), and Van der Linde (1992). The advantages of micropropagation have been reviewed by Hussey (1978), Withers (1988), Chu and Kurtz (1990), Sagawa and Kunisaki (1990), and Harper (1991). Micropropagation may be used with ornamental geophytes in several areas: (1) rapid and mass multiplication of new cultivars, species, selections, and recalcitrant species; (2) production, proliferation, and maintenance of disease-free stocks; (3) rejuvenation of cultivars; (4) year-round production of plantlets; (5) storage of plantlets in a small space; (6) supply of a wide range of plant products, such as microcuttings and cultured (Text continues on p. 107.)

co 0

Table 2.1. Tissue culture multiplication systems for geophytes. Plant materials used Genus

Initial

Intermediate

DICOTYLEDONAE Aconitum Axillary buds

Final

Response obtained

References

Shoot

Plantlet

Watad et al. 1995

Anemone

Seed

Seedling

Shoot

Plantlet

Mensuali-Sodi et al. 1993

Begonia

Leaf Petiole

Callus

Shoot Shoot

Plantlet Plantlet

Peck & Cumming 1984 Viseur & Lievens 1987

Tuber

Callus

Callus

Leaf

Leaf

Shoot Plantlet Plantlet

Leaf

Callus Callus Callus Callus Leaf

Plantlet Plantlet-O,E Plantlet Plantlet-O,E Plantlet Plantlet Plantlet-O,E Plantlet-E Plantlet

Loewenberg 1969 Geier 1977 Wainwright & Harwood 1985; Hawkes & Wainwright 1987 Geier 1977 Wicart et al. 1984 Geier 1977 Wicart et al. 1984 Wainwright & Harwood 1985; Hawkes & Wainwright 1987 Murasaki & Tsurushima 1988 Wicart et al. 1984 Kiviharju et al. 1992 Wainwright & Harwood 1985; Hawkes & Wainwright 1987 Wainwright & Harwood 1985; Hawkes & Wainwright 1987 Schwenkel & Grunewaldt 1988 Kiviharju et al. 1992 Kiviharju et al. 1992

Cyclamen

Leaf-blade Anther Petiole Shoot Cotyledon

Leaf

Shoot Callus Callus Leaf

Root

Leaf

Leaf

Plantlet

Peduncle

Shoot

Shoot Callus Callus

Plantlet Plantlet-E Plantlet-E

Ovary

Embryo

Dahlia

Leaf

Gloxinia

Shoot tip Leaf

Bud

Liatris

Flower stem

Oxalis

Shoot tip Internode

Callus

Gavinlertvatana et al. 1979,1982

Shoot Bud

Plantlet Plantlet

Murashige 1974 Takayama 1990

Shoot

Shoot

Plantlet

Stimart & Harbage 1989

Shoot

Shoot

Callus

Cell

Plantlet Plantlet Plantlet Plantlet

Ochatt et al. 1986 Khan et al. 1988 Crouch et al. 1993 Khan et al. 1988

Lamina Cotyledon

Callus Callus Callus Callus Callus Callus

Callus Cell Stem Cell Callus Cell Callus

Hypocotyl

Callus Callus

Shoot Callus

Leaf blade Thalamus

Callus Callus

Callus Callus

Plantlet-E Plantlet-E Plantlet-E Plantlet-E Plantlet-E Plantlet-E Plantlet-E Plantlet-E, bulblet Plantlet Plantlet-E Plantlet-E, bulblet Plantlet-E Plantlet-E

Konar & Nataraja 1964 Konar & Nataraja 1965a Konar et al. 1972 Poli et al. 1989 Kim et al. 1991b Poli et al. 1989 Kim et al. 1991b Kim et al. 1991c Pugliesi et al. 1992 Kim et al. 1991b Kim et al. 1991c Kim et al. 1991b Beruto & Debergh 1992

Plantlet

Hussey 1980b

Callus Callus Plantlet, bulblet

Mullin 1970 Mullin 1970 Hussey 1978

Petiole

Ranunculus

Flower bud Flower stem Petiole

MONOCOTYLEDONAE Agapanthus Rhizome

Allium CD f-'

Root Scale

Callus Callus

Callus Callus Shoot

to

N

Table 2.1.

Continued. Plant materials used

Genus

Allium (continued)

Alstroemeria

Initial Shoot tip with basal plate Umbel Callus Embryo Leaf Flower stem

Intermediate

Final

Vegetative buds

Caladium

Bud Leaf

Ziv et al. 1983

Bulblet Plantlet Plantlet-E

Ziv et al. 1983 Buiteveld & Creemers-Molenaar 1994 Buiteveld et al. 1994

Shoot Bud Shoot

Shoot Bud Shoot

Rhizome

Rhizome

Plantlet Plantlet Plantlet Plantlet Plantlet, rhizome

Callus

Plantlet-O,E

Shoot

Shoot

Shoot, rhizome Plantlet

Hussey 1980b Ziv et al. 1973 Hussey 1980b Lin & Monette 1987 Pierik et al. 1988; Elliott et al. 1993, Han et al. 1993,1994 Gonzales-Benito & Alderson 1990,1992 Gabryszewska & Hampel 1985 Blakesley & Constantine 1992

Bulblet Bulblet

Bulblet Bulblet

Bulblet Plantlet Plantlet

Murashige 1974 De Bruyn et al. 1992 De Bruyn et al. 1992

Plantlet

McIntyre & Whitehone 1974 Ellyard 1979 McComb & Newton 1981

Plantlet Plantlet Plantlet

Cooper & Cohen 1982 Han et al. 1991a Zhu et al. 1993a,b

Rhizome

Anigozanthos (Macropidia)

Bulblet

Cell

Embryo

Scale Twin scale Flower stem

References

Callus

Rhizome tip

Amaryllis belladonna

Response obtained

Shoot

Shoot Callus Shoot

Canna

Rhizome Meristem tip

Clivia

Meristem tip Fruit Seed/embryo Petal Ovary Pedicel

Colchicum

Flower stem

Convallaria

Flower bud Rhizome

Crocus

Corm

Shoot

Callus

Bud Cormlet Ovary Intact corm Apical bud Shoot tip

co w

Endymion

Bulb

Eucharis

Scale

Eucomis

Bulb Leaf

Shoot

Callus

Callus

Shoot Cormlet Callus Callus

Callus

Callus

Bulblet

Plantlet Plantlet

Kromer 1979 Kromer & Kukulczanka 1985

Plantlet Plantlet Plantlet Plantlet Plantlet Plantlet

Finnie Finnie Finnie Finnie Finnie Finnie

Callus

Hayashi et al. 1988,

Plantlet Plantlet

Verron et al. 1995 Verron et al. 1995

Plantlet Cormlet Cormlet Plantlet, cormlet Plantlet Cormlet Cormlet Cormlet Plantlet-E

Kromer 1985 Sutter 1986 Homes et al. 1987 Ilahi et al. 1987 Isa & Ogasawara 1988 Fakhari & Evans 1989,1990 Plessner et al. 1990 Plessner et al. 1990 Ahuja et al. 1994

Callus, shoot

Mullin 1970

Plantlet

Pierik et al. 1983

Plantlet Plantlet

De Lange et al. 1989 De Lange et al. 1989

& & & & & &

van van van van van van

Staden Staden Staden Staden Staden Staden

1995 1995 1995 1995 1995 1995

co ~

Table 2.1.

Continued. Plant materials used

Genus

Freesia

Initial

Interme diate

Final

Callus

Plantlet Plantlet Plantlet Plantlet Plantlet-E Plantlet-E Plantlet Plantlet Plantlet Plantlet Plantlet Plantlet Plantlet Callus, plantlet Plantlet-E Plantlet-E

Shoot Shoot Shoot Bulblet Bulblet

Plantlet Plantlet Plantlet Bulblet Bulblet

Hussey 1976a Hussey 1976a Hussey 1976a Kukulczanka et al. 1989 Kukulczanka et al. 1989

Shoot

Hussey 1980b

Plantlet Plantlet Shoot

Shoot Callus

Shoot

Shoot

Leaf Callus Pedicel Callus Callus Shoot

Callus Callus Shoot

Shoot Shoot

Shoot Shoot

Flower stem Axillary bud Ovary Inflorescence

Fritillaria

Flower stem Leaf Scale Seed

Galanthus

Scale

Shoot Shoot Shoot Bulblet Bulblet

References Bajaj & Pierik 1974 Bajaj & Pierik 1974; Hussey 1975a Hussey 1976a,1977a,1980b Hussey 1975a Bajaj & Pierik 1974 Hussey 1976a,1977a Wang et al. 1990 Wang et al. 1990 Bajaj & Pierik 1974 Stimart & Ascher 1982 Bajaj & Pierik 1974 Pierik & Steegmans 1975,1976 Bajaj & Pierik 1974 Hussey 1976a,1977a,1980b Hussey 1980b Bach 1987 Wang et al. 1990 Wang et al. 1990

Anther Corm

Flower bud

Response obtained

Galtonia

Scale Twin scale

Bulblet Bulblet

Yanagawa & Sakanishi 1980a,b Yanagawa & Sakanishi 1980a,b

Gladiolus

Flower stem

Plantlet, cormlet

Ziv et aI. 1970, Hussey 1975a; Bajaj et aI. 1983 Hussey 1976a,1977a,1980b Kama et aI. 1990 Simonsen & Hildebrandt 1971; Bajaj et aI. 1983; Kim & Goo 1991 Rao et aI. 1991 Ginzburg & Ziv 1973 Hussey 1975a Hussey 1976a,1977a Sutter 1986 Hussey 1975a Bajaj et aI. 1983 Hussey 1977a,b,1979,1980; Ziv 1979; Dantu & Bhojwani 1987; Lilien-Kipnis & Kochba 1987; Steinitz & Lilien-Kipnis 1989; Steinitz et aI. 1991 Bajaj et aI. 1983 De Bruyn & Ferreira 1992 Bajaj et aI. 1983 Kim et aI. 1988,1991a; Kim & Lee 1993 Kim & Han 1993; Goo & Kim 1994 Kama et aI. 1990 Kim & Kang 1992

Shoot

Shoot Callus

Plantlet, cormlet Plantlet Plantlet, cormlet

Shoot

Shoot

Leaf

Shoot Bud Shoot

Shoot Shoot Shoot

Axillary bud

Shoot

Shoot

Plantlet, cormlet Carmel Plantlet Plantlet Plantlet, cormlet Plantlet Plantlet Plantlet, cormlet

Carmel tip

Stolon tip Corm

Callus

Callus

Plantlet Plantlet, cormlet Plantlet Plantlet, cormlet

Callus

Shoot

Plantlet, cormlet

Callus Callus

Cell Callus

Plantlet-E Plantlet-E

Shoot Carmel

co CJ1

r.o

Cl'l

Table 2.1.

Continued. Plant materials used

Genus

Gladiolus (continued)

Gloriosa

Initial

Intermediate

Final

References

Response obtained

Shoot Bud Shoot

Shoot Bud,PLB Shoot

Plantlet Plantlet Plantlet Plantlet Cormlet Plantlet, cormlet

Shoot base

Callus

Cell

Plantlet-E

Bajaj et al. 1983 Bajaj et al. 1983 Bajaj et al. 1983 LHien-Kipnis & Kochba 1987 Ziv 1989,1990a,b Steinitz & LHien-Kipnis 1989; Steinitz et al. 1991 Kama et al. 1990

Seedling

Callus Callus Callus Shoot Callus

Callus Tuberlet Tuberlet Shoot Tuberlet

Shoot Tuberlet Tuberlet Plantlet, tuberlet Tuberlet

Finnie Finnie Finnie Finnie Finnie

Plantlet, bulblet

Kromer 1985

Root Plantlet Plantlet Plantlet Plantlet Plantlet Plantlet Plantlet-E Plantlet Plantlet-E

Mullin 1970 Heuser & Apps 1976 Heuser & Harker 1976 Heuser & Harker 1976 Heuser & Harker 1976 Meyer 1976 Krikorian et al. 1981 Krikorian & Kann 1981 Smith et al. 1989 Smith & Krikorian 1991

Inflorescence Bract Perianth Apical bud

Embryo Tuber

Haemanthus

Twin scale

Hemerocallis

Tuberous root Sepal Filament Flower stem Shoot tip

Callus Callus Callus Callus Callus Callus Callus

Callus Callus Shoot Shoot Shoot Callus Callus Cell Cell Cell

& & & & &

Van Van Van Van Van

Staden Staden Staden Staden Staden

1989 1989 1989 1989 1989

Hippeastrum

Scale

Bud, root Plantlet, bulblet PLB Shoot

PLB Shoot

Bulblet Plantlet Bulblet

Shoot

Shoot 1/4 Bulblet

Plantlet Bulblet Plantlet, bulblet

Shoot Shoot

Shoot Shoot

Plantlet Plantlet Plantlet Plantlet Plantlet

Twin scale

Flower stem Leaf Ovary Peduncle

Hyacinthus

Callus

Scale

Plantlet, bulblet

Shoot Callus

Shoot Callus

Bulblet

Bulblet

Callus

Plantlet

Bulblet Flower bud Basal plate

'" ~

Plantlet Plantlet Bulblet Bulblet Bulblet Plantlet, bulblet Bulblet Bulblet

Mii et a1. 1974 Seabrook & Cumming 1977; Yanagawa & Sakanishi 1977,1980b Okubo et a1. 1990; Blakesley & Constantine 1992 Hussey 1975a; Okubo et a1. 1990; Huang et a1. 1990 Hussey 1976a Han et a1. 1991b Hussey 1975a; Seabrook & Cumming 1977 Hussey 1976a,1980 Hussey 1976a,1980 Seabrook & Cumming 1977 Seabrook & Cumming 1977 Seabrook & Cumming 1977 Pierik & Woets 1971; Pierik & Ruibing 1973; Pierik & Post 1975; Hussey 1975b, Tamura 1978; Amaki et a1. 1984; Chung et a1. 1981a,1983a Hussey 1977a Bae et a1. 1983 Paek et a1. 1983,1987; Saniewski et a1. 1974 Saniewski 1975 Kim et a1. 1981 Hussey 1975b Hussey 1975b

co 0:>

Table 2.1.

Continued. Plant materials used Initial

Genus

Hyacinthus (continued)

Interme diate

Final

Shoot

Shoot Callus

Shoot Shoot

Shoot Leaf Callus

Shoot

Callus

Shoot Shoot Microscale Leaf Bulblet Callus Plantlet

Callus

Callus

Twin scale Leaf

Flower stem

Bulblet Bulblet Bulblet Ovary

Hymenocallis

Twin scale Scale segments Scale bases

Iris

Flower stem Leaf

Response obtained Bulblet Plantlet Plantlet Plantlet, bulblet Plantlet Plantlet, bulblet Plantlet Plantlet, bulblet

References

Plantlet Bulblet Bulblet Callus Plantlet Plantlet Bulblet Plantlet, bulblet Plantlet, bulblet

Hussey 1975a,b Hussey 1976a Hussey 1975a Hussey 1975a,b; Paek et al. 1987 Hussey 1976a,1977a,1980b Bach & Cecot 1988,1989; Hussey 1975a,1980b Hussey 1975a,b; Kim et al. 1981; Chung et al. 1981a,1983a Hussey 1976a,1977a Paek et al. 1987 Chung et al. 1983b Chung et al. 1983b Bae et al. 1983 Hussey 1975a Hussey 1975b,1980b Hussey 1975a,b Hussey 1975a,b

Bulblet Bulblet Bulblet

Yanagawa and Sakanishi 1980a,b Yanagawa and Sakanishi 1980a.b Yangawa and Ito 1988 Meyer et al. 1975 Hussey 1976a,c,1977a,1980b; Bach 1988; Yabuya et al. 1991 Hussey 1976a,1977a Jehan et al. 1994

Shoot

Callus Shoot

Plantlet Plantlet, bulblet

Shoot Callus

Shoot Callus

Plantlet Plantlet-E

Scale

Shoot

Shoot

Twin scale

Shoot

Shoot

Root Shoot Sepal Petal Ovary

Callus Callus Callus Callus Shoot Callus Callus Shoot Callus Callus Callus

Callus Callus Callus Callus Shoot Callus Callus Shoot Callus Callus Callus

Corm

Bud

Shoot

Rhizome Embryo Shoot tip

Ixia

Callus Leaf Callus

Lachenalia

co co

Flower stem Scale

Bulblet Bulblet

Bulblet Bulblet

Leaf

Bulblet

Bulblet

Plantlet Bulblet Bulblet Bulblet Plantlet, bulblet Plantlet Plantlet-E Plantlet-E Plantlet-O,E Bulblet Plantlet-E Plantlet-E Plantlet Plantlet-E Plantlet-E Plantlet-E

Hussey 1976a Van der Linde et al. 1988 Hussey 1976a,1980b Van der Linde et al. 1988 Kromer 1985 Gozu et al. 1993 Jehan et al. 1994 Radojevic et al. 1987 Radojevic & Subotic 1992 Mielke & Anderson 1989 Reuther 1977 Laublin et al. 1991 Leifert et al. 1992a,b Jehan et al. 1994 Jehan et al. 1994 Jehan et al. 1994

Plantlet, cormlet Plantlet Plantlet Plantlet Plantlet

Sutter Meyer Meyer Meyer Meyer

Plantlet Plantlet Shoot Plantlet

Klesser Klesser Hussey Klesser

1986 & van & van & van & van

Staden Staden Staden Staden

1988 1988 1988 1988

& Nel 1976 & Nel 1976

1980b & Nel 1976

Leucojum

Twin scale

Bulblet

Kromer 1985

Lilium

Scale

Plantlet, bulblet

Robb 1957; Hackett 1969; Stimart & Ascher 1978;

Leaf

Shoot

Shoot

Plantlet Bulblet

Bulblet

Microscale

Bulblet

Ovary

Plantlet

Leaf

Plantlet, bulblet

Seed Peduncle Tepal

Callus

Shoot

Plantlet Bulblet Bulblet

Bulbil Style Filament Anther Pedicel Embryo

Lycoris

Muscari

Bulblet

Bulblet

Bulblet

Microscale

Bulblet Bulblet Bulblet Bulblet Bulblet Plantlet Bulblet

Shoot Shoot

Shoot Shoot

Bulblet Bulblet Plantlet Plantlet

Yanagawa & Sakanishi 19S0a,b Yanagawa & Sakanishi 19S0a,b Huang & Liu 19S9 Huang & Liu 19S9

Bulblet

Bulblet Callus Callus

Plantlet, bulblet Plantlet Plantlet Plantlet, bulblet Plantlet Plantlet

Hussey Kromer Hussey Kromer Hussey Hussey

Scale Twin scale Shoot tip Twin scale

Callus Leaf 0""" """

Hussey 1976a, 1977a Niimi & Onozawa 1979; Niimi 19S4,19S6 Niimi 19S5, Niimi et al. 19S5; Niimi & Saito 1990 Kato & Yasutake 1977; Novak & Petru 19S1; Chung et al. 19S4 Bennici 1979 Takayama & Misawa 1979 Takayama & Misawa 1979; Niimi & Watanabe 19S2; Niimi 19S4; Chung et al. 19S4 Chung et al. 19S1c,19S4 Niimi & Watanabe 1982 Niimi & Watanabe 1982 Paek et al. 19S7 Chung et al. 1984 Liu & Burger 19S6 Maesato et al. 1994

Callus

1975a; Kromer 1985,19S9 19S5 1975a 19S5 1975a 1975a

Scale Disk Mini-chip

Nerine

Ornithogalum

Shoot Shoot

Shoot Shoot Shoot

Plantlet, bulblet Bulblet Bulblet

Shoot

Leaf

Shoot

Twin scale

Bulblet

1/2 Bulblet

Plantlet

Scale Leaf Flower stem

Shoot Shoot Shoot

Peduncle Ovary

Bud Bud

Shoot Shoot Shoot Bulblet Bud,PLB Bud, PLB

Plantlet Plantlet Plantlet Plantlet Plantlet, bulblet Plantlet, bulblet

Twin scale

Bulblet Callus

Leaf Plantlet Bulblet Shoot Callus

Leaf Bulblet Shoot Callus

Shoot Bulblet Callus

Shoot Bulblet Callus

Shoot Callus

Shoot Callus

Flower stem

Ovary

~

0

w

Plantlet Plantlet Plantlet, Plantlet Plantlet, Plantlet Plantlet, Plantlet, Plantlet Plantlet Plantlet, Plantlet Plantlet

bulblet bulblet bulblet bulblet bulblet

Hussey 1977a,1982 Hosoki & Asahira 1980 Squires & Langton 1990; Squires et al. 1991; Chow et al. 1992a,b Chow et al. 1993 Pierik & IppeI1977; Grootaarts et al. 1981 Hussey 1980b Hussey 1980b Hussey 1980b Pierik & Steegmans 1986 Ziv 1990a Ziv 1990a Hussey 1975a; Yanagawa & Sakanishi 1980a,b Hussey 1975a Hussey 1975a Hussey 1976a,b Klesser & Nel 1976 Hussey 1976b, Nel1981 Hussey 1975a,1976b Hussey 1975a,1976a,b Hussey 1976a,b Klesser & Nel1976 Hussey 1975a,1976a,b Hussey 1975a,1976b Hussey 1976b Hussey 1975a,1976a

Ovary Callus Scale Anther

Sparaxis

Callus Callus

Callus Callus

Shoot

Shoot

Shoot Shoot

Shoot Shoot Seedling

Corm Flower stem Leaf Seed

Tu lip a

Scale

Plantlet Plantlet Bulblet Plantlet Plantlet-E

Hussey 1975a Hussey 1975a Yanagawa & Sakanishi 1980a,b Chakravarty & Sen 1987 Chakravarty & Sen 1989

Plantlet Plantlet Plantlet Plantlet Plantlet Cormlet

Hussey 1975a Hussey 1976a,1980b Hussey 1975a Hussey 1976a,1980b Hussey 1976a,1980b Hauser & Horn 1991

Bulblet

Nishiuchi & Myodo 1976; Nishiuchi 1979,1986; Riviere & Muller 1979 Wright & Alderson 1980; Alderson et a1. 1983 Kim et a1. 1989 Wright & Alderson 1980 Hussey 1980b Paterson & Harper 1986 Hussey 1975a; Wright & Alderson 1980 Alderson et a1. 1983,1986; Rice et a1. 1983; Taeb & Alderson 1987,1990a,b; Le Nard 1989; Le Nard et a1. 1987; Baker et a1. 1990; Alderson & Taeb 1990a,b Kim et a1. 1989

Shoot Callus

Callus

Shoot

Shoot

Axillary bud Flower stem

,...., 0

CJl

Shoot Shoot Plantlet, bulblet Shoot

Shoot

Shoot

Plantlet, bulblet

Callus

Callus

Shoot

>-'

o

O'l

Table 2.1.

Continued. Plant materials used

Genus

Initial

Intermediate

Final

Response obtained

References

Tu lip a (continued)

Flower bud Leaf

Shoot

Shoot

Bulblet Plantlet

Paek 1982 Paterson & Harper 1986

Urginea

Scale

Callus

Bulblet

Plantlet, bulblet

Flower stem

Callus Callus Bulblet Callus

Callus Shoot Shoot Shoot

Plantlet-O,E Plantlet Plantlet Plantlet

Jha et a1. 1984,1991; Jha and Sen 1986b Jha & Sen 1986a, Jha et a1. 1991 Jha & Sen 1987b EI Grari & Backhaus 1987 Jha et a1. 1991

Zantedeschia

Apical bud

Shoot

Shoot

Plantlet

Cohen 1981

Zephyranthes

Twin-scale

Bulblet

Bulblet

Plantlet

Furmanowa & Oledzka 1981

Note. Plantlet-E ; Plantlets obtained by somatic embryogenesis; Plantlet-O,E, plantlets obtained by organogenesis and somatic embryogenesis; PLB, protocorm-like bodies.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

107

shoot clumps; (7) easy international exchange of plant materials; and (8) production of secondary metabolites. A. Production Stages

Many ornamental geophytes have a dormant period. Their micropropagation is thus complicated and generally requires one more step than conventional tissue culture techniques (Murashige 1974). Therefore, a stage V is included with the procedures for obtaining plantlets by callus culture and somatic embryogenesis. 1. Stage 0: Selection and Preparation of Mother Plants. The primary objective of" Stage 0" is to select and grow mother plants that are healthy and true to type. Since most geophytes are grown in soil, care must be taken to minimize bacterial, fungal, and virus infection in the mother plants and to produce highly responsive explants (Hussey 1975a; Debergh and Maene 1981; Niimi and Watanabe 1982). Contamination can often be reduced by using low humidities (75%), no overhead waterings, and forced shoots (Hosoki and Sagawa 1977; Debergh and Read 1991). In addition, mother plants can be exposed to specific temperature (Lilium, Stimart and Ascher 1981b; Tulipa, Baker et al. 1990) or light treatments (Dahlia, Gavinlertvatana et al. 1979) that reduce infections. Cytokinins applied during "Stage I" can produce highly responsive explants (Debergh 1987). Lastly, Amaki et al. (1984) reported that long-term storage and silica gel desiccation of hyacinth bulbs in vivo effectively increased the number of bulblets formed from scale explants. 2. Stage I: Establishment of the Aseptic Culture. Many plant organs of ornamental geophytes can be used as explants (Table 2.1), but juvenile ones are usually the most responsive. As indicated previously, contamination is a major problem in "Stage I" and can result in a significant loss of cultures (Paterson and Harper 1986; Leifert et al. 1989a,b; 1992a,b; Leifert and Waites 1992). To remove contaminants from explants of geophytic storage organs, a thorough sterilization is required (Hussey 1975a; Geier 1977; Niimi and Watanabe 1982). Also, browning caused by oxidation can be prevented by antioxidants such as ascorbic acid (Ziv et al. 1970; Debergh and Read 1991). Environmental conditions that can increase the success level are nutrient composition, plant growth regulators (PGRs), light, temperature, composition of the atmosphere and culture methods (Read 1990).

108

K. W. KIM AND A. A. DE HERTOGH

3. Stage II: Multiplication ofPropagules. Multiplication of the shoots (buds and bulblets) obtained from explants can be achieved by (1) enhancement of axillary bud growth, (2) proliferation of adventitious shoots, (3) induction of adventitious buds from callus, and (4) plantlet regeneration by somatic embryogenesis. Genetic stability and the multiplication rate must be considered when selecting the multiplication system. Generally, supplementation of the culture medium with cytokinin (Hussey 1976a; Kromer 1985) or plant growth retardants (Ziv 1989,1990a,b), and the use of liquid shake culture (Takayama et al. 1982; Ziv 1989) and bioreactors (Ziv 1990a,b) maximize shoot proliferation. Cytokinins may produce negative effects on micropropagation (Hussey 1976a; Taeb and Alderson 1987; Pierik et al. 1988; Ziv 1990a; Maesato et al. 1991). Some examples are (1) a decrease in rooting in "Stage III," (2) physiological disorders and malformations such as hyperhydricity (Debergh et al. 1992), which was previously known as vitrification, and (3) decreased survival of the plantlets when transferred from in vitro to in vivo conditions. Generally, a subculture cycle requires 4 to 18 weeks (Paterson and Harper 1986). "Stage II" takes 1 to 3 years depending on the species, and its ability to produce shoots decreases as the culture period is extended (Jehan et al. 1994). 4. Stage III: Obtaining, Hardening, and Bulbing of Plantlets. The objective of this stage is to obtain plantlets and bulblets from shoots for transfer to soil. To reduce losses of small bulblets and to prevent dormancy, plantlets are often directly transplanted (Ziv 1979; LilienKipnis and Kochba 1987). Rooting can be enhanced with the following procedures: (1) adding auxin and/or activated charcoal (AC) in the culture medium (Cumming and Peck 1984; Yabuya et al. 1991), (2) increasing the auxin to cytokinin ratio, and (3) using half-strength salts and sucrose (Seabrook et al. 1976; Ziv 1979; Cumming and Peck, 1984). Hardening, which increases tolerance to moisture stress and prevents hyperhydricity, is required for acclimatization from the heterotrophic to autotrophic state. Also, the agar concentration and light intensity should be increased (Ziv 1979). For some genotypes, it is desirable to directly induce bulblet formation (Hosoki and Asahira 1980; Hussey 1982; Steinitz and Yahel 1982; Van Aartrijk and van der Linde 1986; Hauser and Horn 1991; Kim and Han 1993). The benefits to this procedure are (1) the elimination of in vitro rooting, (2) the prevention of hyperhydricity, (3) the elimination of hardening, (4) increased survival rates, and (5) a shorter bulb production period. The following conditions can enhance bulb formation: (1) a high sucrose concentration (Dantu and Bhojwani 1987; Ziv 1989;

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

109

Chow et al. 1992a; Kim and Han 1993), (2) the use of plant growth retardants (Ginzberg and Ziv 1973; Ziv 1989; Steinitz and LilienKipnis 1989; Steinitz et al. 1991; Kim and Han 1993), (3) the application of low temperatures (Alderson et al. 1986; Anderson et al. 1990; Baker et al. 1990), and (4) the use of aged shoots (Baker et al. 1990). Dormancy of in vitro formed bulblets can occur spontaneously (Hussey 1977a,1982; El Grari and Backhaus 1987; Lilien-Kipnis and Kochba 1987; Anderson et al. 1990; Kim 1991). 5. Stage IV: Dormancy Breaking of Bulblets. Most bulblets obtained in "Stage III" are dormant and the level is affected by the sucrose concentration, bulblet age, and culture temperature (Takayama and Misawa 1980; Stirnart and Ascher 1981b; Paffen et al. 1990). High gibberellin (GA) concentration, low sucrose concentrations, and low temperatures have reduced the dormancy level of Lilium bulblets (Aguettaz et al. 1990; Kim 1991; Djilianov et al. 1994). The addition of fluridone, an inhibitor of abscisic acid (ABA), to the culture medium also prevented dormancy in Lilium (Kim 1991; Kim et al. 1994). Storage at 2 to 5°C for 30 to 100 days has been used to break the dormancy of Dutch Iris bulblets (Anderson et al. 1990), lily bulblets (Takayama and Misawa 1980; Stimart et al. 1982; De Klerk et al. 1992), Ixia and Gladiolus cormlets (Sutter 1986), and Narcissus bulblets (Hussey 1982). The use of GA 3 in combination with-low temperature storage was synergistic in breaking dormancy with Lilium rubellum (Niimi et al. 1988). 6. Stage V: Transfer to Natural Environments. Depending on the species, either rooted shoots (Simonsen and Hildebrandt 1971; Pierik and Steegmans 1975; Ziv 1979; Stimart and Ascher 1982; LilienKipnis and Kochba 1987), unrooted shoots (Debergh and Maene 1981; Read and Fellman 1985), or bulblets (Takayama and Misawa 1980; Hussey 1982; Sutter 1986; Anderson et al. 1990; Kim and Han 1993) have been successfully transferred to sterile rooting media. Special care is needed, however, for transfer of plantlets from an in vitro environment to greenhouse conditions (Lilien-Kipnis and Kochba 1987). In the greenhouse, the relative humidity must be high and light intensities low for several days until the plantlets become autotrophic (Read and Fellman 1985; Debergh and Read 1991). Subsequently, plantlets can be grown under lower humidities and higher light intensities. Some bulblets, such as Lilium, do not require these treatments even though they contain leaves and stems (Novak and Petru 1981).

110

K. W. KIM AND A. A. DE HERTOGH

B. Multiplication Systems Multiplication systems for production "Stage II" for ornamental geophytes have been reviewed by Hussey (1978,1980b), Krikorian and Kann (1986), Van Aartrijk and van der Linde (1986), Pierik (1991), and Van der Linde (1992). Rapid multiplication of geophytes involves four systems: (1) enhancement of axillary bud growth, (2) proliferation of adventitious bud/shoots/bulblets, (3) induction of adventitious buds from callus, and (4) plant regeneration by somatic embryogenesis. 1. Enhancement of Axillary Bud Growth. Generally, explants with buds are used to initiate axillary bud growth. Examples include meristem shoot tips, axillary buds, nodes of floral scapes, cormel tips, and rhizome buds (Table 2.1). Cytokinin concentrations can affect the growth rate of axillary buds, but is species dependent. Shoots obtained with cytokinins usually form a miniature cluster, since it usually stimulates development of axillary buds whose growth is inhibited by apical dominance (Hussey 1976a,1977b). Proliferated shoot clusters are separated to initiate other shoot clusters. If the medium produces normal shoot branching, the process can be continued almost indefinitely without deleterious effects. When the desired number of shoots is obtained, the clusters can be either excised and rooted or induced to form bulbs either in vitro or directly in soil. Serial subculture is generally carried out every 4 to 8 weeks and the multiplication rate is generally 5 to 10 times per subculture. Compared to the other methods, the major advantage of this system is genetic stability and callus or disorganized growth can be avoided (Murashige 1974; Hussey, 1980a,b). Theoretically, this system can be applied to any species that produces axillary buds, however, it may be difficult to distinguish between axillary and adventitious buds (Hussey 1976a). Adventitious buds can be formed with high cytokinin levels (Pierik 1991) or after a series of subcultures (Debergh and Read 1991).

2. Proliferation of Adventitious Buds/Shoots/Bulblets. Some geophytes produce few or no axillary buds (Hussey 1977a,1980a,b), and adventitious shoot proliferation is induced directly from the explants without buds. Examples include leaves, petioles, floral scapes, sepals, petals, peduncles, pedicels, ovaries, anthers, scales, basal plates, and rhizome, corm, and tuber sections (Table 2.1). Whenever possible, the tissues or organs selected should be young and small in size because they have a high morphogenic ability (Pierik 1991). With

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

111

most species, adventitious shoots are readily induced by a high cytokinin to auxin ratio in the culture medium (Seabrook et al. 1976; Hussey 1982; Maesato et al. 1994). However, with Lilium scale explants, either PGRs were not needed (Robb 1957; Takayama and Misawa 1979; Novak and Petru 1981; Niimi 1985) or a low ratio of cytokinin to auxin is needed (Van Aartrijk and Blom-Barnhoorn 1981). With Hyacinthus orientalis, shoot formation on leaf and stem explants was induced by low auxin concentrations (Hussey 1975b). Thus, it is critical to establish the optimum ratio of cytokinin to auxin for each culture medium. Adventitious shoots can be multiplied by three methods: (1) Shoot clumps can be dissected into several segments; (2) the apical meristem can be destroyed to remove apical dominance; and (3) shoot clusters, measured from the base of the shoot clump, can be regularly trimmed to about 3 to 5 mm. After using one of these techniques, shoots can be rooted or bulbing can be induced either in vitro or in soil. The major disadvantage of this system is the high probability of genetic and epigenetic variations (Broertjes et al. 1968; Pierik 1991). This occurs because the regenerates originated either from a single cell or a very small group of cells (Van Aartrijk and van der Linde 1986) or high levels of PGRs were used. The level of genetic variation obtained is usually between that of the axillary branching system and the callus system (Hussey 1978). 3. Induction of Adventitious Buds From Callus. Callus is an unorganized mass of proliferating cells that can be induced from explants of various tissues or organs in most geophytes (Table 2.1). The effective concentration of the exogenous PGR varies with the species, but typically callus formation is favored by high levels of an exogenous PGR. Auxin is generally used for callus induction, occasionally in combination with cytokinin (Cumming and Peck 1984). Synthetic auxins like a-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) are widely used (Hussey 1975a; Kromer 1985; Kim et al. 1988). Organogenesis occurs when the callus is transferred to a medium containing either no PGR or a very low concentration (Kim et al. 1988,1991a). In most cases, callus culture used for micropropagation has some disadvantages. It has a tendency not only to accumulate genetically aberrant cells and lose regeneration ability over time (Hussey 1975a,1978; Chakravarty and Sen 1987; Jha and Sen 1987a), but also not to produce totipotent callus (Table 2.1). To minimize genetic variations, the frequency of subculturing should be reduced and the concentration of PGR lowered. Also, plantlet regeneration through callus must be avoided. True-to-type plantlets

112

K. W. KIM AND A. A. DE HERTOGH

have been regenerated from Freesia and Lilium callus (Simmonds and Cumming 1976; Stimart and Ascher 1982; Mii et a1. 1994). Callus culture can be useful for nonconventional techniques for crop improvement, such as, protoplast fusion and genetic transformation. 4. Plantlet Regeneration by Somatic Embryogenesis. Somatic embryogenesis is the regeneration process in which embryos arise from somatic cells. They may be formed directly from explants or indirectly from calli (Table 2.1). For the propagation cycle to be complete, the embryos must be grown into complete plants and established in soil. Somatic embryogenesis has the potential to be the most effective and fastest means of micropropagation (Tisserat et a1. 1979). Theoretically, millions of plants can be produced from a very small volume of cells. The origin of the embryos can be a single cell (Wang et a1. 1990) or multicellular (Konar et a1. 1972). There is always a possibility of somaclonal variation, but Freesia (Wang et a1. 1990) and Crocus (Ahuja et a1. 1994) plants regenerated by somatic embryogenesis were morphologically normal. More research is needed to improve the storage and planting of the embryos. In addition, Redenbaugh et al. (1986) has proposed the development of embryo coating and encapsulation to produce artificial-type seeds.

C. Somaclonal Variation Depending on the genotype and the propagation method, asexually propagated floricultural crops often produce variants and chimeras (Bennici 1979; Stimart et a1. 1980; Qu et a1. 1988) that are due to genetic, epigenetic, and physiological changes (Kim 1987). Genetic variation is the most problematic, since it causes permanent changes (Hussey 1978; Khan et a1. 1988). Plants derived from terminal and axillary buds are less prone to genetic change than those derived from other explants (Hussey 1978; Bennici 1979; Van Aartrijk and van der Linde 1986; Hakkaart and Versluijs 1988; Foxe 1991). However, compared to other plants, the number of apical meristems in most ornamental geophytes is low. Thus, rapid multiplication of shoots in vitro is achieved mainly through the formation of adventitious buds derived from single cells or a small group of cells. It is possible, however, that one of these cells may mutate (Hussey 1976a,1978; Bennici 1979). Some differentiated cells in mature organs are polyploid. Therefore, it is critical to obtain organ explants with either undifferentiated cells or meristematic tissue (Hussey 1980b). No genetic variation was observed in Lilium when plants were regenerated from leaves (Liu and Burger 1986) or scales (Takayama et a1. 1982).

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

113

Callus cultures have produced genotypic and phenotypic changes with Ornithogalum (Hussey 1976a,1980b), Lilium (Bennici 1979; Stimart et a1. 1980; Qu et a1. 1988), Oxalis (Escandon et a1. 1989), Hemerocallis (Griesbach 1989), and Urginea Uha and Sen 1990). Thus, genetic stability must be thoroughly checked after micropropagation by callus systems. Some cultures that have shown genetic stability include Begonia (Viseur and Lievens 1987), Cyclamen (Kiviharju et a1. 1992), Freesia (Hussey 1977a; Stimart and Ascher 1982), Hemerocallis (Heuser and Apps 1976; Krikorian and Kann 1981; Krikorian et a1. 1981), Iris (Meyer et a1. 1975; Reuther 1977; Radojevic et a1. 1987; Radojevic and Subotic 1992; Laublin et a1. 1991; Jehan et a1. 1994), Lilium (Sheridan 1968; Hussey 1977a; Simmonds and Cumming 1976; Mii et a1. 1994), Ranunculus (Beruto and Debergh 1992), Scilla (Chakravarty and Sen 1987), and Urginea Uha and Sen 1987a). With most geophytes, genetic stability is more common than instability and multiplication by axillary branching systems appears to be the most desirable method to minimize variations (Hussey 1978,1980b).

III. VIRUS ELIMINATION About 50 viruses are known to infect geophytes (Moore et a1. 1979; Byther and Chastagner 1993) and new ones are constantly being discovered (Phillips and Brunt 1986; Asjes 1990). Arabis mosaic virus, bean yellow mosaic virus, cucumber mosaic virus, tobacco rattle virus, tobacco mosaic virus, tobacco ringspot virus, tomato spotted wilt virus, and turnip mosaic virus are among the most widely detected. They are transmitted by aphids, thrips, nematodes, and mechanical procedures employed during propagation (Moore et a1. 1979; Byther and Chastagner 1993). Once a stock is infected, all progeny are also infected, because most are asexually propagated. Under certain conditions the virus symptoms may be masked, but most viruses seriously affect bulb growth and development (Asjes 1990). Virus-free plants are generally taller, have more vigor and a longer vase life, and will produce larger bulbs than virus-infected ones. Thus, virusfree plants improve not only the quantity but also the quality of marketable bulbs and bulb flowers. Virus-free ornamental geophytes can be obtained by (1) visual selection of mother plants in the field or greenhouse, (2) selection of noninfected areas within a plant, (3) in vitro culture, (4) thermotherapy, and (5) chemotherapy (Hollings 1965; Nyland and Goheen 1969; Wang and Hu 1980; Horst 1988; Asjes 1990; Lawson 1990).

114

K. W. KIM AND A. A. DE HERTOGH

Visual selection of mother plants is limited because virus symptoms may be masked under some environmental conditions. Since viruses are distributed irregularly throughout the plant, virus-free clones can be obtained by careful selection and vegetative propagation of noninfected explants. For example, spotted wilt virus-free Dahlia plants have been obtained from meristem tip cuttings (Holmes 1955) and Narcissus bulbs free from narcissus tip necrosis virus and narcissus late-season virus were obtained by Mowat (1980) using shoot tips and twin scaling, respectively. Thus, shoot and meristem tip techniques have been widely used for rapid multiplication of virusfree materials (Allen 1974; Allen et a1. 1980). Thermotherapy, which is a specific high-temperature treatment of the plant, inactivates viruses by inhibiting their multiplication and movement (Horst 1988). This method has been used extensively with carnations and Gladiolus. Chemotherapy, in which antiviral agents are used, has been relatively ineffective in producing virus-free plants and is not widely used (Horst 1988). A desirable solution for obtaining virus-free plants will be to produce virus-resistant geophytes by gene transformation (Lawson 1990), but this may never occur. Thus, researchers have focused on the elimination of virus activities in virus-infected plants by in vitro culture. Thermotherapy (30 to 40°C) has been applied either to mother plants or storage organs before explant excision or to in vitro cultures (Nyland and Goheen 1969; Stone 1973; Welvaert et a1. 1980; Read 1990). For chemotherapy, amatadin hydrochlorite, azauracil, guanidine hydrochlorite, vidarabine, and virazole have been used in culture media (Long and Casseles 1986; Phillips 1990). This procedure has been effective with Hyacinthus (Blom-Barnhoorn et a1. 1986), but only slightly effective with Lilium (Blom-Barnhoorn and van Aartrijk 1985; Cohen et a1. 1985). Combinations of thermotherapy and chemotherapy have also been employed (Brants and Vermeulen 1965; Baruch and Quak 1966; Read 1990). Virus-free geophytes have been successfully obtained through in vitro culture (Table 2.2). A. Meristem Tip Culture Since only limited quantities of virus-free geophytes are available, these clones must be rapidly increased. However, tests must initially be carried out to confirm the absence of viruses. Virus-free geophytes obtained from tissue culture can be easily reinfected in the field (Lawson 1990). Thus, special precautions to eliminate vectors and enhance the propagation rate must be carried out. Morel and Martin

Table 2.2.

In vitro culture systems to eliminate viruses from geophytes. Virus eliminated

Materials used

Cucumber mosaic Virus-symptomless z

Meristem tipY Meristem tip,

Virus-symptomless Dahlia mosaic

Meristem tip, grafting Meristem tip Meristem tip, axillary meristem

Morel & Martin 1952 Mori & Hosokawa 1977 Mullin & Schlegel 1978

Tobacco rattle Tomato spotted wilt Another potyvirus

Meristem tip, axillary meristem Meristem tip Meristem tip, axillary meristem Meristem tip Axillary meristem Axillary meristem Axillary meristem

Hakkaart & Versluijs 1985,1988 Van Zaayen et al. 1992 Hakkaart & Versluijs 1985,1988 Van Zaayen et al. 1992 Hakkaart & Versluijs 1985 Hakkaart & Versluijs 1985 Hakkaart & Versluijs 1985

Caladium

Dasheen mosaic

Meristem

Hartman 1974

Freesia

Virus-symptomless Bean yellow mosaic Cucumber mosaic Freesia mosaic

Meristem Meristem Meristem Meristem

Gladiolus

Virus-symptomless Gladiolus mosaic Bean yellow mosaic

Callus Ovary, anther, t'GUUHLlV, Meristem tip, axillary meristem

Cucumber mosaic

Meristem tip, leaf tip, callus Meristem tip

Genus DICOTYLEDONAE Begonia

Dahlia

MONOCOTYLEDONAE Alstroemeria Alstroemeria carla Alstroemeria mosaic

References

meristem

tip tip, axillary meristem tip, axillary meristem tip, axillary meristem

.....

.....

CJl

Welvaert et al. 1980 Samyn et al. 1984

Brants & Vermeulen 1965 Bertaccini et al. 1989; Foxe 1991 Bertaccini et al. 1989 Bertaccini et al. 1989; Foxe 1991 Simonsen & Hildebrandt 1971 Zhen et al. 1984 Logan & Zettler 1985; Bertaccini & Marani 1986 Aminuddin & Singh 1985 Logan & Zettler 1985

Stone et al. 1975

Narcissus Narcissus virus Q Narcissus yellow stripe Tobacco rattle Unidentified filaments

Meristem tip, star-cutting, chipping Meristem Meristem tip Meristem tip, star-cutting, chipping Twin-scale Meristem tip Meristem tip, twin-scale Meristem tip Meristem tip Meristem tip Meristem tip Meristem tip Meristem tip Meristem tip Meristem tip Meristem tip Meristem tip Meristem tip

Nerine

Nerine latent

Meristem tip

Hakkaart et al. 1975

Ornithogalum

Ornithogalum mosaic

Leaf

Vcelar et al. 1992

Polianthes

Virus-symptomless

Meristem tip

Wang & Hu 1980

Narcissus degeneration Narcissus late yellows Narcissus mosaic Narcissus tip necrosis Narcissus white streak Raspberry ringspot Strawberry latent ring spot Tomato black ring

Z

Y

i-' i-'

'1

Virus-symptomless-specific virus symptoms were not detected. Meristem tip-meristems excised from apical shoot, bulb, or rhizome apex.

Mowat 1980; Phillips 1990 Stone 1973 Stone et al. 1975 Mowat 1980 Mowat 1980; Phillips 1990 Mowat 1980 Phillips 1990 Mowat 1980 Mowat 1980 Mowat 1980 Mowat 1980 Brunt 1980 Phillips 1990 Phillips 1990 Phillips 1990 Phillips 1990 Phillips 1990

118

K. W. KIM AND A. A. DE HERTOGH

(1952) used Dahlia and were the first to demonstrate that virus-free clones of geophytes could be produced by meristem tip culture. At present, meristem tip culture of terminal and axillary buds is widely used to obtain virus-free clones (Table 2.2). The major advantage is the ability to eliminate known and unknown viruses. Apical shoot meristems usually consist of an apical dome of less than 0.1 mm in height, lack vascular connections, and are virus-free (Langhans et al. 1977; Mori and Hosokawa 1977; Wang and Hu 1980; Horst 1988). However, since apical meristems are extremely small, they are difficult to isolate and keep viable (Vcelar et al. 1992). Therefore, slightly larger apical shoot tips comprising of a dome with either one or two leaf primordia (Hartmann 1974; Asjes et al. 1974) or several leaf primordia (Kromer and Kukulczanka 1985; Foxe 1991; Logan and Zettler 1985) are used. This produces higher survival rates, but fewer virusfree plants. Thus, it is important to isolate the smallest apical meristem possible in order to achieve high yields of virus-free plants. Due to the limited number of apical meristems in the mother plants, axillary and adventitious buds/bulblets formed on scales are often used (Asjes et al. 1974; Blom-Barnhoorn and van Aartrijk 1985).

B. Organ Culture In addition to apical meristems, virus-free plants have been produced from geophytic organs. Iris mosaic virus and iris severe mosaic virus-free bulbs were obtained from basal plate of Iris hollandica bulbs (Allen and Anderson 1980). Gladiolus mosaic virus-free corms have been produced from young Gladiolus inflorescences (Zhen et al. 1984), while bean yellow mosaic virus-free corms were regenerated from leaf tips of in vitro-derived plantlets of Gladiolus (Aminuddin and Singh 1985). The production of virus-free Lilium bulbs from scales and virus-free Narcissus bulbs from twin scales has also been reported (Table 2.2). C. Callus Culture Virus concentrations can be lowered by repeated subcultures of callus (Mori and Hosokawa 1977). This procedure has been successful with Gladiolus (Simonsen and Hildebrandt 1971; Aminuddin and Singh 1985) and Iris hollandica (Hirata and Kunishige 1982). It is not widely used, however, because of the genetic instability of regenerated plants and low success rates. An exception is Freesia, in which the genetic stability of virus-free clones was maintained over an extended period (Stimart and Ascher 1982).

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

119

IV. BREEDING AND GENETIC IMPROVEMENT In vitro techniques can (1) shorten the generation cycle, (2) overcome interspecific hybridization barriers, (3) improve plant characteristics through genetic variability, and (4) conserve selected genetic material. Most studies reported for ornamental geophytes are on Lilium (Table 2.3). A. In Vitro Pollination and Fertilization Van Tuyl et al. (1991) and Janson (1993) have used several in vitro techniques to overcome prefertilization barriers with Lilium. Among them are (1) cut-style pollination, in which pollen grains were applied to the top of the ovary after removing the style; (2) grafted style pollination, in which the style with germinated pollen was cut 1 to 2 mm above the ovary and then grafted onto the ovary of another plant; and (3) placenta pollination, in which pollen grains were applied to the placenta after cutting the ovary longitudinally into six sectors. Fertilization has been achieved, but the success rates were lower than with normal stigmatic pollination in vivo (Janson 1993). B. Embryo Rescue and Endosperm Culture After fertilization, embryo abortion and/ or endosperm degeneration can produce failures during interspecific hybridization of some geophytes. Most Lilium hybrids obtained from crosses between species tend to be sterile (Asano 1982; Van Tuyl et al. 1986). The endosperm can degenerate at various stages of development and microscopic examinations revealed that abnormal seeds from abortions contained a small spherical or oval embryo (Kim and Sung 1990). Complete embryo development was prevented by the toxin ferulic acid. Thus, embryo rescue has been employed not only to prevent abortion but also to achieve interspecific crosses. It is possible to obtain lily plants by culturing embryos, 40 to 70 days after they were pollinated, on solidified medium in vitro (North 1975; Asano and Myodo 1977a,b; Van Tuyl et al. 1986). Dissecting very young embryos is difficult because of their small size. Asano (1980) rescued embryos of 0.3 to 0.4 mm in length, 35 days after pollination, using a nurse endosperm technique. Kim and Sung (1990) produced plantlets from immature embryos about 0.14 mm length, 4 weeks after intrastylar pollination without using nurse endosperm. The formation of bulblets using embryo culture has been reported in Tulipa by Aubert et al. (1986).

......

N

o

Table 2.3.

In vitro breeding techniques for geophytes.

Genus

Technique used

Response

References

DICOTYLEDONAE Anemone

Anther culture

Embryoid

Johansson et al. 1982; Johansson 1983; Johansson & Eriksson 1984

Oxalis

Protoplast culture

Plantlet

Ochatt et al. 1989

Ranunculus

Anther & pollen culture

Embryoid

Konar & Nataraja 1965b

MONOCOTYLEDONAE Allium Protoplast culture

Plantlet

Buiteveld & Creemers-Molenaar 1994

Crocus

Protoplast culture

Plantlet

Isa et al. 1990

Gladiolus

Gene transformation

Cell colony Transgenic plant

Graves & Goldman 1987 Kamo et al. 1995

Hemerocallis

Protoplast culture

Plantlet

UHu1l1.HOi:H culture Cell & callus culture

Cell division Tetraploid & octaploid Dwarf type

Fitter & Krikorian 1981,1985; Krikorian et al. 1988 Zhou 1989a,b Chen & Goeden-Kallemeyn 1979; Griesbach 1989

Hyacinthus

Gene transformation

Opine synthesis

Hooykaas-van Slogteren (1986)

Iris

Embryo culture

Amphidiploid

Yabuya (1985)

Ulium

In vitro pollination & fertilization

Hybrid

Van Tuyl et al. 1991; Janson 1993

Hybrid

Ovary & ovule culture

Hybrid

Anther & pollen culture "npollinated ovary culture Protoplast culture

Gene transformation Germplasm storage

Haploid Haploid Pollenplast Gametoplast Cell colony Plantlet Polleplast Pollen & gametoplast GUS expression z Regrowth

Narcissus

Gene transformation

Opine synthesis

Hooykaas-van Slogteren et al. 1984; Hooykaas-van Slogteren 1986

Ornithogalum

Ovule culture

Seedling

Niederwieser et al. 1990

Scilla

Protoplast culture

Plantlet

Deumling & Clermont 1989

Tulipa

Embryo culture Ovule culture Protoplast culture Gene transformation

Plantlet Bulblet Pollenplast fusion GUS expression

Niimi 1978,1980; Aubert et al. 1986 Custers et al. 1992 Maeda et al. 1979 Wilmink et al. 1992

Zephyranthes

Ovule & ovary culture

Protoplast fusion

..... N

.....

North 1975; Asano & Myodo 1977b; Asano 1980,1982; Van Tuyl et al. 1986,1990a; Kim & Sung 1990 Kanoh et al. 1988; Van Tuyl et al. 1990b,1991; Yoon 1991 a,b Sharp et al. 1971; Qu et al. 1988; Prakash & Giles 1986 Ito 1973; Tanaka et al. 1987 Tanaka 1988 Simmonds et al. 1979 Mii et al. 1994 Maeda et al. 1979 Ueda et al. 1990 Van der Leede-Plegt et al. 1992 Bouman & de Klerk 1990

Embryo culture

z

GUS-f3-Glucuronidase .

Sachar & Kapoor 1959

122

K. W. KIM AND A. A. DE HERTOGH

Endosperm cultures have produced triploid plants with several species (Srivastava and Johri 1992), but no reports were found with ornamental geophytes. C. Ovary, Ovule, Anther, and Pollen Culture After fertilization, ovaries are cut either parallel or longitudinal to the seeds. Plantlets were produced from 3- to 4-mm ovary slices with Lilium hybrids (Van Tuyl et al. 1990a, 1991; Yoon 1991a,b). Van Tuyl et al. (1990a) also reported that the addition of anthers, 1 mg/L NAA, 10% sucrose, at pH 6, promoted embryo development in ovary and ovule culture of Lilium. With Tulipa, 2- to 3-mm slices were taken from embryos, 9 to 10 weeks after pollination. The optimal conditions were (1) storage at 5°C, (2) 6% sucrose, (3) the use of a small volume of culture medium, (4) continuous darkness, and (5) a culture temperature of 12 to 15°C (Custers et al. 1992). Plantlets have been produced by using ovule culture in Zephyranthes (Sachar and Kapoor 1959). Homozygous diploid lines can be produced by doubling haploids induced in vitro by either androgenic or gynogenic pathways. Haploid plants from anthers have been regenerated from Lilium (Sharp et al. 1972) and Ranunculus (Konar and Nataraja 1965b). In Lilium, haploid plants have also been produced directly from cultures of unpollinated ovaries and ovules (Prakash and Giles 1986). Callus from anthers of Freesia (Bajaj and Pierik 1974) and Gladiolus (Bajaj et al. 1983) was believed to be haploid. D. Protoplast Culture and Fusion Plantlet regeneration from somatic protoplasts has been reported for Hemerocallis (Fitter and Krikorian 1981) and Lilium x formolongi (Mii et al. 1994). Protoplasts have been obtained from microsporocytes and pollen of Lilium longiflorum (Ito 1973; Tanaka et al. 1987; Tanaka 1988; Ueda et al. 1990). In L. longiflorum, fusion of pollen protoplasts was obtained by Maeda et al. (1979) and cell colonies developed. Ueda et al. (1990) showed that cell fusions were readily induced within pollenplasts, within gametoplasts, and between pollenplasts and gametoplasts in L. longiflorum. Only small cell colonies were derived from protoplasts of L. speciosum x henryi (Simmonds et al. 1979). There are no reports on the production of somatic hybrid plants in geophytes by protoplast fusion.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

123

E. Callus and Cell Culture

Few reports are available on ornamental geophytes. Tetraploid or polyploid plants have been obtained from callus and cell cultures of Hemerocallis (Chen and Goeden-Kallemeya 1979) and rhizomatous irises (Laublin et al. 1991). F. Gene Transformation

Monocotyledonae species, which include many geophytes (Table 2.3), tend to be insensitive to Agrobacterium infection and subsequent transformation (Hooykaas-van Slogteren et al. 1984), but some investigators have been successful. Hooykaas-van Slogteren (1986) was able to achieve gene transformation in Hyacinthus with Agrobacterium. Graves and Goldman (1987) transferred the f)-glucuronidase (GUS) gene to flower stem explants of Gladiolus, and Langeveld et al. (1995) used the virulent C58 strain of Agrobacterium and were able to induce tumors on in vitro plantlets of Lilium 'Harmony'. Genetic transformation in Narcissus was achieved by Hooykaas-van Slogteren et al. (1984) and Hooykaas-van Slogteren (1986) using Agrobacterium infection. Wilmink et al. (1992) used young floral stalk explants of Tulipa and transferred the GUS gene not only by Agrobacterium infection but also by particle bombardment. Kama et al. (1995) produced transgenic Gladiolus plants from suspension cells and callus by particle bombardment. Van der Leede-Plegt et al. (1992) achieved gene transformation in Lilium longiflorum using male gametophytes as the vector. G. Cryopreservation

Cryopreservation with liquid nitrogen (-196°C) and subsequent regeneration of lily meristems has been achieved by Bouman and de Klerk (1990) using the following system: (1) freeze-hardening by precuHuring at 5°C; (2) cryoprotection with dimethylsulfoxide (DMSO), glycerol, and/or sugar; (3) freezing the meristems either by directly dipping them into liquid nitrogen or by a staged freezing to -40°C at the rate of 0.2 to 1.5°C per minute followed by dipping in liquid nitrogen; (4) storage in liquid nitrogen; (5) thawing in waterbath at 37°C; and (6) regrowth in vitro. The cryopreserved Lilium meristems grew actively when cultured in vitro.

124

K. W. KIM AND A. A. DE HERTOGH

V. GENERA REVIEWED

The tissue culture of monocotyledonae ornamental geophytes has been reviewed by Hussey (1975a,1980b), Jha and Sen (1984), Kromer (1985), Krikorian and Kann (1986), Van Aartrijk and van der Linde (1986), and Van der Linde (1992). No review was located on dicotyledonae ornamental geophytes. De Hertogh and Le Nard (1993a,b) covered the production and physiology of 61 genera of ornamental geophytes. They were used as the base for this review, and the literature on 52 genera is summarized. No literature was located on the following genera: Acidanthera, Achimenes, Astilbe, Babiana, Camassia, Chionodoxa, Crocosmia, Eranthis, Erem urus, Erythronium, Ixiolirion, Puschkinia, Scadoxus, Tigridia, and Triteleia. Jha and Sen (1984), Hussey (1975a,1976a,1980b), Krikorian and Kann (1986), Kromer (1985), and Yanagawa and Sakanishi 1977,1980a,b) provide limited information on Bowiea, Cordyline, Crinum, Cyrtanthus, Habranthus, Ipheion, Schizostylis, Sprekelia, Sternbergia, Vallota, and Veltheimia. They are not covered in this review, because no additional information was found. A. Dicotyledonae Aconitum. Watad et a1. (1995) developed a micropropagation method to increase A. napellus using floating membrane rafts. They used shoot tips and obtained optimum shoot proliferation on the rafts with 0.25 mg/L of 6-benzyladenine (BA). The propagation rate of this system was 45% higher than a solid medium system.

Anemone. Phenolic substances, which have detrimental effects on morphogenesis, are continually formed in vitro by Anemone species (Johansson 1983; Mensuali-Sodi et a1. 1993). Thus, activated charcoal (AC) was added to promote seedling growth and to prevent browning of the medium (Mensuali-Sodi et a1. 1993). Embryogenesis was promoted with AC in anther cultures of A. canadensis and A. hupehensis (Johansson 1983). With anther cultures of A. canadensis, A. dichotoma, A. hupehensis, and A. vitifolia, CO 2 increased the production of microspore-derived embryos (Johansson et a1. 1982; Johansson and Eriksson 1984). In addition, storage of anthers for 20 days at 7°C increased the number of proembryos with A. canadensis and combination of 7°C storage and subsequent incubation under 2% CO 2 improved the embryo production of A. hupehensis.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

125

Begonia Tuberous Hybrids. Tuberous hybrid begonias are normally increased by sexual propagation, but the multiflora groups are vegetatively propagated by shoot cuttings or tubers (Haegeman 1993). Tissue culture of begonias, including tuberous hybrids, has been reviewed by Takayama (1990). Plantlet regeneration has been obtained directly from leaf explants (Peck and Cumming 1984) and indirectly from petiole-induced callus (Viseur and Lievens 1987), where the cycle from callus induction to flowering of regenerated plants required 6 to 7 months. No phenotypic variations were observed. Multiflora cultivars have been freed of cucumber mosaic virus (CMV) by placing tubers (enlarged hypocotyls) at 38°C for 1 month, which produced CMV-free shoots and meristems (Welvaert et a1. 1980; Samyn et a1. 1984). Cyclamen. Since vegetative propagation is difficult, cyclamens are generally propagated by F 1 hybrid seed. With C. persicum, tubers are formed after germination and clonal multiplication can be important not only for increasing desired stock plants but also for maintenance of F 1 parent lines. Tissue culture of Cyclamen has been reviewed by Geier et a1. (1990). Lowenberg (1969) showed that tuber-derived callus of C. persicum produced roots and shoots after being subcultured for 6 years. Plantlets have been obtained directly from tuber tissues and indirectly from either anthers or leaf blades through organogenesis (Geier 1977) and somatic embryogenesis (Wicart et a1. 1984; Kiviharju et a1. 1992). Wi cart et a1. (1984) showed that callus induced from leaf blades, leaf stalks, and ovaries formed plantlets and tuberlets through organogenesis and somatic embryogenesis. Organogenesis from cotyledons, petioles, tubers, and roots obtained from seed occurred directly on the explants (Wainwright and Harwood 1985; Hawkes and Wainwright 1987) and on peduncles (Schwenkel and Grunewaldt 1988). Shoots developed into plantlets, which were subsequently established in vivo. Etiolated petiole explants differentiated shoots, which formed roots in the dark. Plantlets were subsequently exposed to light for a few weeks and then established in soil (Murasaki and Tsurushima 1988). Using this method, over 10,000 plantlets were obtained from one mother stock plant in a year. Somatic embryos have been produced through callus obtained from anthers, ovaries, and zygotic embryo explants (Kiviharju et a1. 1992). Plants produced from the embryos flowered after being established in soil and were phenotypically identical to the mother plants.

126

K. W. KIM AND A. A. DE HERTOGH

Dahlia. Vegetative reproduction is widely used with dahlias, and viruses are a major problem (Byther and Chastagner 1993). Gavinlertvatana et al. (1979,1982) have produced large calli on leaf segments pretreated with daminozide under short-day conditions in vivo, but plantlets were not regenerated. Dahlia mosaic virus (DMV) has been removed from infected plants by using 0.2- to 1.0-mm apical and axillary meristems (Morel and Martin 1952; Mullin and Schlegel 1978). Plantlets were produced and were subsequently transplanted into greenhouses. Holmes (1955) has succeeded in removing the spotted wilt virus by using shoot tip cuttings. Gloxinia. Plants have been obtained from tubers by promoting axillary bud initiation from shoot tip explants (Murashige 1974) and by inducing adventitious bud formation from leaf explants in vitro (Takayama 1990). Plantlets have been multiplied by subculturing shoots and buds. Liatris. Plants are propagated by seed or divisions, but those from seed are variable and the multiplication rate by corm division is low (Moe 1993). Plantlets have been obtained from stem pieces excised from inflorescences of 1. spicata in vitro (Stimart and Harbage 1989). The addition of indolebutyric acid (IBA), 6-benzylaminopurine (BA), or a mixture of IBA and BA promoted multiple shoot development during the subculture of the shoots. The highest level of rooting was achieved with 5 IlM IBA and no BA. Oxalis. O. tuberosa does not produce seed and must be propagated asexually by tubers. Plantlets have been obtained directly from apical shoot tips and internode and petiole sections and indirectly from callus in vitro (Ochatt et al. 1986). Variations in sugar and oxalic acid content and morphological characteristics were observed (Khan et al. 1988). Escandon et al. (1989) demonstrated that DNA amplification occurred in protoplast-derived callus of O. glaucifolia and O. rhombeo-ovata. Protoplasts isolated from internode-derived callus of these species proliferated to form cell colonies, which subsequently developed into callus. Plantlets have been regenerated from protoplast-derived callus (Ochatt et al. 1989). Crouch et al. (1993) suggested that O. rec1inata callus cultures have potential for the industrial production of anthocyanins, which could be used for food coloring. Ranunculus. Because clonal multiplication is very slow (Meynet 1993), Ranunculus species are generally propagated sexually, but asexual reproduction is needed. For example, R. serbicus produces

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

127

a skin irritant, protoanemonin, which shows antibacterial and antifungal activities (Bonora et al. 1987). Thus, clonal selections are highly desirable. Somatic embryogenesis has been achieved directly from R. asiaticus cotyledons and hypocotyl explants (Kim et al. 1991c) and from stem epidermal cells of R. sceleratus by in vitro plantlets derived from flower shoots (Konar et al. 1972). Indirectly, somatic embryogenesis has been produced not only from R. sceleratus callus derived from flower buds (Konar and Nataraja 1964,1965b) but also from petioles, cotyledons, hypocotyls, leaf blades (Kim et al. 1991b), and receptacles (Beruto and Debergh 1992) of R. asiaticus. Somatic embryos from cell suspension cultures of R. sceleratus have generated plants (Konar and Nataraja 1965a; Poli et al. 1989). Plant regeneration of R. asiaticus has been achieved by organogenesis directly from cotyledonary explants and indirectly from cotyledons-derived callus (Pugliesi et al. 1992). R. asiaticus plants obtained from receptacle-derived callus had chromosome numbers and phenotypic characteristics of the original stock plants (Beruto and Debergh 1992). Haploid plants of R. sceleratus were obtained through somatic embryogenesis from callus derived from mature anthers (Konar and Nataraja 1965b). B. Monocotyledonae

Agapanthus. Hussey (1980b) has reported that plants were readily regenerated from shoots obtained from rhizome explants, but detailed procedures and results were not provided. Allium. Depending on the species, ornamental alliums are propagated by seed, bulblets, or replacement bulbs (De Hertogh and Zimmer 1993). Alkema (1976) has covered the vegetative systems that are used. Unfortunately, very few tissue culture studies have been conducted on the ornamental species. These studies could be useful not only for multiplication and breeding but also for virus elimination. Ziv et al. (1983) studied A. ampeloprasum 1., a species native to Israel that is used as a cut flower. They found that the shoot tip with a piece of the basal plate and the immature umbel had the highest capacity for shoot regeneration. They were able to induce bulblet formation in vitro. Buiteveld et al. 1994 used an edible A. ampeloprasum and obtained somatic embryogenesis in a suspension culture of cells that originated from zygote embryo-derived callus. Plantlet regeneration from protoplasts, which originated from immature embryo-derived callus, was achieved also through embryogenesis and organogenesis (Buiteveld and Creemers-Molenaar 1994).

128

K. W. KIM AND A. A. DE HERTOGH

Alstroemeria. Since Alstroemeria hybrids are usually sterile triploids, slow to asexually multiply, and alstroemeria mosaic virus is seed transmitted, in vitro culture is needed for effective cloning. Rapid multiplication has been achieved by organogenesis directly from flower stems (Ziv et a1. 1973; Hussey 1980b), leaves (Hussey 1980b), rhizome tips bearing shoots and an apical meristem (Lin and Monette 1987; Pierik et a1. 1988; Elliott et a1. 1993; Han et a1. 1993,1994), and rhizome pieces (Gabryszewska and Hempel 1985; Blakesley and Constantine 1992). Plant regeneration was also achieved through organogenesis indirectly from embryo-derived callus and somatic embryogenesis (Gonzalez-Benito and Alderson 1990,1992). Alstromeria carla virus, alstroemeria mosaic virus, tobacco rattle virus, tomato spotted wilt virus, and other potyviruses were eliminated using rhizome tip and axillary meristem culture (Hakkaart and Versluijs 1985,1988; Van Zaayen et a1. 1992). Amaryllis. A. belladonna plants produce few bulblets or other propagules and the in vivo multiplication rate is extremely slow. Murashige (1974) has regenerated bulblets from bulb scales, and De Bruyn et a1. (1992) obtained plantlets by dividing and subculturing bulblets regenerated directly from twin-scales and immature floral scapes. One bulb could be divided into about 80 twin scales, which produced at least two plantlets. These plantlets could be used for further multiplication, usually producing four bulblets from each plantlet. Anigozanthos (Marcropidia). The physiology of Anigozanthos, including a summary of tissue culture techniques, has been reviewed by Goodwin (1993). McIntyre and Whitehone (1974), Ellyard (1979), McComb and Newton (1981) have shown that explants should be made from the lower vegetative buds of the inflorescence. Shoot multiplication rates are about threefold per month and flowering was achieved within a few months after removal from culture. High rates of survival were obtained. Caladium. Plantlets have been regenerated directly from dormant buds (Cooper and Cohen 1982) and leaf explants (Zhu et a1. 1993a,b) and indirectly from bud-derived callus (Han et a1. 1991a). Hartman (1974) developed complete plantlets using shoot tip explants that formed callus and proliferated and redifferentiated producing numerous shoots. They were dasheen mosaic virus-free and true to type. Hartman calculated that if 10 to 20 plantlets were regenerated in

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

129

vitro every 3 months that 40 to 50 million tubers of a new cultivar could be produced in 3 years. In contrast, plants produced by Zhu et al. (1993a,b) had leaf color variations in regenerated plants from nine cultivars. The origin of the explant material greatly affected the leaf variation obtained.

Canna. Plantlets have been sue explants (Kromer 1979) either apical or axillary buds lets have been multiplied in factory.

regenerated directly from rhizome tisand from meristem tips obtained from (Kromer and Kukulczanka 1985). Plantvitro, but the rates are not highly satis-

Clivia. When propagated by seed, Clivia requires 5 to 7 years to flower. Thus, rapid multiplication of clones is very important. Recently, Finnie and van Staden (1995) have demonstrated that plantlets could be regenerated from meristem tip, fruit, seed/embryo, ovary, and pedicel explants. Fruit and floral explants showed the greatest potential for multiplication systems. Colchicum. Some species produce alkaloids, such as colchicine, and these have been produced from cell suspension cultures of C. autumnale (Hayashi et al. 1988). There were no reports, on micropropagation of any species. Convallaria. In vitro organogenesis was studied by Verron et al. (1995) using rhizome pieces, flower stalk disks, flower bud, and apical meristems. Except for apical meristems, all tissues showed organogenesis potential. Floral bud-like, leaf-like, and vegetative buds were produced after six subcultures. Crocus. Many species are important as flowering plants and C. sativus is used as a source of saffron, a valuable spice. They propagate only vegetatively by annual corm replacement. Plantlets have been regenerated through organogenesis directly from corm tissue of C. vernus (Kromer 1985) and C. sativus (Homes et al. 1987), ovaries of C. chrysanthus (Fakhari and Evans 1989), and intact corms and shoot tips of C. sativus (Plessner et al. 1990). Indirectly, plantlets have been obtained from corm-derived callus of C. sativus (Ilahi et al. 1987, Isa and Ogasawara 1988). Ahuja et al. (1994) showed that C. sativus plantlets, regenerated through somatic embryogenesis from shoot tip-derived callus, had a normal chromosome number. Plantlets have been regenerated from protoplasts obtained from corm-de-

130

K. W. KIM AND A. A. DE HERTOGH

rived callus of C. sativus (1sa et a1. 1990). Saffron pigments have been obtained from stigma-like structures induced in vitro from stigmas (Sano and Himeno 1987; Koyama et a1. 1988; Fakhari and Evans 1990; Sarma et a1. 1990), ovaries (Sano and Himeno 1987; Fakhari and Evans 1990; Sarma et a1. 1991), anthers (Fakhari and Evans 1990; Sarma et a1. 1991), and petals (Fakhari and Evans 1990).

Endymion. Mullin (1970) has reported that E. hispanicus bulb explants were morphogenic and would proliferate to form masses of tuberized shoots with callus. Eucharis. This species propagates vegetatively by daughter bulbs, but bulb scale explants have produced adventitious bulblets that developed into complete plants (Pierik et a1. 1983). After being transferred into soil, the plants flowered and were identical to the mother plants. Eucomis. De Lange et a1. (1989) have reported tissue culture procedures for E. autumnalis, E. bicolor, and E. pole-evansii. They successfully produced plantlets from bulb scales, leaf bases, and floral stalks that could be rooted and then transferred to soil. Meyer (personal communication) developed an embryo rescue procedure to make interspecific crosses. Freesia. Since hybrid freesias produce only 3 to 6 daughter corms per year in vivo, it takes 8 to 10 years to introduce a new cultivar. Tissue culture of Freesia was reviewed by Bajaj (1990). Plantlets have been regenerated by organogenesis directly from anthers (Bajaj and Pierik 1974), flower buds (Pi erik and Steegmans 1975,1976), corms (Hussey 1975a,1976a,1977a,1980b), leaves (Hussey 1976a,1977a), flower stems (Hussey 1976a,1977a,1980b), axillary buds (Hussey 1980b), and ovaries (Bach 1987). They were obtained indirectly from the calli formed on flower buds (Bajaj and Pierik 1974), corms and inflorescence stems (Hussey 1975a), and pedicels (Stimart and Ascher 1982). Hussey (1980b) estimated that one plant could produce 500 to 2000 shoots per year. Depending on the exogenous PGRs used (Wang et a1. 1990), somatic embryogenesis in F. refracta was induced either directly from epidermal cells of young inflorescences and leaf explants or indirectly from the callus originated from the explants. Bach (1992) used two cultivars and induced somatic embryogenesis and plant regeneration from zygotic embryos and meristems, but there were differences between the cultivars.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

131

Hussey (1977a), Stimart and Ascher (1982), and Wang et al. (1990) reported that their regenerated plants were normal. The production of virus-free freesias using meristem tip culture was initially carried out by Brants and Vermeulen (1965). Subsequently, meristem tips excised from either apical or axillary buds produced plants that were free from bean yellow mosaic virus (Bertaccini et al. 1989; Foxe, 1991), cucumber mosaic virus (Bertaccini et al. 1989), and freesia mosaic virus (Bertaccini et al. 1989; Foxe 1991).

Fritillaria. F. meleagris forms a small bulb containing only 2 to 3 scales and when propagated under natural conditions multiplies slowly. Plantlets have been obtained through organogenesis directly from the inflorescence stern and leaf and from scale explants (Hussey 1976a). Bulblets have also been regenerated directly from scale and seed explants. The adventitious shoots and bulblets formed could be subcultured every 4 to 5 months for further multiplication (Kukulczanka et al. 1989). Galanthus. Hussey (1980b) reported that adventitious shoots were obtained from leaf, bulb scale, or stern tissue, but low multiplication rates were achieved. Galtonia. Yanagawa and Sakanishi (1980a,b) used G. candicans and obtained plantlets from single bulb scales and twin scales. The percent regeneration was greatest when a segment of the scale was attached to the basal plate. Gladiolus. If asexual propagation procedures are used, it takes about 10 years to produce enough corms of a new cultivar for commercial use, but Bajaj et al. (1983) showed that only 2 years are required using tissue culture techniques. Ziv and Lilien-Kipnis (1990) have reviewed the tissue culture of Gladiolus. Plantlets, cormlets, and cormels have been regenerated by organogenesis from various plant parts. Organogenesis has been achieved directly from the following explants: flower sterns (Ziv et al. 1970; Hussey 1975a; Kamo et al. 1990); cormel tips and axillary buds (Simonsen and Hildebrandt 1971; Bajaj et al. 1983; Kim and Goo 1991; Dantu and Bhojwani 1995); stolon tips (Ginzburg and Ziv 1973); corm tissues (Hussey 1975a,1976a; Sutter 1986); and leaves, axillary buds, cormel tissues, and perianths (Bajaj et al. 1983). For multiplication, subcultures have been carried out using axillary and adventitious shoots (buds). These were ob-

132

K. W. KIM AND A. A. DE HERTOGH

tained from leaves (Hussey 1975a,1977a), inflorescences (Hussey 1976a,1977a,1980b), corm tissues (Hussey 1976a), axillary buds (Hussey 1977a,b,1978,1980b; Dantu and Bhojwani 1987; LilienKipnis and Kochba 1987; Steinitz and Lilien-Kipnis 1989; Steinitz et a1. 1991; De Bruyn and Ferreira 1992), apical buds (Lilien-Kipnis and Kochba 1987; Steinitz and Lilien-Kipnis 1989; Ziv 1989, 1990a,b; Steinitz et a1. 1991), corm tissues (Sutter 1986), and cormel tips (Rao et a1. 1991). Shoots have been multiplied by repeated subcultures at the rate of about 5- to 10-fold per month and ultimately developed to plantlets or cormlets. Cormlets obtained were dormant and required a cold treatment to break rest (Hussey 1977b; Sutter 1986). Ziv (1979) developed a system to form roots on plantlets in order to successfully transfer them to nonaseptic conditions. Cormlets and plants were produced that shortened the time required to produce large corms. Adventitious shoots regenerated from cormel-derived callus were used to develop complete plantlets and cormlets through callus subculture for more than 2 years (Kim et a1. 1988,1991a; Kim and Lee 1993; Kim and Han 1993; Goo and Kim 1994). In these callus studies, shoots were formed exogenously, while roots were formed endogenously. Subsequently, shoots produced complete plantlets either through root formation from shoot bases or by vascular system connections in the callus. Plantlet regeneration through somatic embryogenesis from cormel-derived callus has been demonstrated by Kamo et a1. (1990) and Kim and Kang (1992). Virus-free plants were initially obtained from callus cultures derived from cormel tip explants (Simonsen and Hildebrandt 1971). Subsequently, in vitro regeneration of virus-free plants was obtained from the following tissues: gladiolus mosaic virus-free plants from inflorescences (Zhen et a1. 1984), bean yellow mosaic virus (BYMV)free plants from meristem tips (Bertaccini and Marani 1986), BYMV and cucumber mosaic virus-free plants from meristem tips excised from apical buds (Logan and Zettler 1985), and BYMV-free plants from leaf tips of plantlets obtained in vitro (Aminuddin and Singh 1985). Graves and Goldm2n (1987) showed that Agrobacterium tumefaciens directly induced gene transformation in corm cells. Also, Kamo et a1. (1995) reported that a large number of transgenic plants were produced by particle bombardment of suspension cells and callus.

Gloriosa. Finnie and van Staden (1989) obtained plantlets have been obtained directly from seedling, embryo, and tuber explants and in-

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

133

directly from callus. They used adventitious shoots or tuberlets obtained from tuber tissues for multiplication by subculturing and obtained plantlets and tubers.

Haemanthus. Kromer (1985) obtained bulblets and plantlets from H. katharinae directly from twin-scale explants. Due to their dormant state, only a few bulblets developed into plantlets. Hemerocallis. Propagation by crown division produces only 1 to 2 plants per year and some hybrids are triploid. Thus, rapid multiplication is desired. Tissue culture of Hemerocallis has been reviewed by Krikorian et a1. (1990). Plantlets have been primarily regenerated indirectly from callus through organogenesis and embryogenesis. The following explants have been used for organogenesis; petals (Heuser and Apps 1976; Heuser and Harker 1976), flower stems (Heuser and Apps 1976; Meyer 1976; Leifert et a1. 1992a,b), sepals and filaments (Heuser and Harker 1976), flower buds including half ovary (Krikorian and Kann 1981; Krikorian et a1. 1981), and shoot tips (Smith et a1. 1989). Regenerated plantlets were phenotypically identical to the mother plants (Meyer 1976; Krikorian et a1. 1981; Krikorian and Kann 1981), indicating that genome of Hemerocallis is very stable in callus culture. Griesbach (1989), however, obtained some dwarf plants from cell and callus cultures generated from shoot tip explants. Smith and Krikorian (1991) used shoot tip explants to regenerate plantlets from callus through somatic embryogenesis. Plantlets have also been regenerated through cell and protoplast cultures from various explants (Fitter and Krikorian 1981,1985; Krikorian and Kann 1981; Krikorian et a1. 1988). Zhou (1989a,b) demonstrated that cell colonies could be obtained from pollen protoplasts of H. fulva. Tetraploid and octaploid plants have been obtained from colchicinetreated diploid H. fulva callus by Chen and Goeden-Kallemeyn (1979). Plantlets were regenerated aboard the spaceship Discovery by Levine and Krikorian (1992), who found that primary shoot apices lost their apical dominance and increased axillary growth occurred. Hippeastrum. Plants are propagated by offset bulblets, scale-stem cuttings, and single or twin scales (Okubo 1993). Mii et a1. (1974) first demonstrated that organogenesis could occur on scale explants. Subsequently, plantlets and bulblets have been regenerated through organogenesis directly from the following explants: single scales (Seabrook and Cumming 1977; Yanagawa and Sakanishi 1977, 1980b;

134

K. W. KIM AND A. A. DE HERTOGH

Okubo et al. 1990; Huang et al. 1990; Blakesley and Constantine 1992), twin scales (Hussey 1975a,1976a; Okubo et al. 1990; Huang et al. 1990; Han et al. 1991b), floral scapes (Hussey 1975a,1976a,1980b), leaves (Hussey 1976a,1980b; Seabrook and Cumming 1977), and ovaries and peduncles (Seabrook and Cumming 1977). Up to 450 plantlets have been regenerated from one mother bulb in 8 weeks (Seabrook and Cumming 1977). Okubo et al. (1990) and Huang et al. (1990) subcultured protocorm-like bodies derived from single-bulb scale explants and these produced large numbers of shoots and bulblets.

Hyacinth us. The natural propagation rate of Hyacinthus is low, since a mother bulb produces only a few offset bulblets (Nowak and Rudnicki 1993). Thus, they are multiplied commercially by scooping and scoring (cross-cutting). The tissue culture of Hyacinth us has been reviewed by Paek and Thorpe (1990). Plantlets and bulblets have been obtained through organogenesis directly from the following explants: scales (Pierik and Woets 1971; Pierik and Ruibing 1973; Pierik and Post 1975; Hussey 1975b,1977a; Tamura 1978; Chung et al. 1981a,1983a; Paek et al. 1983,1987; Amaki et al. 1984); bulblets (Saniewski et al. 1974); flower buds (Saniewski 1975; Kim et al. 1981); floral scapes (Hussey 1975a,b,1977a; Kim et al. 1981; Chung et al. 1981a,1983a; Paek et al. 1987); leaves (Hussey 1975a,b,1976a,1977a; Paek et al. 1987; Bach and Cecot 1988,1989); and basal plates, twin scales, and ovaries (Hussey 1975a,b,1976a). Plantlets and bulblets obtained from single-scale explants were regenerated from the abaxial epidermis (Tamura 1978). Plantlets and bulblets have also been regenerated indirectly from callus, which was dedifferentiated not only from scales (Bae et al. 1983) but also from twin scales, leaves, basal plates, floral scapes, and ovaries (Hussey 1975a,b). Multiplication has been carried out by subculturing in vitro-derived plantlets and bulblets from adventitious shoots (Hussey 1976a,1980b), callus (Hussey 1975a,b,1980b; Bae et al. 1983), bulblets (Kim et al. 1981; Bae et al. 1983), leaves (Bach and Cecot 1988,1989), and scales (Chung et al. 1983b). Pierik and Post (1975) used scale segments and produced 240 to 300 bulblets from one mother bulb in 12 weeks. More than 1000 plantlets were produced from one inflorescence in 4 to 6 months by Kim et al. (1981). There were no reports on plantlet regeneration through somatic embryogenesis. Hyacinth mosaic virus-free plants have been obtained by culturing meristem tips from bulblets formed on scooped bulbs (Asjes et al. 1974) and from bulb scale explants (Blom-Barnhoorn et al. 1986). Gene transformation has been carried out by Hooykaas-van Slogteren (1986) using Agrobacterium.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

135

Hymenocallis. Yanagawa and Sakanishi (19S0a,b) reported that twin scale explants of H. speciosa would produce single bulblets and propagation was not affected by the time of the year. When scale segments were used, bulblets readily formed on the abaxial side of the scale segments and adjacent to the main vascular bundle. Yanagawa and Ito (19SS) subsequently demonstrated that the addition of NAA and BA to the medium increased the number of bulblets formed on scale bases. There were no differences between old or young scale bases and their thickness. A difference was obtained only when the explants were taken from the middle of the scale. Iris. Species in this genus are vegetatively propagated either by rhizome divisions or annual bulb replacement (De Munk and Schipper 1993). Since each plant produces only 2 to 5 daughter rhizomes or bulbs per year, about 10 years are needed to introduce a new cultivar. Plantlets and bulblets of 1. hollandica have been regenerated through organogenesis directly from inflorescence stems (Hussey 1976a,c,1977a,19S0b; Bach 19S5), leaves (Hussey 1976a,1977a), single scales (Hussey 1977a; Van der Linde et a1. 19S5), and twin scales (Hussey 1976a,19S0b). With other species, plantlets and/or bulblets have been regenerated through organogenesis and/or somatic embryogenesis either directly from explants or indirectly from callus. Organogenesis has been achieved directly from the following explants: rhizomes of 1. germanica (Kromer 19S5), inflorescence stems of 1. ensata (Yabuya et a1. 1991), shoot tips of 1. hollandica (Mielke and Anderson 19S9), and shoots of 1. germanica (Leifert et a1. 1992a,b). From 11 young floral scapes of 1. ensata, Yabuya et a1. (1991) produced 1000 plantlets, while Hussey (1976c,19S0b) produced more than 100 adventitious shoots per year from one flowering bulb of 1. hollandica. Organogenesis has been obtained indirectly through callus from the following explants: inflorescence stems of tall bearded Iris species (Meyer et a1. 1975), embryos of 1. setosa (Radojevic and Subotic 1992), and rhizomes of 1. pallida (Gozu et a1. 1993). Somatic embryogenesis has occurred indirectly through callus from the following explants: shoot tips of hybrid rhizomatous irises (Reuther 1977); embryos of 1. pumila (Radojevic et a1. 19S7) and 1. setosa (Radojevic and Subotic 1992); roots of 1. pseudocorus, I. versicolor, and 1. setosa (Laublin et a1. 1991); and leaves, rhizomes, sepals, and ovaries of 1. pallida and 1. germanica (Jehan et a1. 1994). Most regenerated rhizomatous Iris plants have exhibited normal phenotypic characteristics (Reuther 1977; Radojevic et a1. 19S7; Laublin et a1. 1991; Radojevic and Subotic 1992; Jehan et a1. 1994),

136

K. W. KIM AND A. A. DE HERTOGH

but Laublin et a1. (1991) reported that 2 of 80 regenerates of rhizomatous irises were tetraploid. Using I. hollandica bulbs, latent mosaic virus-free bulbs (iris mosaic virus) were obtained by meristem tip culture (Baruch and Quak 1966) and iris mild mosaic-free plants were obtained from basal plates (Allen and Anderson 1980). Bulbs free of virus symptoms were also produced by callus culture using meristem tip explants (Hirata and Kunishige 1982). Anderson et a1. (1990) reported that rod-shaped viruses were removed from infected plants by shoot tip culture and that the virus-free plants multiplied by 2.5 to 3 times every 3 weeks during shoot subculture. Yabuya (1985) was possible to obtain fertile amphidiploids between 1. laevigata and 1. ensata by culturing colchicine-treated embryos. Irones, which have been used as a fragrance, have been extracted from embryo-derived callus of I. siberica (Para and Baratti 1992) and from rhizomes of regenerated plants of 1. pallida (Gozu et a1. 1993; Jehan et a1. 1994) and 1. germanica (Jehan et a1. 1994).

Ixia. This species has an extremely slow natural propagation rate. Plantlets have been obtained by organogenesis directly and indirectly from corms (Sutter 1986; Meyer and van Staden 1988) and leaf explants (Meyer and van Staden 1988). Sutter (1986) estimated that about 1000 to 1500 shoots could be produced in a year from one corm, assuming that corm-derived buds generate 3 to 4 shoots every 8 to 10 weeks. To break dormancy, cormlets required 4 weeks at 2 to 3°C. Lachenalia. To produce plants, multiple buds of Lachenalia species and their hybrids have been obtained from flower stems (Klesser and Nel 1976), scales (Klesser and Nel 1976; Hussey 1980b), and leaves (Klesser and Nel 1976; Niederwieser and van Staden 1990; Niederwieser and Vcelar 1990). Although the optimal stage depended on the genotype, the following procedures tended to produce a large number of buds: (1) using small explants of young tissues and (2) placing all leaf explants horizontally with their abaxial side on the surface of the medium. Klesser and Nel (1976) estimated that about 500 bulblets could be produced from one bulb. Leucojum. Kromer (1985) has regenerated bulb lets of L. vernum directly from twin scale explants, but all bulblets obtained were dormant and remained in this condition. Lilium. Propagation systems for Lilium species and hybrids have been intensively researched (Beattie and White 1993). Under natu-

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

137

ral conditions, lilies propagate vegetatively by bulblets, which form on bulb scales or underground portions of the stem or by bulbils, which form in leafaxils and sometimes in floral axils. In vivo scaling is used commercially to increase the multiplication rate, but only 3 to 5 bulblets can be produced per scale. Thus, tissue culture is used for rapid multiplication. Van Aartrijk et a1. (1990) and Takayama et a1. (1991) provide comprehensive reviews on tissue culture techniques used to obtain plantlets and bulblets from various lily organs. The regeneration of plantlets and bulblets can occur by organogenesis directly from explants and indirectly from callus. Unlike many other geophytes, however, plantlets and bulblets have not been obtained through somatic embryogenesis. The regeneration of plantlets and bulblets through direct organogenesis have been reported from many organs (Table 2.1). Plantlets and bulblets have also been regenerated indirectly through callus from the following organs: shoot tips (Sheridan 1968), stamens (Montezuma-de-Carvalho and Guimaraes 1974), scales (Simmonds and Cumming 1976; Stimart et a1. 1980; Novak and Petru 1981), and seed (Bennici 1979). Mass propagation has primarily been achieved by subculture of microscales, that is, scales of bulblets obtained in vitro, and callus. Compared to solid culture, liquid culture appears to accelerate the multiplication rate (Takahashi et a1. 1992a,b). For instance, with scale-derived callus culture, individual bulbs of 12 Oriental hybrid cultivars produced about 60,000 plantlets in 6 months (Simmonds and Cumming 1976). Also, Novak and Petru (1981) showed that one bulb of the Oriental hybrid 'Crimson Beauty' could produce 10,000 to 1,000,000 plantlets in one year with microscale subculture. By culturing 1-mm scale sections of 1. longiflorum, Stimart and Ascher (1978) used 100 scales and produced more than 8000 bulblets in 6 weeks. Wickremesinhe et a1. (1994) used leaves of 1. longiflorum 'Nellie White' to develop callus cultures that were subsequently induced to undergo organogenesis and produce flowering plants. Takayama and Misawa (1979,1983a,b) used liquid shake culture of microscales and estimated that one bulb of 1. speciosum could produce up to 1.2 x 10 1 °bulblets per year, while one bulb of 1. auratum could produce 3.2 x 10 12 bulblets. When regenerated, however, the plants obtained showed various degrees of variation. Using 12 Oriental hybrid cultivars, plants regenerated from scalederived callus were diploid and showed no morphological abnormality (Simmonds and Cumming 1976). Also, no phenotypic variation was observed in the following systems: callus-derived plantlets of 1. longiflorum and 1. pyrenaicum (Hussey 1980b), scale-derived

138

K. W. KIM AND A. A. DE HERTOGH

plantlets of L. longiflorum, L. x formolongi, L. speciosum, and L. auratum (Takayama et al. 1982), or leaf-derived plantlets of L. longiflorum (Liu and Burger 1986). In contrast, Bennici (1979) reported that with L. longiflorum callus, some cells were tetraploid or aneuploid. He also observed that the shoot and root apices of regenerated plants contained a significant number of cells with mixoploid condition (mosaicism). In contrast, Takahashi et al. (1992b) obtained L. longiflorum plants from liquid culture that were normal. With 'Black Beauty', which is an interspecific hybrid (L. speciosum var. rubrum x L. henryi), callus-derived plantlets exhibited phenotypic changes (Stimart et al. 1980). The state of bulblet dormancy obtained by in vitro culture was affected by sucrose concentration, bulblet age, and culture temperature with L. auratum (Takayama and Misawa 1980) and L. longiflorum (Stimart and Ascher 1981a). High GA 3 concentrations, low sucrose concentrations, and low temperatures reduced the level of dormancy ofL. speciosum (Aguettaz et al. 1990; Paffen et al. 1990). Stimart et al. (1982) reported that irradiation with red light significantly reduced the degree of dormancy in L. longiflorum and that when fluridone, an inhibitor of ABA, was added to the culture medium dormancy was completely prevented. The addition of GA 4 + 7 partially prevented dormancy in L. speciosum (Kim 1991; Kim et al. 1994). Development of dormancy in L. speciosum in vitro depended primarily on the heat energy received during culture and dormancy was not induced until bulblets were aged for a period of time. Djilianov et al. (1994) and De Klerk and Paffin (1995) observed no dormancy when L. speciosum bulblets were grown at 15°e, however, at temperatures greater than 20 o e, dormancy was readily induced. They also found that young bulblets aged for less than 6 weeks produced a low level of dormancy with high sprouting indexes. The effects of high temperatures on dormancy were easily eliminated by low-temperature treatments prior to planting (Delvallee et al. 1990; Higgins and Stimart 1990). This has been widely utilized, for example, with L. auratum at 5°e for 50 to 100 days (Takayama and Misawa 1980), with L. longiflorum at 4°e for at least 1 week (Stimart et al. 1982), and with L. speciosum at 2°e for 6 weeks (De Klerk et al. 1992). For L. rubellum, the use of GA 3 in combination with 12 weeks at 4°e were highly effective in breaking dormancy (Niimi et al. 1988). For L.longiflorum, immersion of bulblets in 45°e water after being removed from culture has promoted foliar emergence and axis elongation (Stimart et al. 1982,1983). Virus free-plants have been produced by using meristem tip explants, bulblets formed on scale explants, and meristem tips iso-

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

139

lated from the buds (shoots and bulblets) regenerated adventitiously from scale explants. Viruses that have been eliminated are lily symptomless virus (LSV) (Asjes et al. 1974; Allen 1975; Allen and Anderson 1980; Allen et al. 1980; Van Aartrijk and Blom-Barnhoorn 1982; Blom-Barnhoorn and van Aartrijk 1985; Cohen et al. 1985; Van Zaayen et al. 1992); tulip breaking virus (TBV) (Allen 1975; BlomBarnhoorn and van Aartrijk 1985; Cohen et al. 1985); LSV and TBV (Blom-Barnhoorn and van Aartrijk 1985; Van Zaayen et al. 1992), cucumber mosaic virus (CMV) (Allen 1975); and rod-shaped particles (Allen and Fernald 1972; Allen 1974). In addition, the use of antiviral compounds (Blom-Barnhoorn and van Aartrijk 1985) and heat treatments (Cohen et al. 1985) enhanced the probability of obtaining virus-free plants. Allen and Fernald (1973) and Allen (1974) demonstrated that a single virus-free bulb obtained in vitro could be multiplied to 787 plantlets in a year by scaling. To overcome prefertilization barriers, cut-style pollination (Asano and Myodo 1977a; Van Tuyl et al. 1991; Janson 1993; Li and Niimi 1995); grafted style pollination (Van Tuyl et al. 1991; Janson 1993); and placenta pollination (Janson 1993) have been used. To overcome postfertilization barriers, embryo rescue techniques were developed, which include ovary culture (Kanoh et al. 1988; Yoon 1991a,b), ovaryovule culture (Van Tuyl et al. 1990b,1991), and immature embryo culture (North 1975; Asano and Myodo 1977b; Asano 1980,1982; Van Tuyl et al. 1986,1990a; Kim and Sung 1990). The combination of in vitro pollination and embryo rescue culture has overcome interspecific and incompatibility barriers in Lilium, permitting the production of hybrid plantlets and bulblets. Androgenic haploid plants were obtained from anther-derived callus of L. longiflorum (Sharp et al. 1972; Qu et al. 1988), but phenotypic and cytological variations were observed among regenerated plants. Gynogenic haploid plants were also produced by unpollinated ovary culture in the Oriental lily 'Leraza' (Prakash and Giles 1986). Simmonds et al. (1979) induced small cell colonies, but failed to obtain plantlets from protoplasts originated from scale-derived callus of L. speciosum x L. henryi. However, Mii et al. (1994) reported that regeneration of plantlets was possible from protoplasts using suspension culture of seed-derived callus of L. x formolongi. The regenerated plants showed normal chromosome numbers and the pollen was fertile, but the leaves were smaller than original plants. Pollen protoplasts (pollenplasts) have been obtained from microsporocytes (Ito 1973) and immature pollen grains (Tanaka et al. 1987) of L. longiflorum. Gametoplasts (generative cell protoplasts) have been isolated from pollen of L. longiflorum (Tanaka 1988). Pollenplast fusion occurred spontane-

140

K. W. KIM AND A. A. DE HERTOGH

ously in L. longiflorum, L. speciosum, and the Asiatic hybrid 'Enchantment' (Maeda et al. 1979). Ueda et al. (1990) reported that fusion readily occurred in L. longiflorum between pollenplasts and gametoplasts. The fusion products were cultured for an extended period of time, but failed to develop into plantlets. Van der Leede-Plegt et al. (1992) have demonstrated that gene transformation is possible with L. longiflorum by using a male gametophyte as the transformation vector. Recently, Langeveld et al. (1995) have used the virulent strain of Agrobacterium C58 to induce tumors on in vitro plantlets of 'Harmony', an Asiatic hybrid lily. Bouman and de Klerk (1990) have cryopreserved meristem tips of L. speciosum, but the procedures need improvement.

Lycoris. Normally, L. aurea plants produce only 1 to 2 bulbs per year and seedlings require about 4 years to flower. Bulblets have been obtained from single scales and twin scales by Yanagawa and Sakanishi (1980a,b). Huang and Liu (1989) obtained up to 30,000 plantlets from one bulb in a year using twin-scale and shoot tip explants. They developed a four-step multiplication procedure: (1) induction of adventitious buds on scale explants, (2) establishment of multiple shoots, (3) rapid multiplication of the shoots, and (4) rooting of individual shoots. Muscari. Most Muscari species are propagated asexually by small offset bulblets and scoring or scooping, but a few species propagate sexually (Rudnicki and Nowak 1993). Plantlets and bulblets have been obtained by organogenesis either directly or indirectly from twin scales (Hussey 1975a; Kromer 1985,1989); scales (Cumming and Peck 1984; Peck and Cumming 1986); and leaves, floral scapes, and ovaries (Hussey 1975a). Peck and Cumming (1986) found that after an initial culture, more than 100 bulblets could be regenerated from one bulb in 12 weeks by using scale explants. Narcissus. Under natural conditions, Narcissus increase approximately 1.6-fold per year and about 16 years are needed to produce 1000 bulbs from one bulb (Rees 1969). Thus, twin scaling and chipping have been developed to increase the multiplication rate. These methods, however, do not provide commercially acceptable multiplication rates (Hanks 1993). Tissue culture of Narcissus has been reviewed by Seabrook (1990). Plantlets and bulblets have been regenerated by organogenesis di-

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

141

rectly from explants or indirectly from callus. No reports were found on the production of plantlets and bulblets by somatic embryogenesis. Plantlets and bulblets have been obtained directly from scales (Hussey 1977a,1982), twin scales (Hussey 1975a,1976a,1982; Steinitz and Yahel 1982; Paek et al. 1987), mini-chips (Squires and Langton 1990; Squires et al. 1991; Chow et al. 1992a,b,1993), basal plates (Hosoki and Asahira 1980), leaves (Hussey 1976a, 19 77a, 1982; Seabrook et al. 1976; Seabrook and Cumming 1982; Hosoki and Asahira 1980), floral scapes (Hussey 1976a,1977a,1982; Seabrook et al. 1976; Seabrook and Cumming 1982; Hosoki and Asahira 1980; Paek et al. 1987), and ovaries (Seabrook et al. 1976; Hosoki and Asahira 1980). Plantlets and bulblets have been regenerated indirectly through callus originated from ovaries (Hussey, 1975a; Seabrook et al. 1976) and leaves (Seabrook et al. 1976; Hussey 1980b). By subculturing shoots, Seabrook et al. (1976) produced up to 2620 unrooted shoots in 5 months from two leaf base explants. In contrast, Hosoki and Asahira (1980) obtained about 140 bulblets from one floral scape in 2 months after the initial culture. By using leaf, scale, or floral scape explants, Hussey (1982) obtained about 500 to 2000 bulblets from one bulb in less than 18 months. Recently, Bergonon et al. (1992) have developed a "shake liquid culture" for N. papyraceus, a species that does not produce large bulbs suitable for chipping. Steinitz and Yahel (1982) cultured twin scales from one N. tazetta bulb and produced 200 to 300 bulblets in less than 6 months. Squires and Langton (1990) produced mini-chip-derived shoots from one 'Yellow Cheerfulness' bulb and obtained about 1200 flowering-sized bulbs in 4 to 5 years. Most Narcissus bulbs produced in vitro have been reported to flower normally. Chow et al. (1992b,1993) showed that severe cutting, such as cutting of shoot clumps close to basal region, and the use of basal parts of leaves excised from shoot clumps were effective methods to achieve rapid multiplication. These plants were more genetically uniform than those regenerated from callus. Hussey (1982) and Squires and Langton (1990) showed that all shoots that became bulblets were dormant. Thus, before planting, the bulblets required 8 to 10 weeks at 4°C (Hussey 1982). Bulblets obtained from a high sucrose and aBA-free medium produced continuous leaf growth without dormancy in vivo (Chow et al. 1992a). The production of virus-free Narcissus plants has been reviewed by Brunt (1980). Stone (1973) and Stone et al. (1975) eliminated arabis mosaic virus (ArMV) and narcissus degeneration virus from virusinfected plants by meristem tip culture. Phillips (1990) eliminated

142

K. W. KIM AND A. A. DE HERTOGH

narcissus latent virus, narcissus mosaic virus (NMV), narcissus tip necrosis virus (NTNV), narcissus yellow stripe virus, tobacco rattle virus, and an unidentified filamentous virus by meristem tip culture. The combination of meristem tip culture and the use of antiviral compounds removed ArMV and narcissus virus Q from infected plants (Phillips 1990). The combination of meristem tip culture and twin scales incubated at either at 37°C for 18 to 21 days or at staged heating from 30 to 36°C over an 8-week period eliminated the following viruses: ArMV, narcissus late yellow virus, NMV, NTNV, narcissus white strike virus, raspberry ringspot virus, strawberry latent ringspot virus, and tomato black ring virus (Mowat 1980). Virus-free plants flowered true to type and could be multiplied by chipping (Stone 1973) and twin scaling (Mowat 1980). Gene transformation has been achieved by Agrobacterium infection (Hooykaas-van Slogteren et a1. 1984; Hooykaas-van Slogteren 1986).

Nerine. Nerines naturally propagate asexually by offset bulblets, and twin scaling and chipping have been used to increase the multiplication rate (Van Brenk and Benschop 1993). The regeneration of plantlets and bulblets from Nerine species through direct organogenesis has been reported from the following explants: twin scales (Pierik and Ippel 1977; Grootaarts et a1. 1981; Jacobs et a1. 1992), scales (Hussey 1980b), leaves (Hussey 1980b), floral scapes (Hussey 1980b; Pierik and Steegmans 1986; Lilien-Kipnis et a1. 1990), peduncles (Ziv 1990a; Jacobs et a1. 1992), and ovaries (Ziv 1990a). To increase the m ul ti plication rate, bul blets, shoots, buds, and proto corm-like bodies have been regenerated from various explants and subcultured. Grootaarts et a1. (1981) demonstrated that bulblets of N. bowdenii could be formed from either axillary or adventitious tissue. Lilien-Kipnis et a1. (1992) have regenerated Nerine x Mansellii using a liquid culture system with potential for mass propagation. Lastly, nerine latent virus-free plants of N. bowdenii have been produced by meristem tip culture (Hakkaart et a1. 1975). Ornithogalum. All species propagate asexually by bulblets, but the rates vary, depending on species, with most being low. Plantlets and bulblets have been obtained primarily through organogenesis from scales (Hussey 1976b; Klesser and Nel1976; Nel1981; Chung et a1. 1981b; Yanagawa and Sakanishi 1980a,b; Yanagawa and Ito 1988), twin scales (Hussey 1975a; Yanagawa and Sakanishi 1980a,b), shoot tips (Chung et a1. 1981b), leaves (Hussey 1975a,1976b; Klesser and

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

143

Ne11976; NeI1981), flower stems (Hussey 1975a,1976b; Klesser and Nel 1976), ovaries (Hussey 1975a,1976b), sepals (Hussey 1976b), peduncles (Nel 1981), petals (Nel 1981), and stigmas and styles (Chung et al. 1980). Hussey (1975a,1976b) obtained plantlets and bulblets from callus derived from explants of twin scales, leaves, flower stems, ovaries, and scales. By subculturing shoots every 6 to 8 weeks, Nel (1981) obtained 9165 plantlets from a single leaf of Ornithogalum hybrids in 9 months. He reported that the leaf-derived plantlets flowered true-to-type within 4 months after transplanting. Although plants derived directly from stem and leaf explants were diploid, Hussey (1976b) found that those derived from callus exhibited a high level of tetraploidy. Klesser and Nel (1976) suggested that virus elimination is possible using leaf, scale, and stem cultures. By using antiviral compounds, Vcelar et al. (1992) obtained ornithogalum mosaic virusfree plants from meristem tips excised from leaf-regenerated adventitious buds. Also, embryos rescued by ovule culture were developed into seedlings by Niederwieser et al. (1990).

Polianthes. This species naturally propagates by offset bulblets and seeds (Benschop 1993), but the multiplication rate by bulblets is low and double-flowered lines are sterile. The regeneration of plantlets has been accomplished through direct or indirect organogenesis from scale-stem explants (Bose et al. 1987). Regenerated plantlets produced normal growth in pot plant culture. Virus-free plants have been regenerated from meristem tip cultures (Wang and Hu 1980). Sandersonia. Finnie and van Staden (1989) used explants from tubers, leaves, stems, flower buds, and ovaries of dormant and nondormant tubers. When explants were taken from specific parts of the tuber, shoots were formed. These could be divided for further shoot multiplication. When 2,4-D was removed from the medium, roots or tuber formation occurred. Scilla. Using S. siberica, Hussey (1975a,1980b) obtained the regeneration of plantlets and bulblets through direct and indirect organogenesis from twin scales, leaves, flower stems, and ovaries. Plantlets and bulblets have also been obtained from scales and twin scales of S. hyacinthoides by Yanagawa and Sakanishi (1980a,b) and from leaves of S. indica by Turakhia and Kulkarni (1988). Plantlets of S. indica have been regenerated through callus originating from young leaf tips and scale leaves by Chakravarty and Sen (1987). They re-

144

K. W. KIM AND A. A. DE HERTOGH

ported that even though there were heterogeneous callus cell populations, the regenerated plants were diploid and had morphological characteristics of the mother plants. Somatic embryogenesis has been induced from anther-derived callus of S. indica (Chakravarty and Sen 1989). The formed embryos germinated into plantlets, which were successfully transferred to field conditions. Deumling and Clermont (1989) reported that plantlets can be produced from protoplasts originating from leaf scale-derived callus of S. siberica. Nuclear and chromosomal aberrations, such as chromosome elimination and chromatin diminution, have been observed during callus culture.

Sparaxis. Plantlets were obtained through direct organogenesis from corms, floral scapes, and leaf explants of mixed hybrids of S. bicolor (Hussey 1975a,1976a,1980b). Rapid multiplication was achieved by subculturing the shoots regenerated from the explants. For easy transplanting into soil, Hauser and Horn (1991) produced corms in vitro from seedlings and plantlets of Sparaxis hybrids. Tulipa. This species has a juvenile phase of about 5 years and there are many pre- and postfertilization barriers between species (Le Nard and De Hertogh 1993a). Asexually, a mature bulb produces only 2 to 6 daughter bulbs per year, and about 10 to 20 years are required to introduce a new cultivar. Plantlets and bulblets have been obtained mainly through direct organogenesis from scales (Nishiuchi and Myodo 1976; Nishiuchi 1979,1986; Riviere and Muller 1979; Wright and Alderson 1980; Alderson et a1. 1983), axillary buds (Hussey 1980b; Paterson and Harper 1986; Hulscher et a1. 1992), leaves (Paterson and Harper 1986), floral scapes (Hussey 1975a; Wright and Alderson 1980; Alderson et a1. 1983,1986; Rice et a1. 1983; Taeb and Alderson 1987,1990a,b; Le Nard et a1. 1987; Le Nard 1989; Le Nard and Chanteloube 1992; Alderson and Taeb 1990a,b; Baker et a1. 1990; Hulscher et a1. 1992), and flower buds (Paek 1982). The number of shoots obtained from initial explants was increased significantly by shoot subculture by Paterson and Harper (1986). They estimated that 2500 and 150,000 shoots can be produced after 1 and 2 years, respectively. Kim et a1. (1989) demonstrated that shoots could be regenerated indirectly from callus that originated from scale and flower stem explants. Bulbing in subcultured shoots has been promoted by low temperatures, long photoperiods, high light intensities, high sucrose concentrations, GA 3 , 1-aminocyclopropane-l-carboxylic acid (ACC), ethylene, NAA, and aged shoots (Nishiuchi 1979; Alderson

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

145

et a1. 1983,1986; Rice et a1. 1983; Le Nard 1989; Baker et a1. 1990; Alderson and Taeb 1990a; Taeb and Alderson 1990a). No reports of somatic embryogenesis were located. Embryos have been developed into seedlings that formed bulblets (Niimi 1978,1980; Aubert et a1. 1986). Also, seedlings and bulblets have been obtained from immature ovules containing embryos from self-pollinated T. gesneriana (Custers et a1. 1992). Van den Bulk et a1. (1994) used isolated microspores of two tulip cultivars and was able to induce embryo formation. These embryos subsequently developed a primary root and other organs such as "droppers." Gene transformation was successfully carried out by particle bombardment and Agrobacterium gene transfer using young flower stem explants (Wilmink et a1. 1992).

Urginea. Plantlets and bulblets have been produced through direct organogenesis (EI Grari and Backhaus 1987) and indirect organogenesis (Iha et a1. 1984,1991; Jha and Sen 1986b) from scale explants. Plantlets were also regenerated through somatic embryogenesis from callus originating from scales and inflorescences (Iha and Sen 1986a; Jha et a1. 1991). About 400 bulblets were produced by liquid culture in 18 weeks from one outer scale of a bulblet formed from scalederived callus (Iha et a1. 1984). Although the plants produced were predominately diploid (Iha and Sen 1987a), variability in chromosome number and morphology was observed (Iha and Sen 1987b). Jha and Sen (1986b) estimated that 64 x 10 6 bulbs can be produced per year from each in vitro-induced bulblet using shake culture. Scale-derived callus exhibited a lower karyological heterogeneity than inflorescence-derived callus (Iha and Sen 1990). Embryogenic calli obtained from scale-derived callus by long-term culture developed into embryos and complete bulbous plantlets. Also, embryogenesis from the leaf surface of the formed bulbous plantlets has been observed (Iha and Sen 1986a). Zantedeschia. These plants propagate by seed, offsets, and divisions in vivo and have low multiplication rates (Funnell 1993). Cohen (1981) obtained plantlets through direct organogenesis from apical bud explants. He found that the proliferation rate could be increased by about five times per month by splitting the shoots obtained from apical buds. Zephyranthes. The in vitro culture of Zephyranthes has been reviewed by Furmanowa and Oledzka (1990). Plants of Z. robusata

146

K. W. KIM AND A. A. DE HERTOGH

have been regenerated through direct organogenesis from twin scale explants by Furmanowa and Oledzka (1981). They found that for rapid multiplication, twin scale-derived bulblets should be divided into four sections and subcultured on solid medium. Sachar and Kapoor (1959) used pollinated ovules and ovaries and produced embryos, which subsequently germinated into seedlings in vitro. VI. CONCLUSIONS

For ornamental geophytes, it is essential to have propagation systems with high multiplication rates that are commercially profitable (De Hertogh and Le Nard 1993b). Unfortunately, most commercially used systems have low multiplication rates and are labor intensive. Also, the bulbs produced must be true to type, pest-free, and not readily susceptible to physiological disorders. Tissue culture system can satisfy these basic requirements, but, with the exception of lilies, they have had a minimal impact on the flower bulb industry. Very often, they are labor intensive and costly. Computer- and robotic-aided mass production systems are needed. Research has been carried out on many genera, but it has been concentrated on a few genera, such as Freesia, Gladiolus, Hyacinth us, Iris, Narcissus, Lilium, and Tulipa (Table 2.1). There is a need to expand the number of genera and selections used. In addition, special attention should be given to recent introductions and selections, breeding lines, and endangered species. The studies have demonstrated that almost all bulb tissues can be used as explants and that morphogenesis can occur either directly or indirectly (Table 2.1). Virus-free plants have been primarily regenerated from meristem tips (Table 2.2). They have been obtained from in vivo plants, in vitro plantlets, and organ and callus cultures. The use of heat treatments, chemicals, and antiviral compounds prior to explant removal has produced only limited success for obtaining virus-free plants. This approach needs additional research. Depending on the species, there has been success with in vitro pollination and fertilization; embryo rescue, ovary and ovule culture, anther, pollen, unpollinated ovary (ovule) culture, protoplast culture and fusion, callus and cell culture, gene transformation, and germplasm storage have also been successfully utilized for geophyte breeding and genetic improvement. No reports were found on endosperm culture. Continued investigations in all these areas is needed for all species.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

147

Le Nard and De Hertogh (1993b) have listed several areas for future research on ornamental geophytes, including plant breeding and genetics. One major goal is to obtain cultivars with improved horticultural characteristics. To accomplish this goal, increased research is needed not only on breeding and biotechnology but also in the area of physiological-genetics. Clearly, many desirable genes will have to be identified, isolated, and then transformed into the species. Some of the desirable traits are (1) increased flower life, (2) enhanced disease resistance, (3) enhanced insect resistance, (4) protection mechanisms against physiological disorders, (5) enhanced nutrient utilization, (6) enhanced water utilization, (7) increased flower color ranges for species with limited colors, (7) the biosynthesis of specific metabolites that have economic value, (9) enhanced natural propagation rates, and (10) shortened juvenile periods. In addition, improved technologies are required for (1) standard breeding techniques, (2) cryopreservation of germplasm, (3) the coating of plantlets and embryos for transferring to soil growing conditions, and (4) improved large-scale commercial tissue culture systems that meet the needs of the flower bulb industry. There are many challenges for creative researchers. LITERATURE CITED Aguettaz, P., A. Paffen, 1. Delvallee, P. van der Linde, and G. J. de Klerk. 1990. The development of dormancy in bulb lets of Ulium speciosum generated in vitro, I: the effects of culture conditions. Plant Cell Tissue Organ Culture 22:167-172. Ahuja, A., S. Koul, G. Ram, and B. L. Kaul. 1994. Somatic embryogenesis and regeneration of plantlets in saffron, Crocus sativus L. Indian J. Exp. BioI. 32:135-140. Alderson, P. G., and A. G. Taeb. 1990a. Influence of culture environment on shoot growth and bulbing of tulip in vitro. Acta Hort. 266:91-94. Alderson, P. G., and A. G. Taeb. 1990b. Effect of bulb storage on shoot regeneration from floral stems of tulip in vitro. J. Hort. Sci. 65:65-70. Alderson, P. G., R. D. Rice, and N. A. Wright. 1983. Towards the propagation of tulip in vitro. Acta Hort. 131:39-47. Alderson. P. G., A. G. Taeb, and R. D. Rice. 1986. Micropropagation of tulip: bulbing of shoots in culture. Acta Hort. 177:291-298. Alkema, H. Y. 1976. Vegetatieve vermeerdering van Allium species. Weekblad Bloembollencultuur 86:981-982. Allen, T. C. 1974. Production of virus-free lilies. Acta Hort. 36:235-239. Allen, T. C. 1975. Viruses of lilies and their control. Acta Hort. 47:69-75. Allen, T. c., and W. C. Anderson. 1980. Production of virus-free ornamental plants in tissue culture. Acta Hort. 110:245-251. Allen, T. c., and K. Fernald. 1972. Elimination of viruses in hybrid lilies. Lily Yearb. 25:53-55.

148

K. W. KIM AND A. A. DE HERTOGH

Allen, T. C., and K. Fernald. 1973. Multiplication of a virus-free 'Enchantment' lily in culture tubes. Lily Yearb. 26:69-72. Allen, T. c., W. C. Anderson, O. Ballantyne, W. Lin, and J. Goodell. 1980. Recent advances in research on lily symptomless virus. Acta Hort. 109:479-484. Amaki, W., Y. Shinohara, Y Hayata, H. Sano, and Y. Suzuki. 1984. Effects of bulb desiccation and storage on the in vitro propagation of hyacinth. Sci. Hort. 23:353360. Aminuddin, and B. P. Singh. 1985. In vitro gladiolus propagation for virus elimination through tissue culture. Indian Phytopathol. 38:375-377. Anderson, W. c., K. A. Mielke, P. N. Miller and T. Allen. 1990. In vitro bulblet propagation of virus-free Dutch iris. Acta Hort. 266:77-81. Asano, Y. 1980. Studies on the crosses between distantly related species of lilies, IV: the culture of immature hybrid embryos 0.3-0.4 mm long. J. Jpn. Soc. Hort. Sci. 49:114-118. Asano, Y 1982. Overcoming interspecific hybrid sterility in Ulium. J. Jpn. Soc. Hort. Sci. 51:75-81. Asano, Y, and H. Myodo. 1977a. Studies on crosses between distantly related species of lilies, I: for the intrastylar pollination technique. J. Jpn. Soc. Hort. Sci. 46:59-65. Asano, Y., and H. Myodo. 1977b. Studies on crosses between distantly related species of lilies, II: the culture of immature hybrid embryos. J. Jpn. Soc. Hort. Sci. 46:267-273. Asjes, C. J. 1990. Production for virus freedom of some principle bulbous crops in The Netherlands. Acta Hort. 266:517-529. Asjes, C. J., M. H. Bunt, and D. H. M. van Slogteren. 1974. Production of hyacinth mosaic virus-free hyacinths and lily symptomless virus-free lilies by meristemtip culture. Acta Hort. 36:223-228. Aubert, B., G. Weber, N. Dorion, M. Le Nard, and C. Bigot. 1986. Etude preliminaire sur l'organogenese adventive a partir d'embryons de tulipe cultives in vitro (Tulipa gesneriana). Can. J. Bot. 64:1837-1842. Bach, A. 1987. The capability of in vitro regeneration of various cultivars of Freesia hybrida. Acta Hort. 212:715-718. Bach, A. 1988. Effect of activated charcoal on in vitro propagation of Iris x hollandica. Bul. Pol. Acad. Sci. (BioI. Sci.) 36:107-111. Bach, A. 1992. Somatic embryogenesis from zygotic embryos and meristems of Freesia hybrida. Acta Hort. 325:429-434. Bach, A., and A. Cecot. 1988. Micropropagation of Hyadnthus orientalis cv. Carnegie in a double-phase culture medium, I: effect of cytokinins on growth and development. Acta Hort. 226:607-610. Bach, A., and A. Cecot. 1989. Application of double-phase culture media in micropropagation of Hyacinthus orientalis cv. Carnegie, II: Effect of auxins on growth and development. Acta Hort. 251:215-221. Bae, S. K., B. K. Lee, and J. S. Eun. 1983. Clonal propagation of hyacinth by in vitro culture, III: bulblets culture, subculture of callus and organ formation from excised tissues. J. Kor. Soc. Hort. Sci. 24:249-257. Bajaj, Y. P. S. 1990. Freesia. p. 413-428. In: P. V. Ammirato, D. R. Evans, W. R. Sharp, and Y. P. S. Bajaj (eds.), Handbook of plant cell culture, Vol.5, Ornamental species. McGraw-Hill, New York. Bajaj, Y. P. S., and R. L. M. Pierik. 1974. Vegetative propagation of Freesia through callus cultures. Neth. J. Agr. Sci. 22:153-159. Bajaj, Y. P. S., M. M. S. Sidhu, and A. P. S. Gill. 1983. Some factors affecting the in vitro propagation of Gladiolus. Sci. Hart. 18:269-275.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

149

Baker, C. M., H. F. Wilkins and P. D. Ascher. 1990. Comparisons of precultural treatments and cultural conditions on in vitro response of tulip. Acta Hort. 266:8390. Baruch, E. R., and F. Quak. 1966. Virus-free plants of iris 'Wedgwood' obtained by meristem culture. Neth. J. Plant Path. 72:270-273. Beattie, D. J., and J. W. White. 1993. Ulium-hybrids and species. p. 423-454. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. Bennici, A. 1979. Cytological chimeras in plants regenerated from Ulium longiflorum tissues grown in vitro. Z. Pflanzenzucht. 82:349-353. Benschop, M. 1993. Polianthes. p. 589-601. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. Bergonon, S., C. Codina, J. Bastida, F. Viladomat, and E. Mele. 1992. The shake liquid culture as an alternative way to the multiplication of Narcissus plants. Acta Hort. 325:447-452. 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. Beruto, M., and P. Debergh. 1992. Somatic embryogenesis in Ranunculus asiaticus 1. hybr. thalamus cultivated in vitro. Plant Cell Tissue Organ Culture 29:161165. Blakesley, D., and D. Constantine. 1992. Uptake and metabolism of 6-benzyladenine in shoot cultures of a range of species. Plant Cell Tissue Organ Culture 28:183186. Blom-Barnhoorn, G. J., and J. van Aartrijk. 1985. The regeneration of plants free of LSV and TBV from infected Ulium bulb-scale explants in the presence ofVirazole. Acta Hort. 164:163-168. Blom-Barnhoorn, G. J., J. van Aartrijk, and P. C. G. van der Linde. 1986. Effect of virazole on the production of hyacinth plants free from hyacinth mosaic virus (HMV) by meristem culture. Acta Hort. 177:571-574. Bonora, A., G. Dall'Olio, A. Donini, and A. Bruni. 1987. An HPLC screening of some Italian Ranunculaceae for the lactone protoanemonin. Phytochemistry 26:22772279. Bose, T. K., B. K. Jana, and S. Moulik. 1987. A note on the micro-propagation of tuberose from scale stem section. Indian J. Hort. 44:100-101. Bouman, H., and G. J. de Klerk. 1990. Cryopreservation of lily meristems. Acta Hort. 266:331-337. Brants, D. H., and H. Vermeulen. 1965. Production of virus-free Freesia's by means of meristem culture. Neth. J. Plant Path. 71:25-27. Broertjes, C., B. Haccius, and S. Weidlich. 1968. Adventitious bud formation on isolated leaves and its significance for mutation breeding. Euphytica 17:321-344. Brunt, A. A. 1980. A review of problems and progress in research on viruses and virus diseases of Narcissus in Britain. Acta Hort. 110:23-30. Buiteveld, J., and J. Creemers-Molenaar. 1994. Plant regeneration from protoplasts isolated from suspension cultures of leek (Allium ampeloprasum L.). Plant Sci. 100:203-210. Buiteveld, J., P. F. Fransz, and J. Creemers-Molenaar. 1994. Induction and characterization of embryogenic callus types for the initiation of suspension cultures of leek (Allium ampeloprasum L.). Plant Sci. 100:195-202. Byther, R. S., and G. A. Chastagner. 1993. Diseases. p. 71-99. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam.

150

K. W. KIM AND A. A. DE HERTOGH

Chakravarty, B., and S. Sen. 1987. In vitro regeneration from callus cultures of Scilla indica (Roxb.) Baker. Cur. Sci. 56:960-962. Chakravarty, B., and S. Sen. 1989. Regeneration through somatic embryogenesis from anther explants of Scilla indica (Roxb.) Baker. Plant Cell Tissue Organ Culture 19:71-75. Chen, C. H., and Y. C. Goeden-Kallemeyn. 1979. In vitro induction of tetraploid plants from colchicine-treated diploid daylily callus. Euphytica 28:705-709. Chow, Y. N., C. Selby, and B. M. R. Harvey. 1992a. Stimulation by sucrose of Narcissus bulbil formation in vitro. J. Hart. Sci. 67:289-293. Chow, Y. N., C. Selby, and B. M. R. Harvey. 1992b. A simple method for maintaining high multiplication of Narcissus shoot cultures in vitro. Plant Cell Tissue Organ Culture 30:227-230. Chow, Y. N., C. Selby, T. W. Fraser, and B. M. R. Harvey. 1993. Basal plate tissue in Narcissus bulbs and in shoot clump cultures: its structure and role in organogenic potential of single leaf cultures. Ann. Bot. 71:437-443. Chu, I. Y. K, and S. L. Kurtz. 1990. Commercialization of plant micropropagation. p. 126-164. In: P.V. Ammirato, D. R. Evans, W. R. Sharp, and Y. P. S. Bajaj (eds), Handbook of plant cell culture, Vo1.5, Ornamental species. McGraw-Hill, New York. Chung, J. D., C. K. Chun. Y. K. Suh, D. W. Lee, S. Y. Byun, and J. K. Park. 1980. Studies on tissue culture of Ornithogalum thyrsoides in vitro, I: effect of plant growth regulators on callus formation and organogenesis from tissues of female organ (stigma, style and ovary). J. Kor. Soc. Hart. Sci. 21:198-203. Chung, J. D., C. K. Chun, K M. Rhee, and K. Y. Paek. 1981a. In vitro propagation of Hyacinthus orientalis L, II: effect of auxins on callus formation and bulblet differentiation from bulb scale segments. Kor. J. Plant Tissue Culture 8:45-48. Chung, J. D., C. K. Chun, Y. K. Suh, and S. Y. Byun. 1981b. Studies on the tissue culture of Ornithogalum thyrsoides in vitro, II: effect of plant growth regulators on callus formation and organogenesis from tissue of shoot apical meristem and bulb scale. J. Kor Soc. Hart. Sci. 22:37-43. Chung, J. D., C. K. Chun, Y. K. Suh, and J. K. Park. 1981c. In vitro culture of bulbil scale of Lilium lancifolium, I: effect of auxins on bulblet formation and growth of seedlings. J. Kor. Soc. Hart. Sci. 22:131-138. Chung, J. D., C. K. Chun, Y. K. Suh, and K M. Rhee. 1983a. In vitro propagation of Hyacinthus orientalis L, III: effect of auxin concentrations and light or dark incubation on organogenesis from flower stalk tissue, and comparison of totipotency between bulbscale and flower stalk tissue. J. Kor. Soc. Hort. Sci. 24:76-85. Chung, J. D., C. K. Chun, Y. K. Suh, and E. M. Rhee. 1983b. In vitro propagation of Hyacinthus orientalis L, IV: organ redifferentiation through the subculture of bulblet scale and leaf tissue segments originated from flower stalk tissue culture. J. Kor. Soc. Hort. Sci. 24:162-168. Chung, J. D., J. K. Chun, and J. A. Shin. 1984. In vitro propagation of Lilium longiflorum through floral organ culture. J. Kor. Soc. Hart. Sci. 25:170-181. Cohen, D. 1981. Micropropagation of Zantedeschia hybrids. Proc. Int. Plant Prop. Soc. 31:312-316. Cohen, D., K. S. Milne, and M. J. Hyland. 1985. In vitro manipulation of virus concentrations in hybrid lilies. Acta Hart. 164:319-324. Cooper, P. A., and D. Cohen. 1982. Caladium-A candidate for micropropagation? Proc. Int. Plant Prop. Soc. 32:363-367.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

151

Crouch, N. R., L. F. van Staden, J. van Staden, F. E. Drewes, S. E. Drewes, and H.J. Meyer. 1993. Accumulation of cyanidin-3-glucoside in callus and cell cultures of Oxalis rec1inata. J. Plant Physiol. 142:109-111. Cumming, B. G., and D. E. Peck. 1984. Tissue culture of grape hyacinth. HortScience 19:723-724. Custers, J. B. M., W. Eikelboom, J. H. W. Bergervoet, and J.P. van Eijk. 1992. In ovulo embryo culture of tulip (Tulipa L.); effects of culture conditions on seedling and bulblet formation. Sci. Hort. 51:111-122. Dantu, P. K., and S. S. Bhojwani. 1987. In vitro propagation and corm formation in Gladiolus. Gartenbauwissenschaft 52:90-93. Dantu, P. K., and S. S. Bhojwani. 1995. In vitro corm formation and field evaluation of corm-derived plants of Gladiolus. Sci. Hort. 61:115-129. Debergh, P. 1987. Recent trends in the application of tissue culture to ornamentals. p. 383-393. In: C. E. Green, D. A. Somers, W. P. Hackett, and D. D. Biesboer (eds.), Plant tissue and cell culture. Alan R. Liss, New York. Debergh, P. C.,and L. J. Maene. 1981. A scheme for commercial propagation of ornamental plants by tissue culture. Sci. Hort. 14:335-345. Debergh, P. c., and P. E. Read. 1991. Micropropagation. p. 1-13. In: P. C. Debergh and R. H. Zimmerman (eds.), Micropropagation: technology and application. Kluwer Academic, Dordrecht, The Netherlands. Debergh, P. c., J. Roggemans, and R. Standaert-De Metsenaere. 1990. Tissue culture in relation to ornamental plants. p. 161-189. In: S.S. Bhojwani (ed.), Plant tissue culture: applications and limitations. Elsevier Science, Amsterdam. Debergh, P. c., J. Aitken-Christie, K. Cohen, B. Grout, S. von Arnold, R. Zimmerman, and M. Ziv. 1992. Reconsideration of the term 'vitrification' as used in micropropagation. Plant Cell Tissue Organ Culture 30:135-140. De Bruyn, M. H., and D. 1. Ferreira. 1992. In vitro corm production of Gladiolus dalenii and G. tristis. Plant Cell Tissue Organ Culture 31:123-128. De Bruyn, M. H., D. 1. Ferreira, M. M. Slabbert, and J. Pretorius. 1992. In vitro propagation of Amaryllis belladonna. Plant Cell Tissue Organ Culture 31:179-184. De Hertogh, A. A., and M. Le Nard. 1993a. Botanical aspects of flower Bulbs. p. 720. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. De Hertogh, A. A., and M. Le Nard. 1993b. World production and horticultural utilization of flower bulbs. p. 21-28. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. De Hertogh, A. A., and M. Le Nard. 1993c. Production systems for flower bulbs. p. 45-52. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. De Hertogh, A. A., and K. Zimmer. 1993. Allium: ornamental species. p. 187-200. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. De Klerk, G. J., and A. Paffen. 1995. The effects of environmental conditions on sprouting of micropropagated lily bulblets with various levels of dormancy. Acta Bot. Neerl. 44:33-39. De Klerk, G. J., 1. Delvallee, and A. Paffen. 1992. Dormancy release of micropropagated bulblets of Lilium speciosum after long culture in soil. HortScience 27:147-148. De Lange, H. S. Tennant, P. Botha, C. Klein, and G. Nichols. 1989. Micropropagation and the trade in indigenous medicinal plants. Veld & Flora 75:60-61.

152

K. W. KIM AND A. A. DE HERTOGH

Delvallee, I., A. Paffen, and G. J. de Klerk. 1990. The development of dormancy in bulblets of Lilium speciosum generated in vitro, II: the effects of temperature. Physiol. Plant. 80:431-436. De Munk, W. J., and J. Schipper. 1993. Iris: bulbous and rhizomatous. p. 349-379. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. Deumling, B., and L. Clermont. 1989. Changes in DNA content and chromosomal size during cell culture and plant regeneration of Scilla siberica: selective chromatin diminution in response to environmental conditions. Chromosoma 97:439448. Djilianov, D., M. M. Gerrits, A. Ivanova, H. A. van Onckelen, and G.J.M. de Klerk. 1994. ABA content and sensitivity during the development of dormancy in lily bulblets regenerated in vitro. Physiol. Plant. 91:639-644. EI Grari, R, and R. A. Backhaus. 1987. In vitro propagation of red squill, Urginea martima Baker. Plant Cell Tissue Organ Culture 10:65-71. Elliott, G. C., M. A. Smith, and M. P. Bridgen. 1993. Growth response of Alstroemeria 'Parirgo Pink' to phosphate supply in vitro. Plant Cell Tissue Organ Culture 32:199204. Ellyard, R K. 1979. In vitro propagation of Anigozanthos manglesii, A. flavidus, and Macropidia fulginosa. HortScience 13:662-663. Escandon, A. S., H. E. Hopp, and G. Hahne. 1989. Differential amplification of five selected genes in callus cultures oftwo shrubby Oxalis species. Plant Sci. 63:177185. Fakhrai, F., and P. K. Evans. 1989. Morphogenic potential of cultured explants of Crocus chrysanthus Herbert. cv. E. P. Bowles. J. Exp. Bot. 40:809-812. Fakhrai, F., and P. K. Evans. 1990. Morphogenic potential of cultured floral explants of Crocus sativus 1. for the in vitro production of saffron. J. Exp. Bot. 41:47-52. Finnie, J. F., and J. van Staden. 1989. In vitro propagation of Sandersonia and Gloriosa. Plant Cell Tissue Organ Culture 19:151-158. Finnie, J. F., and J. van Staden. 1995. In vitro culture of Clivia miniata. Plant Cell Tissue Organ Culture (in press). Fitter, M. S., and A. D. Krikorian. 1981. Recovery of totipotent cells and plantlet production from daylily protoplasts. Ann. Bot. 48:591-597. Fitter, M. S., and A. D. Krikorian. 1985. Mature phenotype in Hemerocallis plantlets fortuitously generated in vitro. J. Plant Physiol. 121:97-101. Foxe, M. J. 1991. Rapid in vitro propagation of virus-indexed Freesia. p. 205-208. In: J. Prakash and R. L. M. Pierik (eds.), Horticulture: new technologies and applications. Kluwer Academic, Dordrecht, The Netherlands. Funnell, K. A. 1993. Zantedeschia. p. 683-704. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science. Amsterdam. Furmanowa, M., and H. Oledzka. 1981. Plant regeneration from excised bulb scale segments of Zephyranthes robusta Baker. Acta Soc. Bot. Pol. 50:399-404. Furmanowa, M., and H. Oledzka. 1990. Zephyr: lily. p. 800-819. In: P. V. Ammirato, D. R. Evans, W. R Sharp, and Y. P. S. Bajaj (eds), Handbook of plant cell culture, Vol.5, Ornamental species. 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. Gavinlertvatana, P., P. E. Read, H. F. Wilkins, and R. Heins. 1979. Influence of photoperiod and daminozide stock plant pretreatments on ethylene and CO 2 levels and callus formation from dahlia leaf segment cultures. J. Am. Soc. Hort. Sci. 104:849-852.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

153

Gavinlertvatana, P., P. E. Read, H. F. Wilkins, and R. Heins. 1982. Ethylene levels in flask atmospheres of Dahlia pinnata Cay. leaf segments and callus cultured in vitro. J. Am. Soc. Hort. Sci. 107:3-6. Geier, T. 1977. Morphogenesis and plant regeneration from cultured organ fragments of Cyclamen persicum. Acta Hort. 78:167-174. Geier, T., H. W. Kohlenbach, and G. Reuther. 1990. Cyclamen. p. 352-374. In: P. V. Ammirato, D. R. Evans, W. R. Sharp, and Y. P. S. Bajaj (eds.), Handbook of plant cell culture, Vol.5, Ornamental species. McGraw-Hill, New York. Ginzburg, C., and M. Ziv. 1973. Hormonal regulation of cormel formation in Gladiolus stolons grown in vitro. Ann. Bot. 37:219-224. Gonzalez-Benito, E., and P. G. Alderson. 1990. Regeneration from Aistroemeria callus. Acta Hort. 280:135-138. Gonzalez-Benito, M. E., and P. G. Alderson. 1992. Callus induction and plant regeneration in Aistroemeria. J. Exp. Bot. 43:205-211. Goo, D. H., and K. W. Kim. 1994. Influence of sucrose, ABA, and daylength on cormlet formation of Gladiolus in vitro: histological observation. J. Kor. Soc. Hort. Sci. 35:400-405. Goodwin, P. B. 1993. Anigozanthos (Macropidia). p. 219-226. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. Gozu,Y., M. Yokoyama, M. Nakamura, R. Namba, K. Yomogida, M. Yanagi, and S. Nakamura. 1993. In vitro propagation of Iris pallida. Plant Cell Rep. 13:12-16. Graves, A. C. F., and S. L. Goldman. 1987. Agrobacterium tumefaciens-mediated transformation of the monocot genus Gladiolus: detection of expression of T-DNAencoded genes. J. Bacteriol. 169:1745-1746. Griesbach, R. J. 1989. Selection of a dwarf Hemerocallis through tissue culture. HortScience 24:1027-1028. Grootaarts, H., J. H. N. Schel, and R. L. M. Pierik. 1981. The origin of bulblets formed on excised twin scales of Nerine bowdenii W. Watts. Plant Cell Tissue Organ Culture 1:39-46. Hackett, W.P. 1969. Aseptic multiplication of lily bulblets from bulb scales. Proc. Int. Plant Prop. Soc. 19:105-108. Haegeman, J. 1993. Begonia: tuberous hybrids. p. 227-238. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. Hakkaart, F. A., and J. M. A. Versluijs. 1985. Viruses of Aistroemeria and preliminary results of meristem culture. Acta Hort. 164:71-75. Hakkaart, F. A., and J. M. A. Versluijs. 1988. Virus elimination by meristem-tip culture from a range of Aistroemeria cultivars. Neth. J. Plant Path. 94:49-56. Hakkaart, F. A., D. Z. Maat, and F. Quak. 1975. Viruses and meristem culture of Nerine. Acta Hart. 47:51-53. Halevy, A. H. 1990. Recent advances in control of flowering and growth habit of geophytes. Acta Hort. 266:35-42. Han, B. H., J. S. Kim, S. L. Choi, and K. Y. Paek. 1991a. In vitro masspropagation of Begonia rex Putz. and Caladium bicolor Vent. Korean J. Plant Tissue Culture 18:95101. Han, B. H., J. S. Kim, and K. Y. Paek. 1991b. Effect of growth regulators on the bulblet formation through twin-scale segment culture in Hippeastrum hybridum 'Red Lion'. Korean J. Plant Tissue Culture 18:355-360. Han, B. H., Y. J. Kim, and T. 1. Kim. 1993. In vitro micropropagation of Aistroemeria spp. cvs Yellow Prince and Cyprus through rhizome culture. Korean J. Plant Tissue Culture 20:351-354.

154

K. W. KIM AND A. A. DE HERTOGH

Han, B. H., Y. J. Kim, and J. K. Choi. 1994. Micropropagation of Alstroemeria spp. through rhizome tip culture. J. KaT. Soc. Hort. Sci. 35:172-177. Hanks, G. R. 1993. Narcissus. p. 463-558. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. Harper, P. C. 1991. Micropropagation of bulb crops. p. 161-168. In: J. Prakash and R. L. M. Pierik (eds.), Horticulture: new technologies and applications. Kluwer Academic, Dordrecht, The Netherlands. Hartman, R. D. 1974. Dasheen mosaic virus and other phytopathogens eliminated from caladium, taro, and cocoyam by culture of shoot tips. Phytopathology 64:237240. Hartmann, H. T., D. E. Kester, and F. T. Davies. 1990. Plant propagation: principles and practices, 5th ed. Prentice Hall, Englewood Cliffs, NJ. Hauser, B., and W. Horn. 1991. In vitro corm formation of Sparaxis hybrids. Acta Hort. 300:169-172. Hawkes, H. Y., and H. Wainwright. 1987. In vitro organogenesis of Cyclamen persicum Mill. seedling tissue. Acta Hort. 212:711-714. Hayashi, T., K. Yoshida, and K. Sana. 1988. Formation of alkaloids in suspensioncultured Colchicum autumnale. Phytochemistry 27:1371-1374. Heuser, C. W., and D. A. Apps. 1976. In vitro plantlet formation from flower petal explants of Hemerocallis cv. Chipper Cherry. Can. J. Bot. 54:616-618. Heuser, C. W., and J. Harker. 1976. Tissue culture propagation of daylilies. Comb. Proc. Int. Plant Prop. Soc. 26:269-272. Higgins, W. S., and D. P. Stimart. 1990. Influence of in vitro generation temperature and post-in vitro cold storage duration on growth response of Ulium longiflorum bulblets. J. Am. Soc. Hort. Sci. 115:930-933. Hirata, Y., and M. Kunishige. 1982. Studies on the virus disease of bulbous iris (Iris hollandica H.): obtaining and multiplication of virus-free bulbs through tissue culture, and bulb production by using its. Bull. Veg. Ornam. Crops Res. Stan. Ser. C (Kurume, Japan) 6:83-99. Hollings, M. 1965. Disease control through virus-free stock. Ann. Rev. Phytopathol. 3:367-396. Holmes, F. O. 1955. Elimination of spotted wilt from Dahlia by propagation of tip cuttings. Phytopathology 45:224-226. Homes, J., M. Legros, and M. Jaziri. 1987. In vitro multiplication of Crocus sativus L. Acta Hort. 212:675-676. Hooykaas-van Slogteren, G. M. S. 1986. Can Agrobacterium tumefaciens be used for genetic manipulation of bulbous crops and other monocotyledons. Acta Bot. Neerl. 35:527. Hooykaas-van Slogteren, G. M. S., P. J. J. Hooykaas, and R. A. Schilperoort. 1984. Expression of Ti plasmid genes in monocotyledonous plants infected with Agrobacterium tumefaciens. Nature 311:763-764. Horst, R. K. 1988. Production of plants free of virus and prevention of reinfection. Acta Hart. 234:393-402. Hosoki, T., and T. Asahira. 1980. In vitro propagation of Narcissus. HortScience 15:602-603. Hosoki, T., and Y. Sagawa. 1977. Clonal propagation of ginger (Zingiber officinale Roscoe) through tissue culture. HortScience 12:451-452. Huang, C. W., H. Okubo, and S. Uemoto. 1990. Comparison of bulblet formation from twin scales and single scales in Hippeastrum hybridum cultured in vitro. Scientia Hort. 42:151-160.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

155

Huang, L. C., and D. M. Liu. 1989. Clonal multiplication of Lycoris aurea by tissue culture. Sci. Hort. 40:145-152. Hulscher, H., H. T. Krijgaheld, and P. C. G. van der Linde. 1992. Propagation of shoots and bulb growth of tulip in vitro. Acta Hart. 325:441-446. Hussey, G. 1975a. Totipotency in tissue explants and callus of some members of the Liliaceae, lridaceae, and Amaryllidaceae. J. Exp. Bot. 26:253-262. Hussey, G. 1975b. Propagation of hyacinths by tissue culture. Sci. Hort. 3:21-28. Hussey, G. 1976a. In vitro release of axillary shoots from apical dominance in monocotyledonous plantlets. Ann. Bot. 40:1323-1325. Hussey, G. 1976b. Plantlet regeneration from callus and parent tissue in Ornithogalum thyrsoides. J. Exp. Bot. 27:375-382. Hussey, G. 1976c. Propagation of Dutch Iris by tissue culture. Sci. Hort. 4:163-165. Hussey, G. 1977a. In vitro propagation of some members of the Liliaceae, Iridaceae and Amaryllidaceae. Acta Hort. 78:303-309. Hussey, G. 1977b. In vitro propagation of Gladiolus by precocious axillary shoot formation. Scientia Hort. 6:287-296. Hussey, G. 1978. The application of tissue culture to the vegetative propagation of plants. Sci. Prog., Oxford 65:185-208. Hussey, G. 1980a. In vitro propagation. p. 51-61. In: D. S. Ingram and J. P. Helgeson (eds.), Tissue culture methods for plant pathologists. Blackwell, Oxford, UK. Hussey, G. 1980b. Propagation of some members of the Liliaceae, Iridaceae and Amaryllidaceae by tissue culture: petaloid monocotyledons. p. 33-42. In: C. D. Brickell, D. F. Cutler, and M. Gregory (eds.), Linnean Soc. Symp. Ser. 8. Academic, London. Hussey, G. 1982. In vitro propagation of Narcissus. Ann. Bot. 49:707-719. Ilahi, 1., M. Jabeen, and N. Firdous. 1987. Morphogenesis with saffron tissue culture. J. Plant. Physiol. 128:227-232. Isa, T., and T. Ogasawara. 1988. Efficient regeneration from the callus of saffron (Crocus sativus). Jpn. J. Breed. 38:371-374. Isa, T., T. Ogasawara, and H. Kaneko. 1990. Regeneration of saffron protoplasts immobilized in Ca-alginate beads. Jpn. J. Breed. 40:153-157. Ito, M. 1973. Studies on the behavior of meiotic protoplasts, I: Isolation from microsporocytes of liliaceous plants. Bot. Mag. (Tokyo) 86:133-141. Jacobs, G., M. Richard, 1. A. Allderman, and K. 1. Theron. 1992. Direct and indirect organogenesis in tissue cultures of Nerine bowdenii W. Watts. Acta Hort. 325:475479. Janson, J. 1993. Placental pollination in Lilium longiflorum Thunb. Plant Sci. 90:105115. Jehan, H., D. Courtois, C. Ehret, K. Lerch, and V. Petiard. 1994. Plant regeneration of Iris pallida Lam. and Iris germanica L. via somatic embryogenesis from leaves, apices and young flowers. Plant Cell Rep. 13:671-675. Jha, S., and S. Sen. 1984. Morphogenesis and propagation of medicinally important plant species of family liliaceae: a review. p. 89-100. NSABMATP (Bot. Lab. Pharm. Anat.), Calcutta Univ. India. Jha, S., and S. Sen. 1986a. Development of indian squill (Urginea indica Kunth.) through somatic embryogenesis from long term culture. J. Plant Physiol. 124:431439. Jha, S., and S. Sen. 1986b. Mass propagation of indian squill, Urginea indica Kunth. in vitro. Acta Hort. 188:105-109.

156

K. W. KIM AND A. A. DE HERTOGH

Jha, S., and S. Sen. 1987a. Nuclear changes and organogenesis during callus culture of Urginea indica Kunth., Indian squill. Cytologia 52:433-438. Jha, S., and S. Sen. 1987b. Karyotype variability in regenerated plants of Urginea indica Kunth. Cytologia 52:615-626. Jha, S., and S. Sen. 1990. Induction of mitosis in polytene nuclei and hormonal effect on nuclear changes during callus initiation in diploid Urginea indica Kunth. (Liliaceae). Genetica 80:9-15. Jha, S., G. C. Mitra, and S. Sen. 1984. In vitro regeneration from bulb explants of Indian squill, Urginea indica Kunth. Plant Cell, Tissue Organ Culture 3:91-100. Jha, S., N. P. Sahu, and S. B. Mahato. 1991. Callus induction, organogenesis and somatic embryogenesis in three chromosomal races of Urginea indica and production of bufadienolides. Plant Cell, Tissue Organ Culture 25:85-90. Johansson, 1. 1983. Effects of activated charcoal in anther cultures. Physiol. Plant. 59:397-403. Johansson, L., and T. Eriksson. 1984. Effects of carbon dioxide in anther cultures. Physiol. Plant. 60:26-30. Johansson, L., B. Andersson, and T. Eriksson. 1982. Improvement of anther culture technique: activated charcoal bound in agar medium in combination with liquid medium and elevated CO 2 concentration. Physiol. Plant. 54:24-30. Kamo, K., J. Chen, and R. Lawson. 1990. The establishment of cell suspension cultures of Gladiolus that regenerate plants. In Vitro Cell. Dev. BioI. 26:425-430. Kamo, K., A. Blowers, F. Smith, J. Van Eck, and R. Lawson. 1995. Stable transformation of Gladiolus using suspension cells and callus. J. Am. Soc. Hort. Sci. 120:347352. Kanoh, K., M. Hayashi, Y. Serizawa, and T. Konishi. 1988. Production of interspecific hybrids between Lilium longiflorum and L. x elegance by ovary slice culture. Jpn. J. Breed. 38:278-282. Kato, Y., and Y. Yasutake. 1977. Plantlet formation and differentiation of epidermal tissues in green callus cultures from excised leaves of Lilium. Phytomorphology 27:390-396. Kawarabayashi, W. 1993a. Effects of several conditions in aerated liquid culture on in vitro multiplication of bulbs of Lilium japonicum Thunb. J. Jpn. Soc. Hort. Sci. 62:197-205. Kawarabayashi, W. 1993b. Mass in vitro production of bulblets of Lilium japonicum Houtt. J. Jpn. Soc. Hort. Sci. 62:611-618. Khan, M. R. I., J. K. Heyes, and D. Cohen. 1988. Plant regeneration from oca (Oxalis tuberosa M.): the effect of explant type and culture media. Plant Cell Tissue Organ Culture 14:41-50. Kim, K. S. 1991. The effect of growth regulators, temperature and sucrose on the dormancy in Lilium speciosum bulblets cultured in vitro. Korean J. Plant Tissue Culture 18:103-111. Kim, K. S., E. Davelaar, and G. J. De Klerk. 1994. Abscisic acid controls dormancy development and bulb formation in lily plantlets regenerated in vitro. Physiol. Plant. 90:59-64. Kim, K. W. 1986. Rapid multiplication of plants through micropropagation. Symposium on agricultural application of plant tissue culture and its industrialization. Kyungpook National Univ. (Taegu, Korea) Special Suppl. 40:85-98. Kim, K. W. 1987. Variability in micropropagation of higher plants. J. Resource Dev. Yeungnam Univ. (Kyongsan, Korea) Suppl. p. 61-68. Kim, K. W., and D. H. Goo. 1991. Sprouting and dedifferentiation of excised gladiolus cormel tip in vitro. J. Kor. Soc. Hort. Sci. 32:388-393.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

157

Kim, K. W., and S. Y. Han. 1993. Cormlet formation in gladiolus shoot base by growth retardants in vitro. J. KoT. Soc. Hort. Sci. 34:136-144. Kim, K. W., and M. S. Kang. 1992. Somatic embryogenesis and plant regeneration from Gladiolus callus in vitro. J. Kor. Soc. Hort. Sci. 33:87-94. Kim, K. W., and J. S. Lee. 1993. Differences in cultivar on formation and growth of the Gladiolus callus in vitro. J. Kor. Soc. Hort. Sci. 34:301-307. Kim, K. W., and S. K. Sung. 1990. Obtaining of plantlets through immature embryo culture of lilies. J. Kor. Soc. Hort. Sci. 31:423-431. Kim, K. W., J. B. Choi, and K. Y. Kwon. 1988. Rapid multiplication of gladiolus plants through callus culture. J. Kor. Soc. Hort. Sci. 29:312-318. Kim, K. W., Y. S. Kim, C. K. Woo, O. H. Kim, and J. S. Kim. 1989. Formation of adventitious bud from Tulipa gesneriana 'Red Matador' callus. J. Kor. Soc. Hort. Sci. 30:157-162. Kim, K. W., M.S. Kang, and D. H. Goo. 1991a. External and histological characteristics of organogenesis from gladiolus callus. J. Kor. Soc. Hort. Sci. 32:125-130. Kim, Y. J., P. M. Hasegawa, and R. A. Bressan. 1981. In vitro propagation of hyacinth. HortScience 16:645-647. Kim, Y. S., E. H. Park, S. O. Yoo, J. S. Eun, and W. S. Song. 1991b. In vitro propagation of ranunculus (Ranunculus asiaticus L.), I: embryogenic callus induction and somatic embryogenesis. J. Kor. Soc. Hort. Sci. 32:401-410. Kim, Y. S., E. H. Park, S. O. Yoo, J. S. Eun, and W. S. Song. 1991c. In vitro propagation of ranunculus (Ranunculus asiaticus L.), II: somatic embryogenesis and plantlet development: anatomical observations. J. Kor. Soc. Hort. Sci. 32:560-567. Kiviharju, E., U. Tuominen, and T. Tormala. 1992. The effect of explant material on somatic, embryogenesis of Cyclamen persicum Mill. Plant Cell Tissue Organ Culture 28:187-194. Klesser, P. J., and D. D. Nel. 1976. Virus diseases and tissue culture of some South African flower bulbs. Acta Hort. 59:71-76. Konar, R. N., and K. Nataraja. 1964. In vitro control of floral morphogenesis in Ranunculus sceleratus L. Phytomorphology 14:558-563. Konar, R. N., and K. Nataraja. 1965a. Experimental studies in Ranunculus sceleratus L. plantlets from freely suspended cells and cell groups. Phytomorphology 15:206211. Konar, R. N., and K. Nataraja. 1965b. Production of embryoids from the anthers of Ranunculus sceleratus L. Phytomorphology 15:245-248. Konar, R. N., E. Thomas, and H. E. Street. 1972. Origin and structure of embryoids arising from epidermal cells of the stem of Ranunculus sceleratus L. J. Cell Sci. 11:77-93. Koyama, A., Y. Ohmori, N. Fujioka, H. Miyagawa, K. Yamasaki, and H. Kohda. 1988. Formation of stigma-like structures and pigment in cultured tissues of Crocus sativus. Planta Medica 54:375-376. Krikorian, A. D., and R. P. Kann. 1981. Plantlet production from morphogenetically competent cell suspensions of daylily. Ann. Bot. 47:679-686. Krikorian, A. D., and R. P. Kann. 1986. Regeneration in Liliaceae, Iridaceae, and Amaryllidaceae. p. 187-205. In: I. K. Vasil (ed.), Cell culture and somatic cell genetics of plants, Vol. 3, Plant regeneration and genetic variability. Academic Press, New York. Krikorian, A. D., S. A. Staicu, and R. P. Kann. 1981. Karyotype analysis of a daylily clone reared from aseptically cultured tissues. Ann. Bot. 47:121-131.

158

K. W. KIM AND A. A. DE HERTOGH

Krikorian, A. D., S. S. Cronauer-Mitra, and M. S. Fitter Corbin. 1988. Protoplast culture of perennials. Sci. Hort. 37:277-293. 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, and Y. P. S. Bajaj (eds.), Handbook of plant cell culture, Vol.5, Ornamental species. McGraw-Hill, New York. Kromer, K. 1979. Biological activity of endogenous and influence of exogenous growth regulators on Canna indica regeneration in vitro. Acta Hort. 91:295-300. Kromer, K. D. 1985. Regeneration of some monocotyledonous plants from subterranean organs in vitro. Acta Agrobotanica 38:65-87. Kromer, K. 1989. The effect of light conditions on regeneration and level of endogenous growth regulators in Muscari racemosum (L.) Mill. bulb-scale sections cultured in vitro. Acta Hort. 251:173-181. Kromer, K. D., and K. Kukulczanka. 1985. In vitro cultures of meristem tips of Canna indica (L.). Acta Hort. 167:279-285. Kukulczanka, K., K. Kromer, and B. Czastka. 1989. Propagation of Fritillaria meleagris L. through tissue culture. Acta Hort. 251:147-153. Langeveld, S. A., A. F. L.M. Derks, M.M. Gerrits, P. M. Boonekamp and J. F. Bol. 1995. Transformation of lily by Agrobacterium. IAPTC Congr., Florence, Italy. Langhans, R. W., R. K. Horst, and E. D. Earle. 1977. Disease-free plants via tissue culture propagation. HortScience 12:149-150. Laublin, G., H. S. Saini, and M. Cappadocia. 1991. In vitro plant regeneration via somatic embryogenesis from root culture of some rhizomatous irises. Plant Cell Tissue Organ Culture 27:15-21. Lawson, R. H. 1990. Production and maintenance of virus-free bulbs. Acta Hort. 266:25-34. Leifert, c., and W. M. Waites. 1992. Bacterial growth in plant tissue culture media. J. Appl. Bacteriol. 72:460-466. Leifert, c., W. M. Waites, and J. R. Nicholas. 1989a. Bacterial contaminants of micropropagated plant cultures. J. Appl. Bacteriol. 67:353-361. Leifert, C., W. M. Waites, H. Camotta, and J. R. Nicholas. 1989b. Lactobacillus plantarum; a deleterious contaminant of plant tissue cultures. J. Appl. Bacteriol. 67:363-370. Leifert, c., H. Camotta, and W. M. Waites. 1992a. Effect of combinations of antibiotics on micropropagated Clematis, Delphinium, Hosta, Iris and Photinia. Plant Cell Tissue Organ Culture 29:153-160. Leifert, C., S. Pryce, P. J. Lumsden, and W. M. Waites. 1992b. Effect of medium acidity on growth and rooting of different plant species growing in vitro. Plant Cell Tissue Organ Culture 30:171-179. Le Nard, M. 1989. In vitro adventitious bud formation on floral stem explants of active growing tulips (Tulipa gesneriana L.). Acad. Sci. Paris. 308:389-394. Le Nard, M., and F. Chanteloube. 1992. In vitro culture of explants excised from growing stems of tulip (Tulipa gesneriana L.): problems related to bud and bulblet formation. Acta Hort. 325:435-440. Le Nard, M., and A. A. De Hertogh. 1993a. Tulipa. p. 617-682. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. Le Nard, M., and A. A. De Hertogh. 1993b. Research perspectives for flower bulbs. p. 777-782. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

159

Le Nard, M., C. Ducommun, G. Weber, N. Dorion, and C. Bigot. 1987. In vitro multiplication of tulip (Tulipa gesneriana L.) from flower stem explants taken from stored bulbs. Agronomie 7:321-329. Leshem, B., H. Lilien-Kipnis, and B. Steinitz. 1982. The effect of light and of explant orientation on the regeneration and subsequent growth of bulblets on £ilium longiflorum Thunb. bulb-scale sections cultured in vitro. Sci. Hart. 17:129-136. Levine, H. G., and A. D. Krikorian. 1992. Shoot growth in aseptically cultivated daylily and haplopappus plantlets after a 5-day spaceflight. Physiol. Plant. 86:349359. Li, T. H., and Y. Niimi. 1995. A comparison of seed sets in se1£-, intraspecific and interspecific pollination of Lilium species by stigmatic and cut-style pollination methods. J. Jpn. Soc. Hort. Sci. 64:143-148. Lilien-Kipnis, H., and M. Kochba. 1987. Mass propagation of new Gladiolus hybrids. Acta Hort. 212:631-638. Lilien-Kipnis, H., M. Ziv., and S. Kahany. 1990. Proliferation and plantlet regeneration from inflorescence derived Nerine explants cultured in vitro. p. 28-33. In: Proc. Eucarpia, Symp. Sect. Ornamentals. Lilien-Kipnis, H., M. Ziv., and S. Kahany. 1992. Proliferation and regeneration of Nerine in liquid culture. Acta Hort. 325:467-473. Lilien-Kipnis, H., N. Azizbekova, and M. Ziv. 1994. Scaled-up proliferation and regeneration of Nerine in liquid cultures, II: ontogeny of somatic embryos and bulblet regeneration. Plant Cell Tissue Organ Culture. 39:117-123. Lin, W. c., and P. L. Monette. 1987. In vitro propagation of Alstroemeria 'Alsaan'. Plant Cell, Tissue Organ Culture 9:29-35. Liu, L., and D. W. Burger. 1986. In vitro propagation of easter lily from pedicels. HortScience 21:1437-1438. Loewenberg, J. R. 1969. Cyclamen callus culture. Can. J. Bot. 47:2065-2067. Logan, A. E., and F. W. Zettler. 1985. Rapid in vitro propagation of virus-indexed gladioli. Acta Hort. 164:169-180. Long, R. D., and A. C. Cassells. 1986. Elimination of viruses from tissue cultures in the presence of antiviral chemicals. p. 239-248. In: L. A. Withers and P. G. Alderson (eds.), Plant tissue culture and its agricultural applications. Butterworths, London. Maeda, M., M. Yoshioka, and M. Ito. 1979. Studies on the behavior of meiotic protoplasts. IV. Protoplasts isolated from microsporocytes of liliaceous plants. Bot. Mag. Tokyo 92:111-121. Maesato, K., K. S. Sarma, H. Fukui, and T. Hara. 1991. In vitro bulblet induction from shoot apices of £ilium japonicum. HortScience 26:211. Maesato, K., K. Sharada, H. Fukui, T. Hara, and K. S. Sarma. 1994. In vitro bulblet regeneration from bulbscale explants of £ilium japonicum Thunb. Effect of plant growth regulators and culture environment. J. Hort. Sci. 69:289-297. McComb, J. A. and S. Newton. 1981. Propagation of kangaroo paws using tissue culture. HortScience 56:181-183. McIntyre, D. K., and G. J. Whitehone. 1974. Tissue culture in the propagation of Australian plants. Comb. Proc. Int. Plant Prop. Soc. 24:262-265. Mensuali-Sodi, A., M. Panizza, G. Serra, and F. Tognoni. 1993. Involvement of activated charcoal in the modulation of abiotic and biotic ethylene levels in tissue cultures. Sci. Hort. 54:49-57. Meyer, H. J., and J. van Staden. 1988. In vitro multiplication of Ixia flexuosa. HortScience 23:1070-1071.

160

K. W. KIM AND A. A. DE HERTOGH

Meyer, M. M., Jr., 1976. Propagation of daylilies by tissue culture. HortScience 11:485-487.

Meyer, M. M., Jr., 1. H. Fuchigami, and A. N. Roberts. 1975. Propagation of tall bearded irises by tissue culture. HortScience 10:479-480. Meynet, J. 1993. Ranunculus. p. 603-610. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. Mielke, K. A., and W. C. Anderson. 1989. In vitro bulblet formation in Dutch iris. HortScience 24:1028-1031. 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. Mii, M., Y. Yuzawa, H. Suetomi, T. Motegi, and T. Godo. 1994. Fertile plant regeneration from protoplasts of a seed-propagated cultivar of Ulium x formolongi by utilizing meristematic nodular cell clumps. Plant Sci. 100:221-226. Moe, R. 1993. Uatris. p. 381-390. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. Montezuma-de-Carvalho, J., and M. L. 1. Guimaraes. 1974. Production of buds and plantlets from the stamen's filament of Ulium regale cultivated in vitro. BioI. Plant. 16:472-473. Moore, W. c., A. A. Brunt, D. Price, A. R. Rees, and J. S. W. Dickens. 1979. Diseases of bulbs. Ministry of Agriculture, Fisheries and Food, Her Majesty's Stationery Office, London. Morel, G., and C. Martin. 1952. Guerison de Dahlias atteints d'une maladie a virus. C. R. Acad. Sci. 235:1324-1325. Mori, K., and D. Hosokawa. 1977. Localization of viruses in apical meristem and production of virus-free plants by means of meristem and tissue culture. Acta Hart. 78:389-396. Mowat, W. P. 1980. The production of virus-free Narcissus stocks in Scotland. Acta Hort. 109:513-521. Mullin, M. 1970. Tissue culture of some monocotyledonous plants. Austral. J. BioI. Sci. 23:473-477. Mullin, R. H., and D. E. Schlegel. 1978. Meristem-tip culture of Dahlia infected with dahlia mosaic virus. Plant Dis. Rep. 62:565-567. Murasaki, K., and H. Tsurushima. 1988. Improvement on clonal propagation of Cyclamen in vitro by the use of etiolated petioles. Acta Hart. 226:721-724. Murashige, T. 1974. Plant propagation through tissue cultures. Ann. Rev. Plant Physiol. 25:135-166. Nel, D. D. 1981. Rapid propagation of Ornithogalum hybrid in vitro. Agroplantae 13:83-84.

Niederwieser, J. G., and J. van Staden. 1990. The relationship between genotype, tissue age and endogenous cytokinin levels on adventitious bud formation on leaves of Lachenalia. Plant Cell Tissue Organ Culture 22:223-228. Niederwieser, J. G., and B. M. Vcelar. 1990. Regeneration of Lachenalia species from leaf explants. HortScience 25:684-687. Niederwieser, J. G., H. A. van de Venter, and P. J. Robbertse. 1990. Embryo rescue in Ornithogalum. HortScience 25:565-566. Niimi, Y. 1978. Influence of low and high temperatures on the initiation and the development of a bulb primordium in isolated tulip embryos. Sci. Hart. 9:61-69. Niimi, Y. 1980. Histological observations on the initiation of the vegetative apex in tulip seeds cultured under low temperatures. Scientia Hart. 13:161-171. Niimi, Y. 1984. Bulblet-productivity of explants from scales, leaves, stems and tepals of Ulium rubellum Baker. Scientia Hart. 22:391-394.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

161

Niimi, Y 1985. Factors affecting the regeneration and growth of bulblets in bulbscale cultures of Ulium rubellum Baker. J. Jpn. Soc. Hart. Sci. 54:82-86. Niimi, Y. 1986. Application of leaf-segment cultures to in vitro bulblet production of six Lilium species. Acta Bot. Neerl. 35:189-194. Niimi, Y., and T. Onozawa. 1979. In vitro bulblet formation from leaf segments of lilies, especially Ulium rubellum Baker. Scientia Hart. 11:379-389. Niimi, Y., and 1. Saito. 1990. Production of bulbs of Lilium rubellum Baker: an attempt to improve in vitro growth of bulblets regenerated from cultured bulb scales. J. Jpn. Soc. Hart. Sci. 59:635-640. Niimi, Y., and H. Watanabe. 1982. In vitro propagation of Lilium rubellum Baker; especially on bulblet formation of stem segments. J. Jpn. Soc. Hart. Sci. 51:344349. Niimi, Y, Y. Endo, and E. Arisaka. 1988. Effects of chilling- and GA 3 -treatments on breaking dormancy in Lilium rubellum Baker bulblets cultured in vitro. J. Jpn. Soc. Hart. Sci. 57:250-257. Nishiuchi, Y. 1979. Studies on vegetative propagation of tulip, II: formation and development of adventitious buds in the excised bulb scale cultured in vitro. J. Jpn. Soc. Hart. Sci. 48:99-105. Nishiuchi, Y. 1986. Multiplication of tulip bulb by tissue culture in vitro. Acta Hart. 177:279-283. Nishiuchi, Y, and H. Myodo. 1976. Studies on vegetative propagation of tulip, I: organ formation from excised bulb scales in vitro. J. Jpn. Soc. Hart. Sci. 45:59-64. North, C. 1975. Embryo culture as an aid to breeding Lilium. Acta Hart. 47:187-192. Novak, F. J., and E. Petru. 1981. Tissue culture propagation of Lilium hybrids. Scientia Hart. 14:191-199. Nowak, J., and R. M. Rudnicki. 1993. Hyacinthus. p. 335-347. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. Nyland, G., and A. C. Goheen. 1969. Heat therapy of virus diseases of perennial plants. Ann. Rev. Phytopathol. 7:331-354. Ochatt, S. J., A. A. Ciai, and O. H. Caso. 1986. Tissue culture techniques applied to native American crops: micropropagation of oca (Oxalis tuberosa Moll.) an Andean tuber- bearing species. Turrialba 36:187-190. Ochatt, S. J., A. S. Escandon, and A. J. Martinez. 1989. Isolation, culture, and plant regeneration of protoplasts of two shrubby Oxalis species from South America. J. Exp. Bot. 40:493-496. Okubo, H. 1993. Hippeastrum (Amaryllis). p. 321-334. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. Okubo, H., C. W. Huang, and S. Uemoto. 1990. Role of outer scale in twin-scale propagation of Hippeastrum hybridum and comparison of bulblet formation from single- and twin-scales. Acta Hart. 266:59-66. Paek, K. Y. 1982. Studies on in vitro culture of Tulipa gesneriana. II: effects of chilling treatment on organogenesis and callus formation from meristems and bulb scale segments. J. Kor. Soc. Hart. Sci. 23:348-356. Paek, K. Y, and C. K. Chun. 1982. In vitro propagation of bulb scale sections of Ulium longiflorum Thunb. J. Kor. Soc. Hart. Sci. 23:230-239. Paek, K. Y., and T. A. Thorpe. 1990. Hyacinth. p. 479-508. In: P. V. Ammirato, D. R. Evans, W. R. Sharp, and Y. P. S. Bajaj (eds.), Handbook of plant cell culture, Vol.5, Ornamental species. McGraw-Hill, New York. Paek, K. Y., C. K. Chun, and J. D. Chung. 1983. Factors influencing regeneration of bulblets and effect of chilling treatment of parent bulblets on scale segments of hyacinth in vitro. J. Kor. Soc. Hart. Sci. 24:68-75.

162

K. W. KIM AND A. A. DE HERTOGH

Paek, K. Y., e. W. Lee, J. K. Choi, and Y. P. Hong. 1987. Effect of explant source, breaking of apical dominance and dormancy on shoot proliferation and bulbing of hyacinth, narcissus and lily in vitro. J. Kor. Soc. Hort. Sci. 28:88-98. Paffen, A. M. G., P. Aguettaz, 1. Delvallee, G. J. de Klerk, and R. J. Bogers. 1990. The development of dormancy in lily bulblets generated in vitro. Acta Hort. 266:5158.

Para, G., and J. Baratti. 1992. Irones and precursors synthesized by Iris siberica tissue culture. Biosci. Biotech. Biochem. 56:1132-1133. Paterson, A. D., and P. C. Harper. 1986. Are we any nearer commercial micropropagation of tulips? Acta Hort. 177:285-289. Peck, D. K, and B. G. Cumming. 1984. In vitro propagation of Begonia x tuberhybrida from leaf sections. HortScience 19:395-397. Peck, D. E., and B. G. Cumming. 1986. Beneficial effects of activated charcoal on bulblet production in tissue cultures of Muscari armeniacum. Plant Cell Tissue Organ Culture 6:9-14. Phillips, S. 1990. The efficacy of four antiviral compounds in the elimination of Narcissus viruses during meristem tip culture. Acta Hort. 266:531-538. Phillips, S., and A. A. Brunt. 1986. Four viruses of Alstroemeria in Britain. Acta Hort. 177:227-233. Pierik, R. 1. M. 1991. Micropropagation of ornamental plants. Acta Hort. 289:45-53. Pierik, R. L. M., and B. J. Ippel. 1977. Plantlet formation from excised bulb scale segments of Nerine. Acta Hort. 78:197-202. Pierik, R. 1. M., and A. J. M. Post. 1975. Rapid vegetative propagation of Hyacinthus orientalis 1. in vitro. Sci. Hort. 3:293-297. Pierik, R. L. M., and M. A. Ruibing. 1973. Regeneration of bulblets on bulb scale segments of hyacinth in vitro. Neth. J. Agr. Sci. 21:129-138. Pierik, R. L. M., and H. H. M. Steegmans. 1975. Freesia plantlets from flower-buds cultivated in vitro. Neth. J. Agr. Sci. 23:334-337. Pierik, R. L. M., and H. H. M. Steegmans. 1976. Vegetative propagation of Freesia through the isolation of shoots in vitro. Neth. J. Agr. Sci. 24:274-277. Pierik, R. 1. M., and H. H. M. Steegmans. 1986. Adventitious plantlet regeneration from floral stem explants of Nerine bowdenii W. Watts. Neth. J. Agr. Sci. 34:217223.

Pierik, R. 1. M., and J. Woets. 1971. Regeneration of isolated bulb scale segments of hyacinth. Acta Hort. 23:423-428. Pierik, R. 1. M., P.A. Sprenkels, and J. van Bragt. 1983. Rapid vegetative propagation of Eucharis grandiflora in vitro. Acta Hort. 147:179-186. Pierik, R. L. M., A. van Voorst, G. Booy, C. A. M. van Acker, C. 1. C. Lelivelt, and J.e. de Wit. 1988. Vegetative propagation of Alstroemeria hybrids in vitro. Acta Hort. 226:81-89.

Plessner, 0., M. Ziv, and M. Negbi. 1990. In vitro corm production in the saffron crocus (Crocus sativus 1.). Plant Cell Tissue Organ Culture 20:89-94. Poli, F., A. Bonora, G. L. Vannini, A. Bruni, and M. P. Fasulo. 1989. Callus formation, cell suspension culture and plant regeneration in Ranunculus serbicus Vis. J. Plant Physiol. 135:637-639. Prakash, J., and K. 1. Giles. 1986. Production of doubled haploids in oriental lilies. p. 335-337. In: W. Horn, C. J. Jensen, W. Odenbach, and O. Schieder (eds.), Genetic manipulation in plant breeding. Walter de Gruyter, Berlin. Pugliesi, C., M. Rabaglio, F. Cecconi, and S. Baroncelli. 1992. Plant regeneration from tissue cultures of Persian buttercup (Ranunculus asiaticus 1.). Plant Cell Tissue Organ Culture 28:125-128.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

163

Qu, Y., M. C. Mok, D. W. S. Mok, and J. R. Stang. 1988. Phenotypic and cytological variation among plants derived from anther cultures of Ulium longiflorum. In Vitro Cell. Dev. BioI. 24:471-476. Radojevic, L., and A. Subotic. 1992. Plant regeneration of Iris setosa Pall. through somatic embryogenesis and organogenesis. J. Plant Physiol. 139:690-696. Radojevic, L. J., O. Sokic, and B. Tucic. 1987. Somatic embryogenesis in tissue culture of iris (Iris pumila L.). Acta Hort. 212:719-723. Rao, T. M., S. S. Negi, and R. D. Swamy. 1991. Micropropagation of Gladiolus. Indian J. Hort. 48:171-176. Raunkiaer, C. 1934. The life forms of plants and statistical plant geography. Clarendon, Oxford, UK. Read, P. E. 1990. Environmental effects in micropropagation. p. 95-125. In: P. V. Ammirato, D. R. Evans, W. R. Sharp, and Y. P. S. Bajaj (eds), Handbook of plant cell culture, Vol.5, Ornamental species. McGraw-Hill, New York. Read, P. E., and C. D. Fellman. 1985. Accelerating acclimation of in vitro propagated woody ornamentals. Acta Hort. 166:15-20. Redenbaugh, K., B. D. Paasch, J. W. Nichol, M. E. Kossler, P. R. Viss, and K. A. Walker. 1986. Somatic seeds: encapsulation of asexual plant embryos. Biotechnology 4:797-801. Rees, A. R. 1969. The initiation and growth of Narcissus bulbs. Ann. Bot. 33:277288. Rees, A. R. 1989. Evolution of the geophytic habit and its physiological advantages. Hertertia 45:104-110. Reuther, G. 1977. Adventitious organ formation and somatic embryogenesis in callus of Asparagus and Iris and its possible application. Acta Hart. 78:217-224. Rice, R. D., P. G. Alderson, and N. A. Wright. 1983. Induction of bulbing of tulip shoots in vitro. Sci. Hort. 20:377-390. Riviere, S., and J. F. Muller. 1979. Etude du bourgeonnement in vitro de l'ecaille du bulbe de Tulipe. Can J. Bot. 57:1986-1993. Robb, S. M. 1957. The culture of excised tissue from bulb scales of Lilium speciosum Thun. J. Exp. Bot. 8:348-352. Rudnicki, R. M., and J. Nowak. 1993. Muscari. p. 455-462. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. Sachar, R. C., and M. Kapoor. 1959. In vitro culture of ovules of Zephyranthes. Phytomorphology 9:147-156. Sagawa, Y., and J. T. Kunisaki. 1990. Micropropagation of floriculture crops. p. 2556. In: P. V. Ammirato, D. R. Evans, W. R. Sharp, and Y. P. S. Bajaj (eds.), Handbook of plant cell culture, Vol. 5, Ornamental species. McGraw-Hill, New York. Samyn, G. L., P. C. Debergh, and D. Vermaerke. 1984. Field performance and phenotypic stability of virus-free tissue-cultured Begonia x tuberhybrida 'Multiflora'. Sci. Hort. 24:185-191. Saniewski, M. 1975. Root and bulblet regeneration by flower buds of Hyacinthus orientalis L. in tissue culture. Bull. Acad. Pol. Sci. BioI. 23:121-123. Saniewski, M., J. Nowak, and R. Rudnicki. 1974. Studies on the physiology of hyacinth bulbs (Hyacinthus orientalis L.), IV: hormonal regulation of induction of roots and bulblets in Hyacinthus orientalis L. grown in culture. Plant Sci. Lett. 2:373-376. Sano, K., and H. Himeno. 1987. In vitro proliferation of saffron (Crocus sativus L.) stigma. Plant Cell Tissue Organ Culture 11:159-166. Sarma, K. S., K. Maesato, T. Hara, and Y. Sonoda. 1990. In vitro production of stigmalike structures from stigma explants of Crocus sativus L. J. Exp. Bot. 41:745-748.

164

K. W. KIM AND A. A. DE HERTOGH

Sarma, K. S., K. Sharada, K. Maesato, T. Hara, and Y. Sonoda. 1991. Chemical and sensory analysis of saffron produced through tissue cultures of Crocus sativus. Plant Cell Tissue Organ Culture 26:11-16. Schwenkel, H. G., and J. Grunewaldt. 1988. In vitro propagation of Cyclamen persicum Mill. Acta Hort. 226:659-662. Seabrook, J. E. A. 1990. Narcissus (Daffodil). p. 577-597. In: P. V. Ammirato, D. R. Evans, W. R. Sharp, and Y. P. S. Bajaj (eds.), Handbook of plant cell culture, Vol. 5, Ornamental species. McGraw-Hill, New York. Seabrook, J. E. A., and B. G. Cumming. 1977. The in vitro propagation of amaryllis (Hippeastrum spp. hybrids). In Vitro 13:831-836. Seabrook, J. E. A., and B. G. Cumming. 1982. In vitro morphogenesis and growth of Narcissus in response to temperature. Sci. Hort. 16:185-190. 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. Sharp, W. R., R. S. Raskin and H. E. Sommer. 1971. Haploidy in Lilium. Phytomorphology 21:334-337. Sheridan, W. F. 1968. Tissue culture of the monocot Ulium. Planta 82:189-192. Simmonds, J. A., and B. G. Cumming. 1976. Propagation of Ulium hybrids, II: production of plantlets from bulb-scale callus cultures for increased propagation rates. Sci. Hort. 5:161-170. Simmonds, J. A., D. H. Simmonds, and B. G. Cumming. 1979. Isolation and cultivation of protoplasts from morphogenic callus cultures ofUlium. Can. J. Bot. 57:512516. Simonsen, J., and A. C. Hildebrandt. 1971. In vitro growth and differentiation of Gladiolus plants from callus cultures. Can. J. Bot. 49:1817-1819. Smith, D. L., and A. D. Krikorian. 1991. Growth and maintenance of an embryogenic cell culture of daylily (Hemerocallis) on hormone-free medium. Ann. Bot. 67:443-449. Smith, D. L., K. Kelly, and A. D. Krikorian. 1989. Ethylene-associated phase change from juvenile to mature phenotype of daylily (Hemerocallis) in vitro. Physiol. Plant. 76:466-473. Squires, W. M., and F. A. Langton. 1990. Potential and limitations of Narcissus micropropagation: an experimental evaluation. Acta Hort. 266:67-76. Squires, W. M., F. A. Langton, and J. S. Fenlon. 1991. Factors influencing the transplantation success of micropropagated narcissus bulbils. J. Hort. Sci. 66:661671. Srivastava, P. S., and B. M. Johri. 1992. Endosperm culture. p. 1-21. In: K. Lindsey (ed.), Plant tissue culture manual, Supplement 2. Kluwer Academic, Dordrecht, The Netherlands. Steinitz, B., and H. Lilien-Kipnis. 1989. Control of precocious gladiolus corm and cormel formation in tissue~ulture. J. Plant Physiol. 135:495-500. Steinitz, B. and H. Yahel. 1982. In vitro propagation of Narcissus tazetta. HortScience 17:333-334. Steinitz, B., A. Cohen, Z. Goldberg, and M. Kochba. 1991. Precocious gladiolus corm formation in liquid shake cultures. Plant Cell Tissue Organ Culture 26:63-70. Stimart, D. P., and P. D. Ascher. 1978. Tissue culture of bulb scale sections for asexual propagation of Ulium longiflorum Thunb. J. Am. Soc. Hort. Sci. 103:182-184. Stimart, D. P., and P. D. Ascher. 1981a. Foliar emergence from bulblets of Ulium longiflorum Thunb. as related to in vitro generation temperatures. J. Am. Soc. Hort. Sci. 106:446-450.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

165

Stimart, D. P. and P. D. Ascher. 1981b. Developmental responses of Lilium longiflorum bulblets to constant or alternating temperatures in vitro. J. Am. Soc. Hort. Sci. 106:450-454. Stimart, D. P. and P. D. Ascher. 1982. Plantlet regeneration and stability from callus cultures of Freesia x hybrida Bailey cultivar 'Royal'. Sci. Hort. 17:153-157. Stimart, D. P., and J. F. Harbage. 1989. Shoot proliferation and rooting in vitro of Uatris spicata. HortScience 24:835-836. Stimart, D. P., P. D. Ascher, and J. S. Zagorski. 1980. Plants from callus of the interspecific hybrid Ulium 'Black Beauty'. HortScience 15:313-315. Stimart, D. P., P. D. Ascher, and H. F. Wilkins. 1982. Overcoming dormancy in Ulium longiflorum bulblets produced in tissue culture. J. Am. Soc. Hort. Sci. 107:10041007. Stimart, D. P., P. D. Ascher, and H. F. Wilkins. 1983. Axis elongation from tissueculture-generated bulblets of Lilium longiflorum Thunb. J. Am. Soc. Hort. Sci. 108:99-101. Stone, O. M. 1973. The elimination of viruses from Narcissus tazetta cv. Grand Soleil d'Or, and rapid multiplication of virus-free clones. Ann. AppI. BioI. 73:4552. Stone, O. M., A. A. Brunt, and M. Hollings. 1975. The production, propagation and distribution of virus-free clones of Narcissus tazetta cv. 'Grand Soleil d'Or'. Acta Hort. 47:77-81. Sutter, E. G. 1986. Micropropagation of Ixia viridifolia and a Gladiolus x homoglossum hybrid. Sci. Hort. 29:181-189. Taeb, A. G., and P. G. Alderson. 1987. Micropropagation of tulip: optimizing shoot production from floral stem explants. Acta Hort. 212:677-681. Taeb, A. G., and P. G. Alderson. 1990a. Effect of photoperiod and quality of light on bulbing of tulip shoots regenerated in vitro. J. Hort. Sci. 65:71-74. Taeb, A. G., and P. G. Alderson. 1990b. Shoot production and bulbing of tulip in vitro related to ethylene. J. Hort. Sci. 65:199-204. Takahasi, S., K. Matsubara, H. Yamagata, and T. Morimoto. 1992a. Micropropagation of virus free bulblets of Ulium longiflorum by tank culture, 1: development of liquid culture method and large scale propagation. Acta Hort. 319:83-88. Takahasi, S., A. Katoh, and T. Morimoto. 1992b. Micropropagation of virus free bulblets of Ulium longiflorum by tank culture, 2: cultivation characteristics of propagated bulblet. Acta Hort. 319:89-94. Takayama, S. 1990. Begonia. p. 253-283. In: P. V. Ammirato, D. R. Evans, W. R. Sharp, and Y. P. S. Bajaj (eds.), Handbook of plant cell culture, Vo1.5, Ornamental species. McGraw-Hill, New York. Takayama, S., and M. Misawa. 1979. Differentiation in Ulium bulbscales grown in vitro: effect of various cultural conditions. Physiol. Plant. 46:184-190. Takayama, S., and M. Misawa. 1980. Differentiation in Ulium bulbscales grown in vitro: effects of activated charcoal, physiological age of bulbs and sucrose concentration on differentiation and scale leaf formation in vitro. Physiol. Plant. 48:121-125. Takayama, S., and M. Misawa. 1982. Regulation of organ formation by cytokinin and auxin in Ulium bulbs cales grown in vitro. Plant Cell Physiol. 23:67-74. Takayama, S., and M. Misawa. 1983a. A scheme for mass propagation of Ulium in vitro. Sci. Hort. 18:353-362. Takayama, S., and M. Misawa. 1983b. The mass propagation of Ulium in vitro by stimulation of multiple adventitious bulb-scale formation and by shake culture. Can. J. Bot. 61:224-228.

166

K. W. KIM AND A. A. DE HERTOGH

Takayama, S., M. Misawa, Y. Takashige, H. Tsumori, and K. Ohkawa. 1982. Cultivation of in vitro-propagated Lilium bulbs in soil. J. Am. Soc. Hort. Sci. 107:S30834. Takayama, S., B. Swedlund, and Y. Miwa. 1991. Automated propagation of microbulbs of lilies. p. 111-131. In: Vasil, 1. K. (ed.), Cell culture and somatic cell genetics of plants, Vol.S, Scale-up and automation in plant propagation. Academic Press, New York. Tamura, S. 1975. Adventitious bud formation from excised hyacinth bulb scale in vitro. J. Jpn. Soc. Hort. Sci. 46:501-50S. Tanaka, 1. 1985. Isolation of generative cells and their protoplasts from pollen of Lilium longiflorum. Protoplasma 142:6S-73. Tanaka, 1., C. Kitazume, and M. Ito. 19S7. The isolation and culture of lily pollen protoplasts. Plant Sci. 50:205-211. Tisserat, B., E. B. Esan, and T. Murashige. 1979. Somatic embryogenesis in angiosperms. Hort. Rev. 1:1-7S. Tomita, M., H. Chono, and K. Tsutsui. 19S5. Interspecific differences in the effects of medium conditions on organogenesis in the micropropagation of some lilies. Memo. Fac. Agr., Hokkaido Univ. (Sapporo, Japan). 16:1-10. Turakhia, D. V., and A. R. Kulkarni. 19S5. In vitro regeneration from leaf explants of Ladebouria hyacinthiana, Roth. (Scilla indica, Bak.). Cur. Sci. 57:214-216. Ueda, K., Y. Miyamoto, and 1. Tanaka. 1990. Fusion studies of pollen protoplasts and generative cell protoplasts in Lilium longiflorum. Plant Sci. 72:259-266. Van Aartrijk, J., and G. J. Blom-Barnhoorn. 19S0. Effects of sucrose, mineral salts, and some organic substances on the adventitious regeneration in vitro of plantlets from bulb-scale tissue of Lilium speciosum 'Rubrum'. Acta Hort. 109:297302. Van Aartrijk, J., and G. J. Blom-Barnhoorn. 19S1. Growth regulator requirements for adventitious regeneration from Lilium bulb-scale tissue in vitro, in relation to duration of bulb storage and cultivar. Sci. Hort. 14:261-26S. Van Aartrijk, J., and G. J. Blom-Barnhoorn. 19S2. Effects of virazole on the regeneration of virus-free plants from bulb-scale explants of Lilium longiflorum "Arai" infected with lily symptomless virus. Acta Bot. Neerl. 31:245-246. Van Aartrijk, J., and G. J. Blom-Barnhoorn. 19S3. Adventitious bud formation from bulb-scale explants of Lilium speciosum Thunb. in vitro: effects of wounding, TIBA, and temperature. Z. Pflanzenphysiol. 110:355-363. Van Aartrijk, J., and G. J. Blom-Barnhoorn. 19S4. Adventitious bud formation from bulb-scale explants of Lilium speciosum Thunb. in vitro: interacting effects of NAA, TIBA, wounding, and temperature. J. Plant. Physiol. 116:409-416. Van Aartrijk, J., and P. C. G. van der Linde. 19S6. In vitro propagation of flower-bulb crops. p. 317-331. In: R. H. Zimmerman, R. J. Griesbach, F. A. Hammerschlag, and R. H. Lawson (eds.), Tissue culture as a plant production system for horticultural crops. Martinus Nijhoff, Dordrecht, The Netherlands. Van Aartrijk, J., G. J. Blom-Barnhoorn, and J. Bruinsma. 19S5a. Adventitious bud formation from bulb-scale explants of Lilium speciosum Thunb. in vitro: effects of aminoethoxyvinyl-glycine, 1-aminocyclopropane-1-carboxylic acid, and ethylene. J. Plant Physiol. 117:401-410. Van Aartrijk, J., G. J. Blom-Barnhoorn, and J. Bruinsma. 19S5b. Adventitious bud formation from bulb-scale explants of Lilium speciosum Thunb. in vitro: production of ethane and ethylene. J. Plant Physiol. 117:411-422.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

167

Van Aartrijk, J., G. J. Blom-Barnhoorn, and P. C. G. van der Linde. 1990. Lilies. p. 535-576. In: Ammirato, P. V., D. R. Evans, W. R. Sharp, and Y. P. S. Bajaj. (eds.), Handbook of plant cell culture, VoL 5, Ornamental species. McGraw-Hill, New York. Van Brenk, G., and M. Benschop. 1993. Nerine. p. 559-588. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flower bulbs. Elsevier Science, Amsterdam. Van den Bulk, R. W., H. P. J. de Vries-van Hulten, J. B. M. Custers, and J. J. M. Dons. 1994. Induction of embryogenesis in isolated microspores of tulip. Plant Sci. 104:101-111. Van der Leede-Plegt, L. M., B. C. E. van de Ven, R. J. Bino, T. P. M. van der Salm, and A. J. van Tunen. 1992. Introduction and differential use of various promoters in pollen grains of Nicotiana glutinosa and Ulium longiflorum. Plant Cell Rep. 11:20-24. Van der Linde, P. C. G. 1992. Tissue culture of flower bulbs: theory and practice. Acta Hort. 352:419-427. Van der Linde, P. C. G., G. M. G. M. HoI, G. J. Blom-Barnhoorn, J. van Aartrijk, and G. J. de Klerk. 1988. In vitro propagation of Iris hollandica Tub. cv. Prof. Blaauw, I: regeneration on bulb-scale explants. Acta Hort. 226:121-128. Van Tuyl, J. M., J. Franken, R. C. Jongerius, c. A. M. Lock, and T. A. M. Kwakkenbos. 1986. Interspecific hybridization in Ulium. Acta Hart. 177:591-595. Van Tuyl, J. M., K. van de Sande, R. van Dien, D. Straathof and H. M. C. van Holsteijn. 1990a. Overcoming interspecific crossing barriers in Ulium by ovary and embryo culture. Acta Hart. 266:317-322. Van Tuyl, J. M., H. M. C. v~. n Holsteijn, and A. A. M. Kwakkenbos. 1990b. Research on polyploidy in inters.. ecific hybridization of lily. Acta Hart. 266:323-329. Van Tuyl, J. M., M. P. van ien, M. G. M. van Creij, T.C.M. van Kleinwee, J. Franken, and R. J. Bino. 1991. Application of in vitro pollination, ovary culture, ovule culture and embryo rescue for overcoming incongruity barriers in interspecific Ulium crosses. Plant Sci. 74:115-126. Van Zaayen, A., C. van Eijk, and J. M. A. Versluijs. 1992. Production of high quality, healthy ornamental crops through meristem culture. Acta Bot. Need. 41:425433. Vcelar, B. M., D. 1. Ferreira, and J. G. Niederwieser. 1992. Elimination of ornithogalum mosaic virus in the Ornithogalum cv. Rogel through meristem tip culture and chemotherapy. Plant Cell Tissue Organ Culture 29:51-55. Verron, P., M. Le Nard, and J. Cohat. 1995. In vitro organogenic competence of different organs and tissues of lily of the valley 'Grandiflora of Nantes'. Plant Cell Tissue Organ Culture 40:237-242. Viseur, J., and C. Lievens. 1987. In vitro propagation and regeneration of plants from calluses of Begonia x tuberhybrida. Acta Hart. 212:705-709. Wainwright, H., and A. C. Harwood. 1985. In vitro organogenesis and plant regeneration of Cyclamen persicum MilL using seedling tissue. J. Hart. Sci. 60:397403. Wang, L., B. Huang, M. He, and S. Hao. 1990. Somatic embryogenesis and its hormonal regulation in tissue cultures of Freesia refracta. Ann. Bot. 65:271-276. Wang, P. J., and C. Y. Hu. 1980. Regeneration of virus-free plants through in vitro culture. Adv. Biochem. Engr. 18:61-99.

168

K. W. KIM AND A. A. DE HERTOGH

Watad, A. A., M. Kochba, A. Nissim, and V. Gaba. 1995. Improvement of Aconitum mapellus micropropagation by liquid culture on floating membrane rafts. Plant Cell Rep. 14:345-348. Welvaert, W., G. Samyn, and E. Van Wymersch. 1980. On the production of virusfree Begonia tuberhybrida Voss cv. multiflora varieties. Acta Hort. 110:253-257. Wicart, G., A. Mouras, and A. Lutz. 1984. Histological study of organogenesis and embryogenesis in Cyclamen persicum Mill. tissue culture: evidence for a single organogenetic pattern. Protoplasma 119:159-167. Wickremesinhe, E. R. M., E. J. Holcomb, and R. N. Arteca. 1994. A practical method for the production of flowering Easter lilies from callus cultures. Sci. Hort. 60:143152. Wilmink, A., B. C. E. van de Ven, and J. J. M. Dons. 1992. Expression of the GUSgene in the monocot tulip after introduction by particle bombardment and Agrobacterium. Plant Cell Rep. 11:76-80. Withers, L. A. 1988. The current status of in vitro culture for the international movement of plant germplasm. p. 47-60. In: IBPGR advisory committee on in vitro storage: conservation and movement of vegetatively propagated germplasm: in vitro culture and disease aspects. IntI. Board Plant Genet. Res., Rome. Wozniewski, T., W. Blaschek, and G. Franz. 1991. In vitro propagation of £ilium testaceum and structural investigation of the storage beta-1 ,4-glucomannan. Plant Cell Rep. 10:457-460. Wright, N. A., and P. G. Alderson. 1980. The growth of tulip tissues in vitro. Acta Hort. 109:263-270. Yabuya, T. 1985. Amphidiploids between Iris laevigata Fisch. and 1. ensata Thunb. induced through in vitro culture of embryos treated with colchicine. Jpn. J. Breed. 35:136-144. Yabuya, T., Y. Ikeda, and T. Adachi. 1991. In vitro propagation of Japanese garden iris, Iris ensata Thunb. Euphytica 57:77-81. Yanagawa, T., and I. Ito. 1988. Differences in the capacity for bulblet regeneration between bulb-scale explants excised from different parts of Hymenocallis and Ornithogalum bulbs. J. Jpn. Soc. Hort. Sci. 57:454-461. Yanagawa, T., and Y. Sakanishi. 1977. Regeneration of bulblets on Hippeastrum bulb segments excised from various parts of a parent bulb. J. Jpn. Soc. Hort. Sci. 46:250-260. Yanagawa, T., and Y. Sakanishi. 1980a. Studies on the regeneration of excised bulb tissues of various tunicated-bulbous ornamentals, I: regenerative capacity of the segments from different parts of bulb scales. J. Jpn. Soc. Hort. Sci. 48:495-502. Yanagawa, T., and Y. Sakanishi. 1980b. Regenerative studies on the excised bulb tissue of various tunicated-bulbous ornamentals, II: morphological observations on bulblet formation from bulb-scale segments. J. Jpn. Soc. Hort. Sci. 49:119-126. Yoon, E. S. 1991a. Ovary slice culture as a technique of hybridization between incompatibility species of £ilium, I: survival rate and germination rate of hybrid embryos. Korean J. Plant Tissue Culture 18:185-193. Yoon, E. S. 1991b. Ovary slice culture as a technique of hybridization between incompatibility species of £ilium, II: anatomical observation of embryonic development and germination. Korean J. Plant Tissue Culture 18:303-311. Zhen, Z. W., H. J. Xiang, G. Y. Lan, H. Z. Qi, X. Z. Xing, X. S. Hwa, C. W. Qi, Z. S. Zhen, and M. K Jiang. 1984. Regeneration of virus-free plants from inflorescences of virus- infected gladiolus. p. 373-374. In: Genetic manipulation in crops. IRRI and Academia Sinica, Beijing, China.

2.

TISSUE CULTURE OF ORNAMENTAL FLOWERING BULBS

169

Zhou, C. 1989a. A study on isolation and culture of pollen protoplasts. Plant Sci. 59:101-108. Zhou, C. 1989b. Cell divisions in pollen protoplast culture of Hemerocallis fulva L. Plant Sci. 62:229-235. Zhu, Y., S. Yazawa, and T. Asahira. 1993a. Varietal differences in leaf color variation of plants regenerated from in vitro culture of leaf blade in Caladium cultures. J. Jpn. Soc. Hort. Sci. 62:431-435. Zhu, Y., T. Takemoto, and S. Yazawa. 1993b. Leaf color of plants regenerated through in vitro culture from variegated leaf segments of Caladium. J. Jpn. Soc. Hort. Sci. 62:619-624. Ziv, M. 1979. Transplanting Gladiolus plants propagated in vitro. Sci. Hort. 11:257260. Ziv, M. 1989. Enhanced shoot and cormlet proliferation in liquid cultured gladiolus buds by growth retardants. Plant Cell Tissue Organ Culture 17:101-110. Ziv, M. 1990a. Morphogenesis of gladiolus buds in bioreactors: implication for scaleup propagation of geophytes. p. 119-124. In: H. J. J. Nijkamp, L. H. W. van der Plas, and J. van Aartrijk (eds.), Current plant science and biotechnology in agriculture: progress in plant cellular and molecular biology. Kluwer Academic, Dordrecht, The Netherlands. Ziv, M. 1990b. The effect of growth retardants on shoot proliferation and morphogenesis in liquid cultured gladiolus plants. Acta Hort. 280:207-214. Ziv, M., and H. Lilien-Kipnis. 1990. Gladiolus. p. 461-478. In: P. V. Ammirato, D. R. Evans, W. R. Sharp, and Y. P. S. Bajaj (eds.), Handbook of plant cell culture, Vol. 5, Ornamental species. McGraw-Hill, New York. Ziv, M., A. H. Halevy, and R. Shilo. 1970. Organs and plantlets regeneration of Gladiolus through tissue culture. Ann. Bot. 34:671-676. Ziv, M., R. Kanterovitz, and A. H. Halevy. 1973. Vegetative propagation of Alstroemeria in vitro. Sci. Hort. 1: 271-277. Ziv, M., S. Kahany, and H. Lilien-Kipnis. 1994. Scaled-up proliferation and regeneration of Nerine in liquid cultures, I: the induction and maintenance of proliferating meristematic clusters by paclobutrazol in bioreactors. Plant Cell Tissue Organ Culture 39:109-115. Ziv, M., N. Hertz, and Y. Biran. 1983. Vegetative reproduction of Allium ampeloprasum L. in vivo and in vitro. Israel J. Bot. 32:1-9.

3

Desiccation-Tolerance of Plant Tissues: A Mechanistic Overview* Melvin J. Oliver Plant Stress Unit, Cropping Systems Research Laboratory United States Department of Agriculture Agricultural Research Service Box 215 Route 3 Lubbock, Texas 79401, USA

J. Derek Bewley Department of Botany, University of Guelph Guelph, Ontario N1G 2W1, Canada

1. II.

Introduction Vegetative Tissues A. The Drying Phase 1. Desiccation-Tolerant Plants 2. Modified Tolerance in Vegetative Plants and Tissues 3. Prokaryotes B. Desiccation-Induced Cellular Damage C. The Dried State D. Rehydration of Dried Vegetative Tissues 1. Tolerant Tissues

* The authors gratefully acknowledge Brent Mishler at the University and Jepson Herbaria at the University of California, Berkeley, for his analysis of the phylogenetic aspects of desiccation tolerance that is included in Fig. 3.1. We thank Andrew Wood, Harlan Scott II, and Cynthia Galloway for their comments on the manuscript, and Carl Leopold and our anonymous reviewers for their stimulating criticisms.

Horticultural Reviews, Volume 18, Edited by Jules Janick ISBN 0-471-57334-5 © 1997 John Wiley & Sons, Inc. 171

172 III. IV.

V.

M.

J. OLIVER AND J. D. BEWLEY

2. Modified-Tolerant Tissues Pollen Seeds A. The Drying Phase B. Metabolic Changes Associated With Seed Drying 1. Proteins 2. Carbohydrates C. The Dried State D. The Consequences of Drying on Seed Metabolism Upon Rehydration Closing Remarks Literature Cited

I. INTRODUCTION

The loss of water from plant cells is an important environmental stress that has a major impact on the area of land available for cultivation. Over 35% of the world's land surface is considered to be arid or semiarid, experiencing precipitation that is inadequate for most horticultural uses. Ramanathan (1988) has argued, based on predictions of global environmental changes, that developing crops that are more tolerant to water deficits while maintaining productivity will become a critical requirement in the near future. Understanding how plant cells tolerate water loss is a vital prerequisite for developing strategies that can impact horticultural and agricultural crop productivity and survival under conditions of decreasing water availability. Much work has been accomplished in this area with the main emphasis on those genes that are expressed during the response of plants to water stress. From this work, our knowledge of stress tolerance has improved immensely but with little success in augmenting the development of breeding programs for increasing drought tolerance of horticultural crops. The approach is restricted in that most crops have a limited capacity for drought tolerance and thus the genetic information necessary for expanding their drought-tolerance may not be present or exploitable. In addition, the response to drought may not be directed at tolerance of the stress directly. In contrast, more may be gained by understanding how stress-tolerant plants or plant structures accomplish tolerance and which genes from these sources contribute directly to this phenotype. In keeping with this philosophy, understanding how plants cells tolerate the severest of water deficits, viz. desiccation, will offer novel perspectives and new insights into water stress-tolerance mechanisms. Native plants that can tolerate desiccation of their vegetative tissues are a potential

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

173

source of genes that can impact water stress or drought tolerance, and the underlying mechanisms (and the genes involved therein) of desiccation tolerance in seeds can be exploited not only to improve vegetative tolerance but also to increase longevity and germinability of future seed stocks. It is with this perspective in mind that this review of desiccation tolerance is written. Earlier comprehensive reviews of desiccation tolerance in plants (Bewley 1979; Bewley and Krochko 1982) established a broad data base for the investigation of all aspects of desiccation tolerance, including the nature of the physiological responses to desiccation and rehydration. Bewley and Oliver (1992) first introduced a review of desiccation tolerance that compared vegetative desiccation-tolerance mechanisms with that of seeds in an attempt to establish an experimental framework for future research. This review extends their approach and develops the idea that desiccation tolerance is a balance between two fundamental processes: cellular protection from desiccation- and rehydration-induced damage and the repair of the damage that does occur. Desiccation tolerance is the ability of cells to revive from the airdried state, a phenomenon that occurs in organisms ranging from the most simple levels of cellular organization, such as bacteria, to those of a much more complex and multicellular nature, such as insects and arthropods. In plants, desiccation tolerance occurs in species that represent most major classes (Fig. 3.1a); the majority of plants exhibiting tolerant vegetative tissues, however, are found in the "lower" taxons that constitute the algae, bryophytes, and lichens. Of the more complex groups of plants, 60-70 species of ferns and fern allies exhibit desiccation tolerance, as do at least 60 species of angiosperms (Bewley and Krochko 1982). The only major class of vascular plants that does not have a representative desiccation-tolerant species is the gymnosperms (a taxonomic group consisting of the phyogenetically distinct cycads, conifers, and gnetophytes), a fact that Bewley and Krochko (1982) postulate may signify a minimum size limitation for desiccation tolerance, which members of this group exceed. Many angiosperms and gymnosperms do produce desiccation-tolerant propagules, of course, in the form of seeds and also pollen. The desiccation-tolerant plants that represent the less complex orders are truly capable of being dried, that is, their internal water content rapidly equilibrates to the water potential of the environment. These plants have been termed poikilohydric (from the greek poikilo, meaning varied), suggesting that they can withstand vary-

174

M.

J. OLIVER AND J. D. BEWLEY gnetophytes

Angiosperm;

Seed Plants

Tracheophytes

(a)

Fig. 3.1. (a) An outline phylogeny of the land plants, based on a consensus of several recent synthetic studies (Crane 1990; Donoghue 1994; Mishler et al. 1994). Names in bold and with an asterisk indicate clades with some known desiccation-tolerant members (data taken from Bewley and Krochko 1982). Parsimony would suggest at least one independent evolution (or re-evolution) of desiccation tolerance in Selaginella, the ferns, and the angiosperms (see Fig. 3.2 for a hypothesis of phylogenetic relationships within the Angiosperms). (b) An outline phylogeny of the angiosperms, based on cladistic analysis of the rbe L gene by Chase et al. (1993). A few selected taxa are shown for orientation. Branches without names represent (in some cases large) clades with no known desiccation-tolerant members. Names in bold and with an asterisk indicate all clades with some known desiccation-tolerant members (data taken from Bewley and Krochko [1982]). Parsimony would suggest at least one independent evolution (or re-evolution) of desiccation tolerance in each clade (Le., at least eight times in the angiosperms).

ing extents of (de)hydration. Since there are no plants that can maintain a constant water content (homeohydric), although many can limit or forestall water loss, the term poikilohydric would appear to be inappropriate (C.A. Leopold, personal communication). Desiccation-tolerant plants experience differing rates of desiccation, depending on the water status of the environment, after free surface water has evaporated. In the case of lichens and algae, desic-

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

175

Cyperaceae*

(b)

cation can occur in minutes, for example, in desert regions following rainfall or in littoral zones as the tide recedes. Bryophytes can also be subjected to rapid desiccation in nature, but many mosses, by growing in complex clump structures, can maintain a localized humidity that can retard water loss. These mosses can still survive rapid drying, but by reducing the drying rate they may increase their recovery rate on rewetting (see below). Even so, drying rates are relatively rapid in these plants when compared to more advanced species and can occur within a few hours. Desiccation-tolerant vegetative tissues of pteridophytes and angiosperms exhibit a modified tolerance, since all employ mechanisms to retain water and thus control their rate of water loss. In most cases, this results in drying in 12 to 48 h or more, depending on the species of plant. More rapid water loss results in the death of such desiccation-tolerant plants. In summarizing earlier studies on desiccation-tolerant plants, Bewley (1979) concluded that a plant or plant structure must meet three criteria to survive severe loss of protoplastic water. It must (1)

176

M.

J.

OLIVER AND

J.

D. BEWLEY

limit the damage incurred to a repairable level, (2) maintain its physiological integrity in the dried state (perhaps for extended periods of time), and (3) mobilize repair mechanisms on rehydration that effect restitution of damage suffered during desiccation (and on the inrush of water back into the cells). These criteria can be simplified into the working hypothesis that desiccation tolerance can be achieved by mechanisms that are based on the protection of cellular integrity or are based on the repair of desiccation- (or rehydration-) induced cellular damage, as described by Bewley and Oliver (1992). As will be pointed out, it is unlikely that anyone plant or tissue relies entirely on either of the two alternatives of the hypothesis, but rather exhibits properties of both the protective and repair processes. Indeed, given that desiccation tolerance has evolved independently on a minimum of 12 separate occasions (Fig. 3.1), one would expect that there exist examples of desiccation-tolerant plants that span the spectrum of possible combinations of the two strategies-from plants or structures that rely heavily on cellular protection mechanisms to those that rely more on repair. In the following narrative we will discuss the evidence for this hypothesis in relation to the effects of drying, maintenance of the dried state, and rehydration/recovery of vegetative and reproductive plant tissues. II. VEGETATIVE TISSUES A. The Drying Phase 1. Desiccation-Tolerant Plants. True tolerant plants lose water at varying rates according to the water status of the environment. Such plants have an opportunistic life strategy and growth and reproduction must take place during the periodic wetting episodes. Such plants may have evolved a mechanism for desiccation tolerance that is based on a constitutive level of cellular protection coupled with a major emphasis on a rapid recovery- (rep air-) based metabolic response. This strategy allows for such plants to vary in their response according to the rate at which water is lost from the cells, but in all cases damage to cells as a result of drying cannot be greater than can be restituted by the repair mechanisms. Under conditions where water loss is rapid, and consequently a metabolic response is precluded, the constitutive protection systems must be sufficient to limit damage such that rehydration-activated repair mechanisms can cope. Such an event would allow survival but extend the time necessary for full recovery, and hence delay growth. When water loss is gradual,

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

177

at higher ambient humidities, the plant can respond even during the drying phase to set in place rehydration-activated repair systems; this would result in faster recovery times and a quicker resumption of growth. Evidence for this repair-based response for desiccation tolerance comes primarily from studies on the moss Tortula ruralis (Hedw.) GaerL, Meyer, and Scherb. during desiccation (at various rates) and rehydration (reviewed in Bewley 1979; Bewley and Krochko 1982; Bewley and Oliver 1992). Desiccation of gametophytic tissues of T. ruralis results in a rapid decline in protein synthesis, as in all desiccation-tolerant and intolerant mosses tested so far (Bewley 1972, 1973; Siebert et al. 1976; Henckel et a1. 1977; Oliver 1983; M. J. Oliver, unpublished data for T. caninervis [MitL] Broth. and T. norvegica. [Web.] Wahlenb.). This loss of protein synthetic capacity is manifested in a loss of polysomes resulting from the run-off of ribosomes from mRNAs, concomitant with their failure to reinitiate protein synthesis (see Bewley [1979]; and Bewley and Oliver [1992] for reviews). Rapid desiccation of T. ruralis, however, leads to the retention of 50% of the polysomes in the dried state, indicative that water loss alone is not the cause of the detachment of ribosomes from mRNAs. This is presumed to be because the loss of water is so fast that mRNAs are trapped on polysomes before runoff is completed. The rapid loss of polysomes during drying (under "natural" drying rates) and the apparent sensitivity of the initiation step of protein synthesis to protoplasmic drying leads us to the conclusion that the induction of synthesis of "protective" proteins during drying is highly unlikely. This is borne out by the observation that no new mRNAs are recruited into the protein synthetic complex even during slow drying (Oliver 1991). The fact that the moss survives rapid desiccation (even when desiccation is achieved in a few minutes in a lyophilizer) indicates that an inducible protection mechanism is not necessary for survival. In addition, if protective proteins (or other compounds) are an important part of the desiccation tolerance of this species they must be present at all times; that is, they must be constitutively expressed. However, as has been shown in numerous studies (see Bewley 1979; Oliver and Bewley 1984a; Bewley and Oliver 1992), T. ruralis gametophytes recover at a much slower rate if rapidly desiccated. This indicates that the moss can activate systems when exposed to slower drying rates that can decrease the time taken to recover from desiccation on subsequent rehydration, either by limiting further damage or by allowing for a quicker repair response on rehydration, or both.

178

M.

J.

OLIVER AND

J.

D. BEWLEY

That an overall strategy such as constitutive protection and rehydration-induced repair exists is strengthened by observations concerning the behavior of two cellular components that are purported to offer protection to desiccation during a wet-dry-wet episode: sucrose (see discussions below and Crowe et al. [1992] for review) and dehydrins (see discussions below,and Close et al. [1993] and Dure [1993] for reviews). Sucrose is the only free sugar available for cellular protection in desiccation-tolerant mosses, including Tortula ruraliformis (Besch.) Grout and T. ruralis (Willis 1964; Bewley et al. 1978; Smirnoff 1992). The amount of this sugar in T. ruralis gametophytic cells is approximately 100/0 dry weight, which apparently is sufficient to offer membrane protection during drying, at least in vitro (Strauss and Hauser 1986). Moreover, neither drying nor rehydration in the dark or light results in a change in sucrose concentration, suggesting that it is important for cells to maintain sufficient amounts of this sugar (Bewley et al. 1978). The lack of an increase in soluble sugars during drying appears to be a common feature of desiccation-tolerant mosses (Smirnoff 1992). Little work has been done concerning dehydrins in desiccationtolerant vegetative tissues of poikilohydric species. Western blots of soluble protein extracts from control, dry, and rehydrated gametophytes using purified antibodies raised against the common carboxyterminus of corn seedling dehydrins (Close et al. 1993) show that T. ruralis produces two major dehydrins (80-90 and 35 kD). These are present in the hydrated state and do not appear to increase during rapid or slow drying (Bewley et al. 1993); in fact, the amount present appears to decrease somewhat during slow drying. A similar result was obtained with the desiccation-tolerant moss Thuidium delacatulum (Hedw.) BSG. (T. L. Reynolds, M. J. Oliver and J. D. Bewley, unpublished data). Thus, for desiccation-tolerant species (in contrast to those that exhibit modified desiccation tolerance), proteins that may help accommodate water loss are constitutive. Both sucrose synthesis and dehydrin expression are inducible in modified poikilohydric desiccation-tolerant plants and seeds and, at least in the case of dehydrins, in nontolerant plant tissues (for a review see Skriver and Mundy [1990]). The constitutive nature of protection is not limited to desiccation-tolerant species, however, since in all plants there is a certain level of constitutive protection against free radicals (e.g., superoxide dismutases [SOD]and glutathione); important agents in stress induced cellular damage (see below). More substantial evidence for the possible accumulation during drying of components for use in recovery from desiccation and sub-

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

179

sequent regrowth comes from recent studies on the molecular aspects of desiccation tolerance in T. ruralis. Using cDNA clones corresponding to transcripts that are preferentially translated during rehydration (Scott and Oliver 1994), it was determined that several "recovery" transcripts accumulate during slow drying (Scott and Oliver, in preparation). Analysis of this accumulation during a time of metabolic decline revealed that transcripts are being sequestered in the polysomal fraction of cell extracts. As shown previously (Dhindsa and Bewley 1977), this fraction is actively losing polysomes during slow drying, and protein synthesis is inhibited. In fact, when transcript accumulation is at its peak there are no polysomes remaining (Fig. 3.2). Sucrose density gradient analysis revealed that

50 (fi. 40

en

E 30 o ~ 20

o

Q.

10 O+----If----+---+----+---+----If-----t

o

0.25

0.5

0.75

1

2

3

4

Drying Time (h) Fig. 3.2. Northern analysis of Tr288 transcripts during drying of T. ruralis gametophytes compared to percentage of remaining polysomes. Polysome data are taken from Dhindsa and Bewley (1977). The dashed line represents the percentage of polysomes in RNAse-treated samples. The major accumulation of the Tr288 transcript in the polysomal fraction of drying gametophytes occurs after polysomes have declined to levels seen in the RNAse controls. This indicates that the transcript is recruited into this fraction at a time when protein synthesis has ceased. Tr288 is a rehydration cDNA described in Scott and Oliver (1994).

180

M.

J.

OLIVER AND

J.

D. BEWLEY

Slow Dried

14

Top

15

16

Bottom Polysome Profile

Fig. 3.3. Sucrose-density-gradient analysis of the polysomal pellet from slow-dried T. ruralis gametophytes, indicating the location of Tr288 transcripts by Northern blot analysis. Fraction 3 contains the majority of the Tr288 transcript, indicating its presence in particles that sediment to a sucrose density slightly less than that of the small ribosomal subunit (fraction 4).

the transcripts accumulate in a pelletable fraction that sediments near the top of a 10 to 50% w/v sucrose gradient above, and spreading into, the region of the gradient occupied by the small ribosomal subunit (Fig. 3.3). This result is consistent with the hypothesis that, during desiccation, mRNA transcripts are sequestered in mRNA particles (mRNPs). The sequestration of "recovery" mRNAs is not required for desiccation tolerance or survival, since rapidly desiccated moss does not accumulate mRNAs during drying. In fact, available evidence suggests that rapid desiccation results in some loss of mRNAs (Oliver and Bewley 1984b). The implication from this work is that the sequestration of mRNAs required for recovery hastens the repair of desiccation/rehydration-induced damage and thus minimizes the time needed to restart growth on rehydration. These findings may also explain, in the absence of an inducible dehydrin and sugar response, the ability of T. ruralis to "harden" during recurring desiccation events (Schonbeck and Bewley 1981a,b).

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

181

As stated earlier, some mosses, although technically tolerant, can limit their rate of water loss by retaining water within clurn ps, and thus it is possible that some bryophytes have developed desiccation-tolerance mechanisms based on the induction of protective components. Such a mechanism may be in place in Funaria hygrometrica Hedw., a widespread desiccation-tolerant moss. Protonema of this moss grown in culture are tolerant of slow desiccation but are killed if water loss is rapid (Werner et al. 1991), a scenario that implies the induction or activation of protective components. The plant growth regulator abscisic acid (ABA) appears to be necessary (and is implicated as the signal) for this response, which leads to desiccation tolerance in this species. ABA increases in the protonema during drying, and when slow-dried moss is rehydrated it can survive a subsequent rapid desiccation. The application of ABA to protonema enables survival of rapid drying also (Werner et al. 1991). Bopp and Werner (1993) report that ABA exerts its influence through the synthesis of specific proteins that are synthesized during drying and indicate that some of these resemble dehydrins. Thus it is possible that tolerant plants have evolved mechanisms for desiccation tolerance similar to those in more advanced species. This possibility has been underscored recently by the demonstration that promoter elements from the wheat Em gene (an ABA-induced gene of the group designated LEAs [see below], thought to be important in desiccation tolerance of seeds) respond to both ABA and osmotic stress in transgenic protonema of the moss Physcomitrella patens Hedw. (Knight et al. 1995). Even though this moss is not desiccation tolerant, this result does point to the highly conserved nature of plant stress responses and the involvement of ABA as a signal for their activation. In light of this work, it is interesting that ABA has not been found in Tortula ruralis, which does not synthesize specific proteins in response to applied ABA (Bewley et al. 1993; M. J. Oliver, unpublished data). 2. Modified Tolerance in Vegetative Plants and Tissues. Since, by

definition, desiccation-tolerant plants that exhibit a modified-tolerant life habit can limit the rate at which water is lost from their tissues, their desiccation tolerance may be based on a metabolic response that is induced by a declining whole-plant water status. Such a mechanism would presumably be evolutionarily favored because the cost of maintaining cells in a state of readiness (as appears to be the case for desiccation-tolerant plants) can be avoided, and resources needed for competitive growth and reproductive rates are channeled for the survival of desiccation only when absolutely necessary. One

182

M.

J.

OLIVER AND

J.

D. BEWLEY

can envision that the induced response is designed to both protect the plant from and repair desiccation/rehydration-induced cellular damage. However, of the modified-tolerant plants that have been studied, all the evidence points toward the induction of protective mechanisms on the onset of drying.

Angiosperms. The most extensively studied desiccation-tolerant plant in this category is the African resurrection plant, Craterostigma plantagineum Hochst., which can survive desiccation of its vegetative tissues if drying is slow, taking 24 to 48 h to reach 15% of fresh weight (Bartels et a1. 1990). Such plants do not survive rapid desiccation (Gaff 1977) and thus it appears that the activation of protective components is necessary for tolerance. Callus derived from the leaf tissue of this plant is not inherently desiccation tolerant but becomes so if treated for 4 days with ABA before drying (Bartels et a1. 1990). In both callus and leaf tissues ABA increases six- to sevenfold during slow drying. Although long implicated as the mediating hormone in responses of plants to water stress, this was the first report of its involvement in the acquisition of desiccation tolerance by vegetative tissues. During drying many new proteins are synthesized in both callus and leaf tissue and many appear when ABA is applied to nonstressed tissues (Bartels et a1. 1990). By using differential screening, cDNAs corresponding to transcripts expressed only in desiccation-tolerant tissues were isolated and characterized (Bartels et a1. 1990; Piatkowski et a1. 1990; Bartels et a1. 1992; Bartels et a1. 1993). The majority of the cDNAs represent transcripts that increase greatly in abundance following ABA treatment; some are expressed within the first 30 min of drying, while others appear later (Bartels et a1. 1990). The differing kinetics of expression during drying and the requirement for ABA for the induction of desiccation tolerance in callus has led to the hypothesis that ABA coordinates the activation of genes leading to cellular tolerance of extreme drying (Bartels et a1. 1993). That ABA can directly effect the transcription of desiccation-specific genes in this plant has been demonstrated for the gene CDeT2 745, a Craterostigma-specific gene of unknown function (Michel et a1. 1993; Nelson et a1. 1994). The promoter of this gene contains sequences that correspond to elements in promoters known to be regu1ated by ABA, the so-called G-box elements, of which the CDeT2745 promoter contains four. However, a promoter deletion containing these elements, but no upstream regions, is not ABA-responsive (Michel et a1. 1994), and protein factors that bind to these elements

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

183

are constitutive (Nelson et al. 1994). The existence of a novel DNAbinding protein (or proteins) that is sequence-specific in the promoter region and is ABA-responsive has been demonstrated (Nelson et al. 1994). The sequence to which this (or these) factor binds appears to be related to sequences present in seed storage protein promoters (which have not been determined to be ABA regulated). These data imply that there are multiple pathways for ABA signal transduction. Interestingly, the promoter of this gene, when expressed in transgenic tobacco, is restricted to those tissues that normally undergo desiccation, that is, maturing seeds and pollen (Michel et al. 1993). In Craterostigma, the gene is expressed in all tissues, but mainly in the leaves. The involvement of ABA in desiccation tolerance may not be universal in the angiosperms, although again the evidence is sparse and often superficial. Detached leaves of the desiccation-tolerant grass Sporobolus stapfianus Gandoger. do not survive equilibration to an atmosphere of below 92 % relative humidity, similar to those of normal nontolerant crop plants (Gaff and Ellis 1974). For detached leaves to exhibit desiccation tolerance their relative water content has to be 61 % .or lower before they are removed from the parent plant. At this level of water stress, endogenous ABA has just started to increase in the leaves; the peak in ABA content is much later, indicating that desiccation-tolerance does not require this compound. In addition, the application of ABA does not appreciably alter the extent of tolerance exhibited by detached leaves (Gaff and Loveys 1994). It is pertinent at this point to mention reports of "desiccation"induced genes isolated from vegetative tissues of Arabidopsis tbaliana (1.) Heynh (Yamaguchi-Shinozaki et al. 1992; YamaguchiShinozaki and Shinozaki, 1993; Kiyosue et al. 1994). These tissues of Arabidopsis do not recover from the air-dried state, and hence Arabidopsis cannot be classified as a desiccation-tolerant plant. These genes, therefore, must be responding to severe water stress (which has mistakenly been reported as desiccation) and as such do not represent ones involved in desiccation tolerance. The central question, at least with regards to the tenets of this review, is what are the roles of the proteins that are encoded in the genes that respond to desiccation and which confer desiccation tolerance? Moreover, do they relate to protective or repair-like functions? Several of the cDNA clones isolated from the desiccation-tolerant Craterostigma tissues are related to late-embryogenesis abundant proteins (LEAs), responsive-to-ABA proteins (RABs), and dehydrins, which have long been implicated in cellular protection

184

M.

J.

OLIVER AND

J.

D. BEWLEY

during seed desiccation and during water stress (see discussion in seed section and Skriver and Mundy [1990], Bray [1993], Chandler et a1. [1993], and Dure [1993] for reviews). Others, however, are specific to Craterostigma (Piatkowski et a1. 1990; Bartels et a1. 1993). Several of these proteins have been immunolocalized to the cytoplasm of leaf cells of Craterostigma and at least one desiccationspecific protein (dsp 15) may undergo post-translational modification (Schneider et a1. 1993). Three other cDNAs from Craterostigma complement messages that are not related to the aforementioned classes of proteins but represent nuclear genes whose protein products (dsps 21, 22, and 34) are localized in the chloroplasts of leaf cells. Dsp 21 and 22 are localized in the stroma and are strongly inducible by ABA in leaves and callus. Dsp 22 appears to be regulated also by light and is closely related to plant early light-inducible genes (Elip) and to a carotene biosynthesis-related gene from a green alga (Bartels et a1. 1992). Dsp 34 is associated with thylakoids and is present only in dried leaf tissue and not in callus, even if the latter is ABA-treated prior to drying. This protein also appears to be glycosylated (Schneider et a1. 1993). The implication from these studies is that there is a protective role for these proteins in the stabilization of both cytoplasmic and chloroplastic components. One of the cold-regulated (COR) proteins (COR15A) of Arabidopsis has recently been shown to be located in chloroplasts (Lin and Tomashow 1992). In nonacclimated transgenic plants that express elevated levels ofCOR15A (equivalent to those in acclimated plants), chloroplasts maintain photosynthetic functions (variable fluorescence) better at freezing temperatures than those from nonacclimated controls. Thus, COR15A may protect the chloroplast from chilling damage in the same manner that the dsps may protect them from desiccation (N. Artus and M. Tomashow, personal communication). This is the first direct evidence of a potential protective function for a stress-induced protein and it will be interesting to determine if the chloroplast proteins induced in Craterostigma have a similar function. Desiccation of Craterostigma also induces a major change in carbohydrate metabolism during water loss, which may be directly related to desiccation tolerance. Under normal hydrated conditions, leaves of Craterostigma contain the unusual carbohydrate 2-octulose, which accumulates to nearly 50% of dry weight (Bianchi et a1. 1991a, 1992). During drying, this sugar is rapidly converted into sucrose, perhaps as a result of activation of aldolases and transketolases. Such increases in sucrose and other sugars, or sugar derivatives, occur in

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

185

several desiccation-tolerant, modified-poikilohydric species, such as sucrose and trehalose in Myrotbamnus flabellifolia Welw. (Bianchi et al. 1993; Drennan et al. 1993), a-trehalose in Selaginella lepidopbylla Hook and Grev. (Gaff 1989), and sucrose in Boea bygroscopica (F.) Meull. (Kaiser et al. 1985; Bianchi et al. 1991b) and the grasses Sporobolus stapbianus and S. festivas Hoscht. (Kaiser et al. 1985). These observations, along with a body of work demonstrating that sugars stabilize membranes during drying (see Crowe et al. [1992] for review), support the idea that the accumulation of sugars during drying is an integral part of vegetative (as well as propagative) desiccation tolerance. However, little work has involved dried vegetative tissues and this has not been demonstrated. In addition, there is a growing body of evidence that, even in seeds, sugars alone do not confer desiccation tolerance on these tissues (see Farrant et al. [1993]). Thus, although the presence of soluble sugars is important, there must be other factors, such as proteins, that also have a major role in cellular protection leading to desiccation tolerance in vascular plants.

Pteridopbytes. Ferns and fern allies that are desiccation tolerant have received only limited attention, but the most extensively studied desiccation-tolerant pteridophyte is the desert resurrection fern Selaginella lepidopbylla. The majority of studies on this fern address ecophysiological questions (see Eickmeier et al. [1992] for review) and thus are outside the scope of this review. Recent studies on the desiccation-tolerant fern Polypodium virginianum 1. have been aimed at understanding the fundamental basis of desiccationtolerance mechanisms in this plant, and have been recently reviewed (Bewley et al. 1993). Polypodium virginianum can survive water loss if desiccated slowly (over 10 days) but not if dried rapidly over silica gel (Reynolds 1992; Reynolds and Bewley 1993a), and thus exhibits modified desiccation tolerance. This is also attested to by the observation that slow-dried fronds contain more water than fronds dried over silica gel (Bewley et al. 1993; Reynolds 1992). This suggests that cellular changes occur during slow drying that result in a greater retention of water in the dried state, a property consistent with a protection-based strategy of desiccation tolerance. Indeed, ion leakage on rehydration is less from slow-dried than rapid-dried fronds, indicating that slow drying results in the retention of a more intact plasma membrane (Reynolds and Bewley 1993b). The exact mechanisms of cell or membrane protection are unknown, but during slow desiccation sucrose increases almost eight-fold.

186

M.

J.

OLIVER AND

J.

D. BEWLEY

Slow drying of Polypodium fronds also induces the synthesis of novel proteins, most noticeably a low-molecular-mass doublet of 19-29 kD (Reynolds and Bewley 1993a). Although these are of a similar molecular mass to corn dehydrins, they are not immunologically related. The fern does synthesize dehydrin-like proteins in response to drying, but they are of higher molecular mass than those of corn, being two proteins of 60 to 70 kD and one of 45 kD, all of which are closer in mass to those of Tortula (Bewley et al. 1993). The same novel proteins are synthesized during rapid drying, but since fronds do not survive this, the newly synthesized proteins per se cannot account for the desiccation tolerance of Polypodium. Unlike in Craterostigma, ABA does not increase in Polypodium tissues during drying; rather it decreases, especially after the fresh weight has declined by 20% (Reynolds and Bewley 1993a). Nevertheless, application of ABA to the fronds results in the synthesis of similar proteins as during desiccation, and pretreatment of Polypodium fronds with ABA allows them to survive rapid desiccation (Reynolds and Bewley 1993a,b), as occurred in Craterostigma callus. Thus, it appears that in its response to ABA, Polypodium is similar to some angiosperms and that this compound may act in a similar way to increase the protective capacity of frond cells. How ABA is involved in the in vivo response remains enigmatic. Polypodium contains a substantial amount of sucrose in the dried state, about 25% of dry weight, but it is not known if this is the result of its increased accumulation in response to drying. Sellaginella is one of the few plants that contains trehalose (Kander 1967), as do the two primitive ferns Botrychium lunaria (1.) Sw. and Ophioglossum vulgartum1. (Kander and Senser 1965; Kander 1967), but again the relationship between the presence of the sugar and desiccation tolerance is not known. The desiccation-tolerant fern Cardiomanes reniforme (Forst.) C. Presl. (also named Trichomanes reniforme Forst.) accumulates a novel glucoside, cardiomanol, in old leaves and rhizomes (presumably as an osmoprotectant) but it is not known if these tissues are more desiccation tolerant than tissues that do not accumulate this compound. 3. Prokaryotes. It is interesting, when considering the evolution of desiccation tolerance, to examine some of the traits of the more primitive photosynthetic organisms (e.g., the cyanobacter, the most studied of which is Nostoc communeHun.). Such studies may shed some light on the relative importance of protection versus recovery in early land plants (with the proviso that desiccation tolerance has evolved more than once, see earlier discussion and Fig. 3.1), and perhaps on

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

187

the importance of the various components and their contributions to this trait (see Potts 1994). Nostoc commune forms characteristic crusts of bacterial cells embedded in a complex water-absorbing carbohydrate sheath (Whitten 1992). Peat et al. (1988) demonstrated that vegetative cells and heterocysts, stored dry for 2 years, are not structurally damaged. Rapidly dried Nostoc does not maintain structurally intact heterocysts (Peat and Potts 1987), however, suggesting that drying rates have to accommodate a period within which protection mechanisms can be activated. When Nostoc colonies are repeatedly desiccated and rehydrated, three acidic proteins, Wsp (water stress proteins) are synthesized and accumulate to high concentrations (Scherer and Potts 1989). These three proteins, 33, 37, and 39 kD, are very closely related. All three have identical N-terminal amino acid sequences, which, along with internal sequences, show close similarity to those of carbohydrate-modifying enzymes, in particular a p-xylosidase (Hill et al. 1994). In addition, antisera to the Wsp cross-react strongly with several enzymes of this group, such as, N-glycosidase F, and purified Wsp have a strong l,4,-P-D-xylanxylanohydrolase (xylanase) activity. It has been suggested that these proteins playa structural role in cell protection (Scherer and Potts 1989) because of their abundance in dried Nostoc mats and the high content of hydroxylated amino acids in the wsp primary sequences that could stabilize them (in a similar way to that proposed for sugars). A more subtle role for Wsp was also revealed (Hill et al. 1994). Immunolocalization studies revealed that Wsp are secreted outside of the bacterial cells and accumulate in the extracellular glycan matrix of the colonial mat. The extracellular glycan sheath also contains UV-A/B-absorbing pigments that protect the bacteria from damaging radiation when desiccated. These pigments form large complexes with Wsp polypeptides. Thus, it is possible that the Wsp are involved in the stabilization of these pigments and, with their associated xylanase activity, play an important role in the modification of the extracellular glycans. The biochemical basis of the protection of desiccated plant material from the effects of damaging UV radiation has not received attention, although the synergistic effects of light and desiccation in cellular damage by free radical production have been well documented. Thus, Wsp proteins may have multiple roles in both desiccation tolerance and survival of other extreme environments, which have important connotations in our studies on other desiccation-related proteins. Recently proteins that exhibit close similarities to those implicated in desiccation and water stress responses have been observed

188

M.

J.

OLIVER AND

J.

D. BEWLEY

in the cyanobacterium Anabaena Hun. Several proteins that are induced in drying Anabaena cultures cross-react with antibodies to wheat group 3 LEA polypeptides (see seed section). The similarity of the Anabaena proteins is also manifested corresponding cDNA sequences, indicating that they are highly conserved (Curry and Walker-Simmons 1993). A dehydrin-like protein that is recognized by an antibody specific to the carboxy-terminal sequences of plant dehydrins (KIKELPG) has also been observed in osmotically stressed cultures of Anabaena (Lammers and Close 1993). Such studies not only show the possible universality of proteins involved in water stress and desiccation responses but may also indicate the early evolution of a cell protective mechanism of desiccation tolerance. B. Desiccation-Induced Cellular Damage

The central tenets of this review on the underlying mechanisms of desiccation tolerance in plants are based on the premise that such mechanisms can impact damage caused to the cell by desiccation (or rehydration) by preventing and/or repairing it. In light of this, it is important to discuss what damage occurs and how effective tissues are in limiting and/or rectifying it when it happens. Although much has been accomplished in certain areas, especially in ultrastructural studies and protection from desiccation-associated oxidative damage, many aspects have not been addressed, such as repair of cellular damage and regeneration of damaged membranes. The role of antioxidant defense mechanisms in limiting the effects of active oxygen species generated as a result of drying, and through the direct inhibition of photosynthesis during drying, has been recently and thoroughly reviewed by McKersie (1991) and Smirnoff (1993). Oxidative damage during desiccation results in several types of cellular damage, the major categories are oxidation of protein sulfhydryl groups leading to denaturation, pigment loss, and photosystem damage (especially in the light), and lipid peroxidation and free fatty acid accumulation in membranes (McKersie 1991; Smirnoff 1993). Protective mechanisms are of two types, which are synergistic (Smirnoff 1993). The first involves mechanisms that are implicated in lowering the amount of free radicals during desiccation. This includes enzymes such as peroxidase, catalase, and SOD, along with antioxidant compounds such as ascorbic acid, a-tocopherol, carotenoids, and glutathione (GSH) The second involves mechanisms that regenerate these antioxidants (glutathione reductase, ascorbate peroxidase, and mono and dehydroreductases).

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

189

Oxidative inactivation of sulfhydryl-containing enzymes as a result of desiccation has been reported in several desiccation-tolerant mosses (Stewart and Lee 1972) and in the fern Selaginella lepidophylla (Lebkuecher and Eickmeier 1992). This damage apparently continues during storage of the tissues in the dried state and can be alleviated by incubation with GSH (Stewart and Lee 1972). GSH is present throughout the cell and in highest amounts in the chloroplast (Smith et al. 1990); it plays an important role in maintaining the appropriate redox level. Slow desiccation of Tortula results in the oxidation of the GSH pool to approximately 30% GSSG (Dhindsa 1987), indicating a decreased ability of the moss to withstand oxidative injury. Interestingly, desiccation itself does not result in an increase in GSH oxidation since GSSG does not increase when the moss is dried rapidly (Dhindsa 1987), but it does increase on rehydration of rapidly dried moss. Desiccation of the tolerant moss T. ruraliformis does not result in a loss of GSH (Seel et al. 1992a) and in the grass S. staphianus GSH actually increases during drying (Sgherri et al. 1994). How these conflicting findings relate to an overall mechanism of desiccation tolerance is enigmatic, but may simply reflect varying capabilities among desiccation-tolerant species to buffer oxidative damage in this way. In S. staphianus (Sgherri et al. 1994) and T. ruraliformis (Seel et al. 1992a) ascorbate decreases during drying; in these plants maintenance of high amounts of GSH may be more important in protection than it is in T. ruralis. T. ruraliformis also maintains an appreciable a-tocopherol content during drying, but this is depleted in a desiccation-intolerant species, Dicranella palustris (Dicks.) Crundw., again this indicates that other antioxidants can be more important than ascorbic acid or GSH (Seel et al. 1992a). In all plant tissues studied to date, light increases the amount of desiccation-induced damage as a result of oxidation (Smirnoff 1993). Oxidative damage incurred during drying of T. ruraliformis and D. palustris increases if the plants are irradiated under high light conditions (1100-1200 Jlmol/m 2 s-l). Neither photosynthetic pigment content nor the ability of T. ruraliformis to recover was affected, but there was a reduction in pigments in the sensitive species D. palustris (Seel et al. 1992a). Similar findings have been reported in desiccation-tolerant ferns (Lebkuecher and Eickmeier 1991, 1992; Muslin and Homann 1992). Loss of pigments during desiccation is common in many modified desiccation-tolerant angiosperms, the so-called poikilohydrous, poikilochlorophyllous plants (Bewley and Krochko 1982), but the mechanism by which this occurs during drying has not been characterized.

190

M.

J.

OLIVER AND

J.

D. BEWLEY

Lipid peroxidation also occurs during desiccation of vegetative tissues in bryophytes. By measurement of malondialdehyde, an indicator of lipid peroxidation, Seel et al. (1992b) demonstrated that the relatively desiccation-intolerant moss D. palustris exhibited increased lipid peroxidation following desiccation, while the tolerant species T. ruraliformis did not. Interestingly, the extent of lipid peroxidation as a whole, whether in hydrated or desiccated gametophytes, was five- to sixfold higher in the intolerant species. This may be indicative of an inherent protection against lipid peroxidation in tolerant species. Stewart and Bewley (1982) recorded a decrease in lip oxygenase activity (a lipid peroxidation enzyme) during desiccation, again indicating a protective mechanism inherent in desiccation-tolerant species. That such a mechanism exists in desiccation-tolerant angiosperms can be inferred from the observation that colneyleic acid, a lipoxygenase inhibitor, accumulates in desiccating leaves of Craterostigma plantagineum (Bianchi et al. 1992). C. The Dried State

There are few studies on the ultrastructure of vegetative tissues in the dried state; of these, the majority concern the bryophytes (see Oliver and Bewley [1984a] for review) and a few describe the leaf structures of dried angiosperms (reviewed by Gaff 1980). Many of the earlier models for desiccation tolerance in vegetative tissues have now been discarded, especially those that postulated certain structural aspects of desiccation-tolerant plant cells that enabled them to withstand protoplasmic dehydration (Oliver and Bewley 1984a). Desiccation-tolerant vegetative tissues exhibit similar types of disruption to all major organelles and the plasmalemma (as shown by leakage studies) due to drying as those occurring in cells of desiccation-intolerant tissues. The difference lies to some extent in the amount of damage that is suffered, but especially in the ability of the tolerant plant tissues to recover from such damage after their cells are rehydrated. The inability to study dried cells at the ultrastructural level has confounded attempts to determine if structural damage occurs during desiccation, since the fixation of tissues, for however brief a time, always results in some rehydration, with possible damage resulting from this latter process. Recently, a partial answer to this difficulty was obtained using freeze-fracture technology directed at establishing the condition of plasma and organellar membranes in dried leaf cells of T. ruralis and Selaginella lepidophylla (Platt et al. 1994). The cell membranes

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

191

of both species in the dried state remain as intact bilayers containing normally dispersed intramembranous particles. The structural organization of the organelles is also maintained in both species and no areas of hexagonal n (H n ) phase, nonbilayer organization were detected in any of the cell membranes. This is significant because it has been suggested that such structures must occur in desiccated cells (Simon 1974, 1978), based on theoretical considerations concerning membrane lipid organization at hydration levels below 20%. Although such structures would be indicative of damage occurring during drying, the H n phase, lipid nonbilayer structure has not been found in any dried plant tissue. The discrepancy between this observation and the theoretical model (bolstered by early work on animal membrane models) is probably due to the composition of plant membranes and the presence of stabilizing factors such as sugars and proteins. Thus, it appears that, at least for Tortula and Selaginella, and perhaps in the vegetative tissues of all desiccation-tolerant plants, membrane disruption may occur not during drying but during subsequent rehydration. Interestingly, and somewhat contradictorily, rapidly dried Tortula exhibits a greater degree of cellular disruption on rehydration than does slowly dried moss (Schonbeck and Bewley 1981a; Oliver and Bewley 1984b) even though the membranes in both remain intact (Platt et al. 1994). Thus, although drying may not cause detectable membrane damage, it does affect the capaci ty of the membranes to withstand the rigors of rehydration. D. Rehydration of Dried Vegetative Tissues In all desiccation-tolerant vegetative plants and tissues, cellular damage is manifested immediately on rehydration, either as a result of the inrush of water or as a consequence of the drying process, or both. The most evident symptom of this damage is the leakage of cellular ions into the surrounding water. Such leakage has been used as a measure of the extent of one aspect of cellular disruption, membrane damage, resulting from the desiccation/rehydration event (see Simon [1978], Bewley [1979], Gaff [1980], Bewley and Krochko [1982], and Simon and Mills [1983], for reviews). These studies, along with ultrastructural observations of extensive cellular disruption in rehydrating cells, such as swollen, misshapen chloroplasts and mitochondria that have lost their internal organization (see Gaff [1980] and Oliver and Bewley [1984a] for reviews), are testament to the fact that cells of desiccation-tolerant plants are not immune to the rigors

192

M.

J.

OLIVER AND

J.

D. BEWLEY

of drying and rehydration. It is their ability to recover from such damage that may make them unique. The metabolic and molecular events involved in the repair of cells of vegetative tissues on rehydration is the least studied of all desiccation-related phenomena. This is unfortunate, since the elucidation of such cellular repair mechanisms may have a fundamental impact on our understanding of desiccation tolerance and may lead to new strategies for breeding stress-resistant crop species. 1. Tolerant Tissues. Desiccation-tolerant vegetative tissues may be the most appropriate for studies of cellular repair mechanisms, particularly those that rely more heavily on efficient recovery strategies to achieve desiccation tolerance than on protecting cells from damage. Because of their simple structure it is perhaps not surprising that the most studied plant vegetative tissues in this regard are bryophytes. Early work (see Bewley and Oliver [1992] for review) established the ability of T. ruralis and other mosses to rapidly recover synthetic metabolism when rehydrated. The speed of this recovery was dependent on the prior speed at which desiccation occurred; the faster the rate of desiccation, the slower the recovery. It was also established that although the pattern of protein synthesis in the first 2 h of rehydration of T. ruralis is distinctly different from that of hydrated controls, novel transcripts were not made in response to desiccation (Oliver and Bewley 1984c, Oliver 1991). Hence, it was suggested that T. ruralis responds to desiccation by an alteration in protein synthesis on rehydration, which is in large measure the result of a change in translational control(s). Some changes in transcriptional activity were observed but these did not result in a qualitative change in the transcript population during desiccation or rehydration. Thus, it appears that T. ruralis relies more on the activation of preexisting repair mechanisms for desiccation tolerance than it does on either preestablished or activated protection systems. The fact that T. ruralis reacts to the stress of desiccation more at the level of translation than transcription to effect a change in gene expression is unusual in stress physiology. However, it is logical for a plant that relies on rapid repair to utilize a rapid mechanism for controlling protein synthesis. In a detailed study of the changes in protein synthesis initiated by rehydration in T. ruralis, Oliver (1991) demonstrated that the synthesis of 25 proteins is terminated, or substantially decreased, and the synthesis of 74 proteins is initiated, or substantially increased, during the first 2 h of hydration. The change in synthesis of these

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

193

two groups of proteins, the former termed hydrins (previously called h or hydration proteins) and the latter rehydrins (previously called r or recovery proteins), is not coordinately controlled. The termshydrin and rehydrin are functional terms and as such do not refer to any common sequence or structural property. The synthesis of hydrins is inhibited on rehydration of gametophytes that were previously dried to 50% of their fresh weight, whereas rehydrin synthesis is initiated or stimulated only by a greater water loss, to between 50 and 20% of their fresh weight. These findings indicate that it takes a certain amount of prior water loss to fully activate the protein-based portion of the recovery mechanisms on rehydration, which may in turn indicate that there is also a mechanism by which the amount of water loss is "sensed" and "translated" into a protein synthetic response. Perhaps this is a strategy that has evolved to link the amount of energy expended in repair to the amount of damage potentiated by differing extents of drying. The time required to return to normal levels of synthesis of the two groups of proteins at longer times of hydration also differs. The synthesis of all hydrins returns to control levels between 2 and 4 h following rehydration, whereas the reduction in synthesis of rehydrins to control levels depends on each individual rehydrin. Some are synthesized only transiently, within the first hour or two, while others are still being synthesized at elevated levels 10 to 12 h after rehydration. A full return to control levels of synthesis for all proteins is evident at 24 h postrehydration. Again, such diversity in synthesis patterns among rehydrins is consistent with a cellular repair based strategy of tolerance; some repair processes take longer than others. However, until the functions of individual rehydrins are elucidated, the notion of a repair-based strategy of desiccation tolerance must remain only as a testable hypothesis. With this hypothesis in mind, Scott and Oliver (1994) isolated 18 cDNAs that represent transcripts preferentially translated during rehydration of dried T. ruralis gametophytic tissue. All 18 rehydrin cDNAs represent mRNAs present in hydrated moss cells; that is, none represent transcripts exclusive to rehydration, but all are present in greater amounts in polysomes of rehydrated gametophytes compared with those from the undesiccated moss. This is indicative of a selection of specific mRNAs into polysomes during rehydration, as would be expected if translational control was occurring. However, several (but not all) of the rehydrin cDNAs represent transcripts that also accumulate in the total RNA pool of rehydrated gametophytes, indicating either an increased transcription of rehydrin genes or an in-

194

M.

J.

OLIVER AND

J.

D. BEWLEY

crease in rehydrin mRNA stability on rehydration (Scott and Oliver 1994). This suggests that although desiccation and rehydration do not effect a qualitative alteration in transcription in T. ruralis, they may alter transcription of certain genes quantitatively. Several of the 18 rehydrin cDNAs have been partially or fully sequenced, but only one, Tr 155, has homologies with a previously documented sequence (Oliver, unpublished data). This clone is similar to two seed dormancy transcripts, one from barley embryos (Aalen et al. 1994) and one expressed during hydration of dormant seeds of Bromus secalinas 1. (Goldmark et al. 1992). The functions of the proteins encoded by these transcripts is unknown but both are related to events associated with seed imbibition (rehydration) and dormancy. 2. Modified-Tolerant Tissues. The majority of research into events

occurring on rehydration of desiccation-tolerant vegetative tissues that exhibit modified tolerance has centered on pteridophytes, in particular Selaginella and Polypodium. Rehydration of slow-dried fronds of P. virginianum results in the rapid decline of proteins synthesized during drying such that they are no longer present after 6 h of rehydration. Several proteins decline in amount during desiccation, including four thylakoid proteins (Reynolds 1992), and increase again after 24 h of rehydration to amounts present in the undesiccated frond. Rehydration also results in the synthesis of specific proteins. Within the first 3 h of rehydration, 18 novel polypeptides are synthesized that are not synthesized during desiccation. The synthesis of these proteins is also transient since it ceases after 6 h of rehydration; however, a new set of at least 22 new proteins is synthesized at later times (up to 24 h) of rehydration (Reynolds and Bewley 1993a). This is somewhat analogous to the synthesis of r proteins in T. ruralis. Indeed, this fern is phlyogenetically situated between bryophytes and angiosperms, and it appears that its response to desiccation and rehydration is also intermediate; that is, novel proteins are synthesized during desiccation, and their synthesis is apparently under the control of ABA (as in higher plants, but not bryophytes), and there is synthesis of rehydration proteins (as in bryophytes). There has been very little research concerning metabolic events related to rehydration of dried tissues of desiccation-tolerant angiosperms. The most extensive studies concern the recovery of the photosynthetic apparatus on rehydration of the "poikilochlorophyllous" resurrection plant Xerophyta scabrida (Pax) Th. Duir. &

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

195

Schinz. (Tuba et al. 1993a,b). As a result of desiccation, this monocotyledonous plant undergoes a complete breakdown of the internal structures of the chloroplast (although this was observed after a 30min fixation procedure that resulted in partial rehydration) concomitant with a loss of chlorophyll and most carotenoids. Full turgor (occurring after 2 h of hydration) and maximum leaf water content (established after 10 h) are prerequisites for the initiation of recovery of pigment content and the photosynthetic apparatus, this being completed by 72 h postrehydration (Tuba et al. 1993a,b). Such a response is obviously a repair-based one and, as such, probably requires rehydration-induced changes in gene expression.

III. POLLEN

Recent reviews of desiccation tolerance in pollen establish a major role for protective mechanisms in limiting desiccation-induced damage both in a general sense (Hoekstra et al. 1992) and for horticultural practices (Hanna and Towill 1995). In light of these comprehensive treatises, we will not detail the research in this area but will briefly comment on the overall nature of the findings presented in the aforementioned reviews. Pollen can be classified into two types, bicellular and tricellular, according to their structure. This classification, with a few exceptions, also separates desiccation-tolerant (bicellular) from desiccation-sensitive (tricellular) pollen (Hanna and Towill 1995). Desiccation tolerance in pollen appears to be achieved by a combination of cellular protective measures designed to stabilize membranes in the drying grains. These measures include an accumulation of sucrose, a possible increase in membrane acyl group unsaturaion (not seen in vegetative cells from desiccation-tolerant tissues), and an accumulation of antioxidants (Hoekstra et al. 1992). However, as pointed out by these authors, these factors only partially explain desiccation tolerance in pollen, and other factors need to be investigated, such as the role of proteins. The possible activation of repair mechanisms on rehydration of dried pollen has not received attention, despite the fact that it is well established that imbibitional injury does occur and has a major impact on pollen longevity (see both reviews).

196

M.

J.

OLIVER AND

J.

D. BEWLEY

IV. SEEDS

Desiccation tolerance of developing and germinating seeds has received a great deal of attention over the past several decades. However, because of the difficulty in separating the developmental programs and their associated metabolism from those directly involved in conferring desiccation tolerance, the underlying mechanisms of protection or repair are by and large still unresolved. Nevertheless, there are some events associated with seed maturation and germination that might be related to protection against and recovery from drying. A. The Drying Phase

The normal terminal event in the development and maturation of the so-called "orthodox" seeds is a decrease in water content, so that the mature seed achieves a desiccated and metabolically quiescent state. This is when most seeds are dispersed from the parent plant, and in the dry state they can retain their viability from several to many years (Priestley 1986). On rehydration in suitable conditions' the seed will imbibe water and commence germination, which is completed with the emergence of the embryo (usually the radicle tip), followed by seedling establishment and growth into the vegetative plant (Bewley and Black 1994). During development of the seed its metabolism is predominantly anabolic, with the resultant synthesis and deposition of polymeric reserves (protein, starch, triacylglycerol) in storage tissues such as the cotyledon or endosperm. Following germination, these stored reserves are mobilized to provide materials to support the growth of the seedling, until such times as it becomes autotrophic. The "switch" from a developmental to a germinative mode, both physiologically and metabolically, is elicited in many seeds by the drying process, or at least a partial loss of water (Dasgupta and Bewley 1982; Kermode and Bewley 1985a; Kermode and Bewley 1989; Kermode et al. 1989a; Kermode 1990). Seeds that develop in a fully hydrated environment, such as in tomato or squash fruits, will germinate without desiccation if removed from the fruit, although they are desiccation tolerant (Welbaum and Bradford 1989; Berry and Bewley 1991). The ability of seeds to withstand desiccation is acquired during their development. This acquisition is usually substantially earlier than the drying event itself. Seeds of some species can withstand premature desiccation well prior to the midpoint of their develop-

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

197

ment (Harlan and Pope 1992; Wellington 1956; Kermode and Bewley 1985a). Seeds undergo metabolic changes during the onset of tolerance, including the synthesis of certain sugars and proteins, although whether these are essentially linked to the acquisition of tolerance is still the subject of debate (see later). Even somatic embryos can be induced to survive desiccation, generally following exposure to ABA (Senaratna et a1. 1990; Janick et a1. 1993), despite the fact that the vegetative tissues from which they are derived are intolerant of drying. Metabolism resumes in germinating seeds within minutes of the commencement of imbibition (Bewley and Marcus 1990). Early metabolic events utilize the components conserved within the dry seed, which are replaced as normal turnover events proceed during germination. The germinating seed itself retains its desiccation tolerance, which is lost at the time of radicle emergence. B. Metabolic Changes Associated With Seed Drying

Several metabolic changes occur within seeds just prior to or during maturation drying. These include the synthesis of two types of products, sugars and proteins, which may have a functional significance as part of the protective mechanisms present in seed tissues to avert desiccation-induced damage. 1. Proteins. A highly abundant set of hydrophilic proteins, some of which are synthesized from about the midpoint of seed development, but are called late embryogenesis abundant (LEA) polypeptides (Galau and Hughes 1987; Galau et a1. 1987; Galau et a1. 1991), have been implicated in the acquisition of desiccation tolerance. The genes encoding these proteins in developing cotton seeds comprise two distinct and coordinately regulated classes. One class contains six different lea transcripts; they appear relatively early in development and exhibit two transient peaks before reaching a maximum about 3 days before the seed begins to dry out (Galau et a1. 1987; Galau and Hughes 1987). The second set is larger, with 12 members, and the transcripts and proteins are produced late in maturation, achieving maximum expression just before and during desiccation. In the mature cotton embryos they make up about 2% of the total soluble protein and about 30% of the nonstorage protein. Within the cells of the embryo, the LEA proteins are uniformly localized throughout the cytoplasm (Roberts et a1. 1993).

198

M.

J.

OLIVER AND

J.

D. BEWLEY

LEA proteins or lea transcripts have now been reported in the mature embryos of many species of monocots, dicots, and gymnosperms. These proteins may act as desiccation protectants, and transcription of lea genes can be elicited by desiccation of early-maturation-stage cotton embryos (Galau et aL 1991). Proteins related to some of the LEAs, such as dehydrins in barley, pea, and maize, and RAB proteins in rice seedlings, can be induced by underwater stress. The resurrection plant (Craterostigma plantagineum), which can withstand desiccation of its vegetative tissues, also produces LEAs during the early stages of drying. On the other hand, seeds of several recalcitrant temperate tree species synthesize a dehydrin protein, yet they are desiccation intolerant (Finch-Savage et aL 1994). The physical nature of LEA proteins is such that they have properties consistent with their having a role in desiccation tolerance; for example, they are extremely hydrophilic and resistant to denaturation (Close et al. 1989; Dure et al. 1989). These proteins may solvate cellular components such as other proteins and membranes, and thus protect them from drying-induced damage or disruption by providing a surrogate water film. Other LEA proteins may form amphiphilic helices, which sequester ions that are concentrated during maturation drying (Baker et al. 1988; Roberts et al. 1993). A set of LEA proteins arises in developing barley and maize embryos about the time that tolerance of desiccation is acquired. A small subset of these proteins is induced when barley embryos at the intolerant stage of their development are cultured in ABA (Bartels et al. 1988; Bochicchio et al. 1991) and a causal relationship between this regulator and lea gene expression has been suggested. Evidence for and against this relationship exists in the literature. In developing cotton embryos, as previously mentioned, there is expression of two sets of lea genes. The first set appears at about the time when embryo ABA content is greatest. High expression of the second set of lea genes, however, occurs at the start of,and during maturation drying itself, generally when the endogenous ABA content is low. A possible explanation for this lack of correlation is an early-regulated, ABA-controlled mechanism that begins to operate only later when drying commences. On the other hand, ABA may not be involved in the synthesis of the second group of LEA proteins. Studies of mutants do implicate ABA in some cases, but sometimes equivocally. Seed dormancy and viability are not affected in ABA-deficient (aba) and ABA-insensitive (abi3) double mutants of Arabidopsis thaliana; but the seeds do not undergo drying on the parent plant, do not tolerate imposed desiccation, and lack several LEA proteins (Koorneef et al. 1989; Meurs et al. 1992). Tolerance of desiccation can be con-

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

199

ferred on the developing seeds by treating the parent plant with an analogue of ABA (LAB 173711) or by placing the developing seed directly in ABA and sucrose. The effect of these treatments on lea transcript expression is not known. Embryos of ABA-deficient vp2 and vp5 maize lines produce RAB 17 and RAB 28 LEA proteins, but the latter is absent from the ABAinsensitive vp1 mutant (PIa et al. 1989, 1991). In contrast, the maize Em LEA is not present in either vp5 or vp1, but, surprisingly, some LEA proteins are produced in vp1 in response to ABA (Butler and Cuming 1993). Reservations about the universal involvement of ABA in LEA protein production can also be raised by the fact that osmotic stress induces synthesis of the Em protein when ABA synthesis is prevented by norfurazon (Morris et al. 1990). Additionally, other barley lea genes are differentially expressed in response to ABA and imposed water stress (Espelund et al. 1992). It is possible that LEA protein production is regulated by different mechanisms in different seeds, but it by no means is clear that ABA plays a direct role in the induction and maintenance of expression of LEA proteins or that they are integrally involved in the imposition of desiccation tolerance. 2. Carbohydrates. In the maturing seeds of several species, concentrations of certain sugars and oligosaccharides increase at the time of the onset of desiccation tolerance (Amuti and Pollard 1977; Koster and Leopold 1988; LePrince et al. 1990a; Chen and Burris 1990; Blackman et al. 1992) and thus are potentially components of a protective mechanism. The disaccharide sucrose and the oligosaccharides raffinose and stachyose increase late in development; during earlier developmental stages the monosaccharides glucose, mannose, fructose, and galactose are more prominent. There is an increase in sucrose, raffinose, and particularly stachyose in soybean embryos induced to become desiccation tolerant by slow drying (Blackman et al. 1992), but not in those maintained in the intolerant state. Seeds of Arabidopsismutants, which are insensitive to ABA, and the double mutants, which are insensitive to and deficient in ABA, have much lower desiccation tolerance than the wild types. Seeds of these mutant types have relatively high amounts of total sugars (mono- and disaccharides) but much lower quantities of the raffinose-series oligosaccharides than the wild type (Ooms et al. 1993). Thus, the relative amounts of the different types of sugars may be important rather than the absolute amounts. However, no temporal correlation between the increase in oligosaccharide content and the onset of tolerance in Arabidopsis can be established in the wild type.

200

M.

J.

OLIVER AND

J.

D. BEWLEY

While changes in carbohydrate metabolism occur in association with the onset of desiccation tolerance, relatively little is known about the source of these sugars. Conversion of existing monosaccharides to sucrose and to oligosaccharides is a possibility, but the breakdown of starch could also be involved (Chen and Burris 1990; LePrince et a1. 1990a), because its hydrolysis occurs in some seeds (e.g., Brassica campestris L., soybean, and white mustard) prior to and during seed drying. Whatever the source of carbohydrate, enzymes for the synthesis of sucrose and oligosaccharides must be present; whether they are programmed to appear at the time leading up to maturation drying remains to be determined. Aldose reductase, an enzyme involved in the synthesis of sorbitol, increases during development and maturation of desiccation-tolerant maize and Arabidopsis seeds (Bartels et a1. 1991; Ooms et a1. 1993). Whether sorbitol is important in tolerance to drying, or is even present in significant quantities, is unknown. The importance of sugars and oligosaccharides in tolerance of drying may be related to their role in the formation of an intracellular glassy matrix at room temperature, and in the protection of membranes and vital proteins/enzymes. C. The Dried State During the final stages of development the water content of a seed declines steadily as the insoluble reserves are deposited. With the progressive loss of water from the seed, the forces resisting water loss increase, and at moisture contents that exist in the mature dry seed, the water is associated with a macromolecular surface (sometimes called "bound" water). This water has very restricted mobility and its thermodynamic properties are different from those of bulk liquid water (Vertucci 1990; Bruni and Leopold 1991). Moreover, this water is not freezable, and hence many dry seeds can withstand being plunged into liquid nitrogen. At least five types of water have been distinguished on the basis of their calorimetric and motional properties (Vertucci 1990, 1993; Vertucci and Farrant 1995). These types are also temperature dependent. Type 1 is absorbed onto macromolecules through ionic bonding. Type 2 has glassy characteristics. Type 3 is bound with negligible energy and may form bridges over hydrophobic sites. The complete removal of type 3 water is associated with membrane structural changes. The cytoplasm of dry seeds exists in a glassy (vitrified) state, and since a glass is a liquid of high viscosity, chemical reactions requiring molecular diffusion are greatly slowed down. This

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

201

prevents damaging interactions between cell components, and denaturation of enzymes, for example, is retarded or averted because the enzymes are held in their stable, folded state. Vitrification also prevents crystallization of solutes in the cytoplasm (Burke 1986; Leopold et al. 1992). On the basis of the above we might not expect metabolism to occur in a dry seed. But so-called dry seeds shed from the parent plant, or in storage, may contain type 3 water, ot have a greater hydration (types 4 and 5), and thus metabolic reactions can proceed, albeit at a low rate. This results in a loss of viability in storage. Nonenzymic reactions can also occur in dry seeds, and free-radical formation increases with decline in moisture content, contributing to the deterioration of the seeds with time (Hendry 1993). D. The Consequences of Drying on Seed Metabolism Upon Rehydration Upon rehydration of the mature dry seed, metabolism resumes and germination processes are initiated. Even in dormant seeds, there is a similar activity of metabolism as in nondormant seeds, but only in the latter is germination completed with the emergence of the radicle (Bewley and Black 1994). Desiccation of developing seeds, after they have acquired a tolerance of drying, promotes germination on subsequent rehydration, and, as in mature seeds, developmental protein synthesis ceases followed by the onset of metabolic events associated with germination and postgerminative growth. This includes mobilization of the storage reserves and the synthesis of enzymes associated with their hydrolysis and utilization (Evans et al. 1975; Armstrong et al. 1982; Dasgupta and Bewley 1982; Adams et al. 1983; Kermode and Bewley 1985a,b, Kermode et al. 1985; Misra and Bewley 1985; Oishi and Bewley 1990). The changes in the mode of synthesis, for example, of proteins, from one that is developmental to one that is germination/growth-oriented is indicative of a switch in genome activity. This results in the permanent off-regulation of developmental protein synthesis, such as storage protein synthesis, and the on-regulation of, for example, hydrolytic enzymes. A switch in the mRNA population is elicited by both premature and maturation drying (Misra and Bewley 1985; Cornford et al. 1986; Kermode et al. 1989b; Bewley and Oliver 1992). It is not known how drying mediates the on- and off-regulation of the many genes that are affected, or if there are one or more intermediates involved in a signaling mechanism that exists within cells. However, it is likely that the gene for

202

M.

J.

OLIVER AND

J.

D. BEWLEY

the storage protein in Phaseolus vulgaris 1. (~-phaseolin) is directly down-regulated by drying (Oliver et a1. 1993). ABA, which maintains storage protein synthesis during the development of many seeds (see Kermode [1990] for references), is no longer capable of this after drying of the seed. But if an immature seed (embryo) is germinated without passing through a desiccation phase, it is still capable of responding to ABA, which can on-regulate the storage protein genes (Xu and Bewley 1994). There is a strong possibility that some repair-based mechanisms come into operation on rehydration of dry seed. There is extensive literature on the repair processes that occur on imbibition of aged dry seeds and it is likely that similar processes will operate during the early stages of germination of unaged seeds, in which there could be more limited damage resulting from maturation drying (e.g., reviewed in Osborne [1983], Priestly [1986], LePrince et a1. [1993], Bewley and Black [1994], and Osborne and Boubriak [1994]). This may include restitution of membrane structures and repair to or replacement of macromolecules; such events may be an integral part of early germination as seeds resume their metabolism. Osmopriming, a common horticultural practice involving the imbibition of seeds in an osmotic solution, can enhance the rate and uniformity of germination (Heydecker et a1. 1975; Hegarty 1978) and improve the germination of dry, aged seed lots (Brocklehurst and Dearman 1983; DellAquila et a1. 1984; Thanos et a1. 1989). Imbibition of seeds in osmotica delays their germination, perhaps allowing a longer period for repair processes to be completed before the critical event of radicle elongation commences (Bray 1995). To conclude this section on the desiccation tolerance of seeds, a brief mention needs to be made of the loss of tolerance that occurs at the very last stages of germination as the radicle emerges. Thus, while tolerance during development may be manifest for several weeks, and in the mature dry state for many years, it may be lost within hours of the start of imbibition of the dry seed (Akalehiywot and Bewley 1980; McKersie and Tomes 1980; Sargent et a1. 1981). The reasons for this are unknown, but loss of tolerance is associated with some major morphological and physiological changes, such as increased vacuolation associated with cell expansion, and replication of DNA as cell division occurs, a decline in protective enzymes (superoxide dismutase and peroxidase), and sucrose and raffinose-series oligosaccharides (Crevecour et a1. 1976; Koster and Leopold 1988; LePrince et a1. 1990b) These and other temporally associated events could be sensitive to desiccation.

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

203

V. CLOSING REMARKS

In our initial comments we proposed that mechanisms of desiccation tolerance spanned a range from tissues reliant on cellular protection to those reliant on repair of desiccation-induced damage. Although the available information is still relatively sparse, it does appear to be consistent with this proposition. Desiccation-tolerant angiosperms and seeds appear to rely mostly on protective strategies. Pteridophytes are perhaps more reliant on protection but still retain a significant repair component, and bryophytes (and perhaps most desiccation-tolerant species) are more reliant on repair mechanisms, although even they must limit desiccation-induced damage to a reparable level. Within each of these groups there will be species that are exceptions to this generalization; for example, the bryophyte Funaria hygrometrica appears to utilize a protection strategy similar to that of angiosperms and pteridophytes. But regardless of how individual species have developed in their protection-repair strategies, the existence of these alternatives should not be overlooked in future studies. First and foremost of future goals is a need to understand the functions and roles of many of the proteins identified that respond to or are associated with desiccation stress. This goal can be approached in several ways. The potentially most productive is the use of transgenic plants. Three desiccation-specific cDNAs from Craterostigma have been used in plant expression vectors for the synthesis of their corresponding desiccation-specific proteins in transgenic tobacco (Iturriaga et al. 1992). Unfortunately, even though significant amounts of each protein were detected in the leaves, there was no detectable change in the phenotype of the transgenic host plants. This result is a testament to the complexity of desiccation tolerance (as outlined in this review) and to the limitations of using heterologous nontolerant host plants for transgenic studies. Nevertheless, there is scope for more key proteins and enzymes to be used in this manner, alone and especially in combinations. As transformation and regeneration methods are developed for desiccation tolerant species, such as Tortula and Craterostigma , limiting the production of specific proteins using antisense technology might lead to an elucidation of their role in desiccation tolerance. For the near future however, research will be more empirical than targeted: molecular technology is again ahead of our understanding of the basic biochemical and cellular processes. The evolutionary aspects of desiccation tolerance as a trait have received little attention. As more plants are investigated and as their

204

M. J. OLIVER AND J. D. BEWLEY

protection versus repair strategies are determined, we will be able to answer questions pertaining to the value of these strategies in the survival of extreme environments. Placing the various plants within the phylogenetic framework presented here will also allow us to identify archetypes, which ultimately may lead to the identification of the fundamental roles of the various cellular and metabolic components in the complex and multifaceted trait of desiccation tolerance.

LITERATURE CITED Aalen, R. B., H. G. Opsahl-Ferstad, C. Linnestad, and O. A. Olsen. 1994. Transcripts encoding an oleosin and a dormancy-related protein are present in both the aleurone layer and in the embryo of developing barley (Hordeum vulgare L.). Plant J. 5:385-396.

Adams, C. A., M. C. Fjerstad, and R. W. Rinne. 1983. Characteristics of soybean seed maturation: necessity for slow dehydration. Crop Sci. 23:265-267. Akalehiywot, T., and J. D. Bewley. 1980. Desiccation of oat grains during and following germination, and its effects on protein synthesis. Can. J. Bot. 58:23492355.

Amuti, K. S, and C. J. Pollard. 1977. Soluble carbohydrates of dry and developing seeds. Phytochemistry 16:529-532. Armstrong, c., M. Black, J. M. Chapman, H. A. Norman, and R. Angold. 1982. The induction of sensitivity to gibberellin in aleurone tissue in developing wheat grains, I: the effect of dehydration. Planta 159:573-577. Baker, J., C. Steele, L. Dure III. 1988. Sequence and characterization of 6 LEA proteins and their genes from cotton. Planta 175:485-492. Bartels, D., M. Singh, and F. Salamini. 1988. Onset of desiccation tolerance during development of the barley embryo. Planta 175:485-492. Bartels, D., K. Schneider, G. Terstappen, D. Piatkowski, and F. Salamini. 1990. Molecular cloning of abscisic acid-modulated genes which are induced during desiccation of the resurrection plant Craterostigma plantagineum. Planta 181:27-34. Bartels, D., K. Engelhardt, R. Roncarati, K. Schneider, M. Rotter, and F. Salamini. 1991. An ABA and GA modulated gene expressed in the barley embryo encodes an aldose reductase related protein. EMBO J. 10:1037-1043. Bartels, D., C. Hanke, K. Schneider, D. Michel, and F. Salamini. 1992. A desiccation related Elip-like gene from the resurrection plant Craterostigma plantagineum is regulated by light and ABA. EMBO J. 11:2771-2778. Bartels, D., R. Alexander, K. Schneider, R. Elster, R. Velasco, J. Alamillo, G. Bianchi, D. Nelson, and F. Salamini. 1993. Desiccation-related gene products analyzed in a resurrection plant and in barley embryos. p. 119-127. In: T. J. Close and E. A. Bray (eds.), Plant responses to cellular dehydration during environmental stress. Current Topics in Plant Physiology: Am. Soc. Plant Physiol. Series Vol. 10. Berry, T., and J. D. Bewley. 1991. Seeds of tomato (Lycopersicon esculentum Mill.) which develop in a fully hydrated environment in the fruit switch from a developmental to a germinative mode without the requirement for desiccation. Planta 186:27-34.

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

205

Bewley, J. D. 1972. The conservation of polyribosomes in the moss Tortula ruralis during total desiccation. J. Exp. Bot. 23:692-698. Bewley, J. D. 1973. Desiccation and protein synthesis in the moss Tortula ruralis. Can. J. Bot. 51:203-206. Bewley, J. D. 1979. Physiological aspects of desiccation-tolerance. Annu. Rev. Plant. Physiol. 30:195-238. Bewley, J. D., and M. Black. 1994. Seeds. Physiology of development and germination. 2d ed. Plenum, New York. Bewley J. D., and J. E. Krochko. 1982. Desiccation tolerance. p. 325-378. In: O. L. Lange, P. S. Nobel, C. B. Osmond, and H. Ziegler. (eds.), Encyclopedia of plant physiology. Vol 12B, Physiological ecology II. Springer, Berlin. Bewley, J. D., and A. Marcus. 1990. Gene expression in seed development and germination. p. 165-193. In: W. E. Cohn and K. Moldave (eds.), Progress in nucleic acid research and molecular biology. Academic, Orlando, FL. Bewley, J. D., and M. J. Oliver. 1992. Desiccation-tolerance in vegetative plant tissues and seeds: protein synthesis in relation to desiccation and a potential role for protection and repair mechanisms. p. 141-160. In: C. B. Osmond and G. Somera (eds.), Water and life: a comparative analysis of water relationships at the organismic, cellular and molecular levels. Springer, Berlin. Bewley, J. D., P. Halmer, J. E. Krochko, and W. E. Winner. 1978. metabolism of a drought-tolerant and a drought-sensitive moss: respiration, ATP synthesis and carbohydrate status. p. 185-203. In: J. H. Crowe and J. S. Clegg (eds.), Dry biological systems. Academic, New York. Bewley, J. D., T. L. Reynolds and M. J. Oliver. 1993. Evolving strategies in the adaptation to desiccation. p. 193-201. In: T. J. Close and E. A. Bray (eds.), Plant responses to cellular dehydration during environmental stress. Current Topics in Plant Physiology: Am. Soc. Plant Physiol. Series Vol. 10. Bianchi, G., A. Gamba, C. Murelli, F. Salamini, and D. Bartels. 1991a. Novel carbohydrate metabolism in the resurrection plant Craterostigma plantagineum. Plant J. 1:355-359. Bianchi, G., C. Murelli, A. Bochicchio, and C. Vazzana. 1991b. Changes in lowmolecular weight substances in Boea hygroscopica in response to desiccation and rehydration. Phytochemistry 30:461-466. Bianchi, G., A. Gamba, C. Murelli, F. Salamini, and D. Bartels. 1992. Low molecular weight solutes in desiccated and ABA-treated calli of Craterostigma plantagineum. Phytochemistry 31:1917-1922. Bianchi, G., A. Gamba, R. Limiroli, N. Pozzi, R. Elster, F. Salamini, and D. Bartels. 1993. The unusual sugar composition in leaves of the resurrection plant Myrothamnus flabellifolia. Physiol. Plant. 87:223-226. Blackman, S. A., R. L. Obendorf, and A. C. Leopold. 1992. Maturation proteins and sugars in desiccation tolerance of developing soybean seeds. Plant Physiol. 100:225-230. Bochicchio, A., C. Vazzana, R. Velasco, M. Singh, and D. Bartels. 1991. Exogenous ABA induces desiccation tolerance and leads to the synthesis of specific gene transcripts in immature embryos of maize. Maydica 36:11-16. Bopp, M., and O. Werner. 1993. Abscisic acid and desiccation-tolerance in mosses. Bot Acta 106:103-106. Bray, C. M. 1995. Biochemical processes during the osmopriming of seeds. p. 767789. In: J. Kigel, and G. Galili (eds.), Seed development and germination. Dekker, New York.

206

M.

J. OLIVER AND J. D. BEWLEY

Bray, E. A. 1993. Molecular responses to water deficit. Plant Physiol. 103:10351040. Brocklehurst, P. A., and J. Dearman. 1983. Interactions between seed priming treatments and nine seed lots of carrot, celery, and onion, I: laboratory germination. Ann. Appl. BioI. 102:577-584. Bruni, F. B., and A. C. Leopold. 1991. Glass transitions in soybean seed: relevance to anhydrous biology. Plant Physiol. 96:660-663. Burke, M. J. 1986. The glassy state and survival of anhydrous biological systems. p. 358-363. In: A. C. Leopold (ed.), Membranes, metabolism and dry organisms. Cornell Univ., Ithaca, NY. Butler, W. M., and A. C. Cuming. 1993. Differential molecular responses to abscisic acid and osmotic stress in viviparous maize embryos. Planta 189:47-54. Chandler, P. M., R. Munns, and M. Robertson. 1993. Regulation of dehydrin expression. p. 159-166. In: T. J. Close and E. A. Bray (eds.), Plant responses to cellular dehydration during environmental stress. Current Topics in Plant Physiology: Am. Soc. Plant Physiol. Series Vol. 10. Chase, M. W., D. E. Soltis, R. G. Olmstead, D. Morgan, D. H. Les, B. D. Mishler, M. R. Duvall, R. A. Price, H. G. Hills, Y. Qiu, K. A. Kron, J. H. Rettig, E. Conti, J. D. Palmer, J. R. Manhart, K. J. Sytsma, H. J. Michaels, W. J. Kress, K. G. Karol, W. D. Clark, M. Hedren, B. S. Gaut, R. K. Jansen, K. Kim, C. F. Wimpee, J. F. Smith, G. R. Fumier, S. H. Strauss, Q. Xiang, G. M. Plunkett, P. S. Soltis, S. M. Swensen, S. E. Williams, P. A. Gadek, C. J. Quinn, 1. E. Eguiarte, E. Golenberg, J. G. H. Learn, S. W. Graham, S. C. H. Barrett, S. Dayanandan and V. A. Albert. 1993. Phylogenetics of seed plants: an analysis of nucleotide sequences from the plastid gene rbcL. Ann. Missouri Bot. Garden 80:528-580. Chen, Y., and J. S. Burris. 1990. Role of carbohydrates in desiccation tolerance and membrane behavior in maturing maize seed. Crop Sci. 30:971-975. Close, T. J., A. A. Kortt, and P. M. Chandler. 1989. A eDNA-based comparison of dehydration-induced proteins (dehydrins) in barley and corn. Plant Mol. BioI. 13:95-108. Close, T. J., R. D. Fenton, A. Yang, R. Asghar, D. A. DeMason, D. E. Crone, N. C. Meyer, and F. Moonan. 1993. Dehydrin: the protein. p. 104-118. In: T. J. Close and E. A. Bray (eds.), Plant responses to cellular dehydration durin environmental stress. Current Topics in Plant Physiology: Am. Soc. Plant Physiol. Series Vol. 10. Cornford, C. A., M. Black, J. M. Chapman, and D. C. Baulcombe. 1986. Expression of a-amylase and other gibberellin-related genes in aleurone tissue of developing wheat grains. Planta 169:420-428. Crane, P. R. 1990. The phylogenetic context of microsporogenesis. p. 11-41 In: S. Blackmore and R. B. Knox (eds.), Microspores: evolution and ontogeny. Academic, London. Crevecour, M., R. Deltour, and R. Bronchart. 1976. Cytological study of water stress during germination of Zea mays. Planta 132:31-41 Crowe, J. H., F. A. Hoekstra, and L. M. Crowe. 1992. Anhydrobiosis. Annu. Rev. Physiol. 54:579-599. Curry, J., and M. K. Walker-Simmons. 1993. Sequence analysis of wheat cDNAs for abscisic acid-reponsive genes expressed in dehydrated wheat seedlings and the cyanobacterium, Anabaena. p. 128-136. In: T. J. Close and E. A. Bray (eds.), Plant responses to cellular dehydration during environmental stress. Current Topics in Plant Physiology: Am. Soc. Plant Physiol. Series Vol. 10.

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

207

Dasgupta, J., and J. D. Bewley. 1982. Desiccation of axes of Phaseolus vulgaris during development causes a switch from a developmental pattern of protein synthesis to a germination pattern. Plant Physiol. 70:1224-1227. Dell'Aquilla, A., D. Pignone, and G. Carella. 1984. Polyethylene glycol 6000 priming effect of germination of unaged wheat lots. BioI. Plant. 26:166-173. Dhindsa, R. 1987. Glutathione status and protein synthesis during drought and subsequent rehydration of Tortula ruralis. Plant Physiol. 83:816-819. Dhindsa, R, and J. D. Bewley. 1977. Water stress and protein synthesis, V: protein synthesis, protein stability and membrane permeability in a drought-sensitive and drought-tolerant moss. Plant Physiol. 59:295-300. Donoghue, M. J. 1994. Progress and prospects in reconstructing plant phylogeny. Ann. Missouri Bot. Garden 81:405-418. Drennan, P. M., M. T. Smith, D. Goldsworthy, and J. van Staden. 1993. The occurrence of trehalose in the leaves of the desiccation-tolerant angiosperm Myrothamnus flabellifolius Welw. J. Plant Physiol. 142:493-496. Dure, L. III. 1993. A repeating ii-mer amino acid motif and plant desiccation. Plant J. 3:363-369. Dure, 1., III, M. Crouch, J. Harada, T-H. D. Ho, J. Mundy, R Quatrano, T. Thomas, and Z.R. Sung. 1989. Common amino sequence domains among LEA proteins of higher plants. Plant Mol. BioI. 12:475-486. Eickmeier, W. G., J. G. Lebkuecher, and C. B. Osmond. 1992. Photosynthetic water oxidation and water stress in plants. p. 223-239. In: C. B. Osmond and G. Somero (eds.), Water and life: a comparative analysis of water relationships at the organismic, cellular and molecular levels. Springer Berlin. Espelund, M., S. Saeboe-Larssen, D. W. Hughes, G. A. Galau, F. Larsen, and K. S. Jakobsen. 1992. Late embryogenesis-abundant genes encoding proteins with different numbers of hydrophilic repeats are regulated differentially by abscisic acid and osmotic stress. Plant J. 2:241-252. Evans, M., M. Black, and J. M. Chapman. 1975. Induction of hormone sensitivity by dehydration is one positive role for drying in cereal seed. Nature 258:144-145. Farrant, J. M., N. W. Pammenter, and P. Berjak. 1993. Seed desiccation in relation to desiccation-tolerance: a comparison between desiccation-sensitive (recalcitrant) seeds of Avencenia marina and desiccation-tolerant types. Seed Sci. Res. 3:1-13. Finch-Savage, W. E., S. K. Pramanik, and J. D. Bewley. 1994. The expression of dehydrin proteins in desiccation-sensitive (recalcitrant) seeds of temperate trees. Planta 193:478-485. Gaff, D. F. 1977. Desiccation-tolerant vascular plants of Southern Africa. Oecologia 31:95-109. Gaff, D. 1980. Protoplasmic tolerance to extreme water stress. p. 207-230. In: N. C. Turner and P. J. Kramer (eds.), Adaptation of plants to water and high temperature stress. Wiley Interscience, New York. Gaff, D. 1989. Responses of desiccation-tolerant "resurrection" plants to water stress. p. 255-268. In: K. H. Krebb, H. Richter, T. M. Hinkley (eds.), Structural and functional responses to environmental stresses. SPB Academic, The Hague, Netherlands. Gaff, D., and R. P. Ellis. 1974. South African grasses with foliage that revives after dehydration. Bothalia 11:305-308. Gaff, D., and B. R Loveys. 1994. Abscisic acid levels in drying plants of a resurrection grass. First Asia-Pacific Conf. Plant Physiol., Kuala Lumpur. Nov. 1992. Trans. Malaysian Soc. Plant Physiol. 3:286-287.

208

M. J. OLIVER AND J. D. BEWLEY

Galau, G. A., and D. W. Hughes. 1987. Coordinate accumulation of homologous transcripts of seven cotton lea gene families during embryogenesis and germination. Dev. BioI. 123:213-221. Galau, G. A., N. Bijaisoradat, and D. W. Hughes. 1987. Accumulation kinetics of cotton late embryogenesis-abundant mRNAs: coordinate regulation during embryogenesis and the role of abscisic acid. Dev. BioI. 123:198-212. Galau, G. A., K. S. Jakobsen, and D. W. Hughes. 1991. The controls of late dicot embryogenesis and early germination. Physiol. Plant. 81:280-288. Goldmark, P. J., J. Curry, C. F. Morris, and M. K. Walker-Simmons. 1992. Cloning and expression of an embryo-specific mRNA up-regulated in hydrated dormant seeds. Plant Mol. BioI. 19:433-441. Hanna, W. W., and L. E. Towill. 1995. Long-term pollen storage. Plant Breeding Rev. 13:179-207. Harlan, H. v., and M. N. Pope. 1992. The germination of barley seeds harvested at different stages of growth. J. Hered. 13:72-75. Hegerty, T. W. 1978. The physiology of seed hydration and dehydration, and the relation between water status and the control of germination: a review. Plant Cell Environ. 1:101-119. Henckel, R. A., N. A. Satarova, and S. V. Shaposnikova. 1977. Protein synthesis in poikiloxerophytes and wheat embryos during the initial period of swelling. Sov. Plant Physiol. 14:754-762. Hendry, G. A. F. 1993. Oxygen, free-radical processes, and seed longevity. Seed Sci. Res. 3:141-153. Heydecker, W., J. Higgins, and Y. J. Turner. 1975. Invigoration of seeds? Seed Sci. Technol. 3:881-882. Hill, D. R., S. L. Hladun, S. Scherer, and M. Potts. 1994. Water stress proteins of Nostoc commune (Cyanobacteria) are secreted with UV-A/B-absorbing pigments and associate with 1,4-13-D-xylanxylanohydrolase activity. J. BioI. Chern. 269:77267734. Hoekstra, F. A., J. H. Crowe, 1. M. Crowe, and D. G. J. 1. Van Bilsen. 1992. Membrane behavior and stress tolerance in pollen. pp. 177-186. In: E. Ottaviano, D. L. Mulcahy, M. Sari Gorla, and G. Bergamini Mulcahy (eds.), Angiosperm pollen and ovules. Springer, New York. Iturriaga, G., K. Schneider, F. Salamini, and D. Bartels. 1992. Expression of desiccation-related proteins from the resurrection plant Craterostigma plantagineum in transgenic tobacco. Plant Mol. BioI. 20:555-558. Janick, J., Y. H. Kim, S. Kitto, and y. Saranga. 1993. Desiccated synthetic seed. p. 11-23. In: K. Redenbaugh (ed.). Synseeds: application of synthetic seeds to crop improvement. CRC, Boca Raton, FL. Kaiser, K., D. Gaff, and W. H. Outlaw, Jr. 1985. Sugar contents of leaves of desiccation sensitive and desiccation-tolerant plants. Naturwissenschaften 72:608-609. Kander, O. 1967. Biosynthesis of poly- and oligosaccharides during photosynthesis in green plants. pp 134-151. In: A. San Pietro, F. A. Greer, and T. J. Army (eds.), Harvesting the sun: Photosynthesis in plant life. Academic, New York. Kander, 0., and M. Senser. 1965. Vorkommen von trehalose in Botrychium lunaria. Z. pflanzenphysiol. 53:157-161. Kermode, A. R. 1990. Regulatory mechanisms involved in the transition from seed development to germination. CRC Crit. Rev. Plant Sci. 9:155-195. Kermode, A. R., and J. D. Bewley 1985a. The role of maturation drying in the transition from seed development to germination, I: acquisition of desiccation toler-

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

209

ance and germinability during development of Ricinus communis L. seeds. J. Exp. Bot. 36:1906-1915. Kermode, A. R., and J. D. Bewley. 1985b. The role of maturation drying in the transition from seed development to germination, II.:post-germinative enzyme production and soluble protein synthetic changes within the endosperm of Ricinus communis L. seeds. J. Exp. Bot. 36:1916-1927. Kermode, A. R, and J. D. Bewley. 1989. Developing seeds of Ricinus communis L., when detached and maintained in an atmosphere of high relative humidity, switch to a germinative mode without the requirement for complete desiccation. Plant Physiol. 90:702-707. Kermode, A. R., D. J. Gifford, and J. D. Bewley. 1985. The role of maturation drying in the transition from seed development to germination, III: insoluble protein synthetic pattern changes within the endosperm of Ricinus communis L. seeds. J. Exp. Bot. 36:1928-1936. Kermode, A. R, M. Y. Oishi, and J. D. Bewley. 1989a. Regulatory roles for desiccation and abscisic acid in seed development: a comparison of the evidence from whole seeds and isolated embryos. Crop Sci. Am. Spec. Publ. 14:23-50. Kermode, A. R, S. K. Pramanik, and J. D. Bewley. 1989b. The role of maturation drying in the transition from seed development to germination, VI: desiccationinduced changes in messenger RNA populations within the endosperm of Ricinus communis L. seeds. J. Exp. Bot. 40:33-41. Kiyosue, T., K. Yamaguchi-Shinosaki, and K. Shinozaki. 1994. Characterization of two cDNAs (ERD10 and ERD14) corresponding to genes that respond rapidly to dehydration stress in Arabidopsis thaliana. Plant Cell Physiol. 35:225-231. Knight, C. D., A. Sehgal, K. Atwal, J. C. Wallace, D. J. Cove, D. Coates, R S. Quatrano, S. Bahadur, P.G. Stockley, and A. C. Cuming. 1995. Molecular responses to abscisic acid and stress are conserved between moss and cereals. Plant Cell 7:499-506 Koorneef, M., C. J. Hanhart, H. W. M. Hilhorst, and C. M. Karssen. 1989. In vivo inhibition of seed development and reserve protein accumulation in recombinants of abscisic acid biosynthesis and responsiveness mutants in Arabidopsis thaliana. Plant Physiol. 90:463-469. Koster, K. L., and A. C. Leopold. 1988. Sugars and desiccation tolerance in seeds. Plant Physiol. 88:829-832. Lammers, P. J., and T. J. Close. 1993. An osmotic stress protein of cyanobacteria is immunologically related to plant dehydrins. Plant Physiol. 101:773-779. Lebkuecher, J. G., and W. G. Eickmeier. 1991. Reduced photoinhibition with stem curling in the resurrection plant Selaginella lepidophylla. Oecologia 88:597604. Lebkuecher, J. G., and W. G. Eickmeier. 1992, Photo inhibition of photophosphorylation, adenosine triphosphate content, and glyceraldehyde-3-phosphate dehydrogenase (NADP+) following high-irradiance desiccation of Selaginella lepidophylla. Can. J. Bot. 70:205-211. Leopold, A. C., F. Bruni, and R J. Williams. 1992. Water in dry organisms. p. 161169. In: G. N. Somero, C. B. Osmond, and C. L. Bolis (eds.), Water and life: comparative analysis of water relationships at the organismic, cellular and molecular levels. Springer, Berlin. LePrince, 0., R. Bronchart, and R. Deltour. 1990a. Changes in starch and soluble sugars in relation to acquisition of desiccation tolerance during maturation of Brassica campestris seed. Plant Cell Environ. 13:339-346.

210

M.

J. OLIVER AND J. D. BEWLEY

LePrince, 0., R. Deltour, P. C. Thorpe, N. M. Atherton, and G. A. F. Hendry. 1990b. The role of free radicals and radical processing systems in loss of desiccationtolerance in germinating maize (Zea mays L.) New Phytol. 116:573-580. LePrince, 0., G. A. F. Hendry, and B. D. McKersie. 1993. The mechanisms of desiccation tolerance in developing seeds. Seed Sci. Res. 3:231-246 Lin, C., and M. F. Thomashow. 1992. DNA sequence analysis of a complementary DNA for cold-regulated Arabidopsis gene cor15 and characterization of the COR 15 polypeptide. Plant Physiol. 99:519-525. McKersie, B. 1991. The role of oxygen free radicals in mediating freezing and desiccation stress in plants. p. 107-118. In: E. Pel!, and K. Staffen (eds.), Active oxygen and oxidative stress and plant metabolism. Current Topics in Plant Physiology: Am. Soc. Plant Physiol. Series Vol. 8. McKersie, B. D., and D. T. Tornes. 1980. Effects of dehydration treatments on germination, seedling vigor and cytoplasmic leakage in wild oats and birdsfoot trefoil. Can. J. Bot. 58:471-476. Meurs, C., A. S. Basra, C. M. Karssen, and 1. C. van Loon. 1992. Role of abscisic acid in the induction of desiccation tolerance in developing seeds of Arabidopsis thaliana. Plant Physiol. 98:1484-1493. Michel, D., F. Salamini, D. Bartels, P. Dale, M. Baga, and A. Szalay. 1993. Analysis of a desiccation and ABA-responsive promoter isolated from the resurrection plant Craterostigma plantagineum. Plant J. 4:29-40. Michel, D., A. Furini, F. Salamini, and D. Bartels. 1994. Structure and regulation of an ABA- and desiccation-responsive gene from the resurrection plant Craterostigma plantagineum. Plant Mol. BioI. 24:249-560. Mishler, B. D., L. A. Lewis, M. A. Buchheim, K. S. Renzaglia, D. J. Garbary, C. F. Delwiche, F. W. Zechman, T. S. Kantz and R. 1. Chapman. 1994. Phylogenetic relationships of the "green algae" and "bryophytes". Ann. Missouri Bot. Garden 81:451-483. Misra, S., and J. D. Bewley. 1985. Reprogramming of protein synthesis from a developmental to a germinative mode induced by desiccation of the axes of Phaseolus vulgaris. Plant Physiol. 78:875-882. Morris, P. c., A. Kumar, B. J. Bowles, and A. C. Cuming. 1990. Osmotic stress and abscisic acid induce expression of the wheat Ern genes. Eur. J. Biochem. 190:625630. Muslin, E. H., and P. H. Homann. 1992. Light as a hazard for the desiccation-resistant 'resurrection' fern Polypodium polypodioides L. Plant Cell Environ. 15:8189. Nelson, D., F. Salamini, and D. Bartels. 1994. Abscisic acid promotes novel DNAbinding activity to a desiccation-related promoter of Craterostigma plantagineum. Plant J. 5:451-458. Oishi, M. Y., and J. D. Bewley. 1990. Distinction between the responses of developing maize kernels to fluridone and desiccation in relation to germinability, aamylase activity and abscisic acid content. Plant Physiol. 94:592-598. Oliver, M. J. 1983. The role of desiccation in the control of transcription and translation in the moss Tortula ruralis. Thesis, Univ. of Calgary, Calgary Canada. Oliver, M. J. 1991. Influence of protoplasmic water loss on the control of protein synthesis in the desiccation-tolerant moss Tortula ruralis: ramifications for a repair-based mechanism of desiccation-tolerance. Plant Physiol. 97:1501-1511. Oliver, M. J., and J. D. Bewley. 1984a. Desiccation and ultrastructure in bryophytes. Adv. Bryol. 2:91-131.

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

211

Oliver, M. J., and J. D. Bewley. 1984b. Plant desiccation and protein synthesis, V: stability of poly(A)- and poly(A)+ RNA during desiccation and their synthesis on rehydration in the desiccation-tolerant moss Tortula ruralis and the intolerant moss Crateneuron filicinum. Plant Physiol. 74:917-922. Oliver M. J., and J. D. Bewley. 1984c. Plant desiccation and protein synthesis, VI: changes in protein synthesis elicited by desiccation of the moss Tortula ruralis are effected at the translational level. Plant Physiol. 74:923-927 Oliver, M. J., J. Armstrong, and J. D. Bewley. 1993. Desiccation and the control of expression of l3-phaseolin in transgenic tobacco seeds. J. Exp. Bot. 44:1239-1244 Ooms, J. J. J., K. M. Leon-Klossterziel, D. Bartels, M. Koornneef, and C. M. Karssen. 1993. Acquisition of desiccation tolerance and longevity in seeds of Arabidopsis thaliana. A comparative study using abscisic acid-insensitive abi3 mutants. Plant Physiol. 102:1185-1191. Osborne, D. J. 1983. Biochemical control of systems operating in the early hours of germination. Can. J. Bot. 61:3568-3577. Osborne, D. J., and 1. 1. Boubriak. 1994. DNA and desiccation tolerance. Seed Sci. Res. 4:175-185. Peat, P., and M. Potts. 1987. The ultrastructure of immobilized desiccated cells of the cyanobacterium Nostoc commune UTEX 584. FEMS Microbiol. Lett. 43:233227. Peat, P., N. Powell, and M. Potts. 1988. Ultrastructural analysis of the rehydration of desiccated Nostoc commune Hun (cyanobacteria) with particular reference to the immunolabeling of NifH. Protoplasma 146:72-80. Piatkowski, D., K Schneider, F. Salamini, and D. Bartels. 1990. Characterization of five abscisic acid-responsive eDNA clones from the desiccation-tolerant plant Craterostigma plantagineum and their relationship to other water-stress genes. Plant Physiol. 94:1682-1688. PIa, M., A. Goday, J. Vilardell, J. Gomez, and M. Pages. 1989. Differential regulation of ABA-induced 23-25 kDa proteins in embryo and vegetative tissues of the viviparous mutants of maize. Plant Mol. BioI. 13:385-394. PIa, M., J. Gomez, A. Goday, and M. Pages. 1991. Regulation of the abscisic acidresponsive gene rab28 in maize viviparous mutants. Mol. Gen. Genet. 230:394400. Platt, K. A., M. J. Oliver, and W. W. Thomson. 1994. Membranes and organelles of dehydrated Selaginella and Tortula retain their normal configuration and structural integrity: freeze fracture evidence. Protoplasma 178:57-65. Potts, M. 1994. Desiccation tolerance of prokaryotes. Microbial. Rev. 58:755-805. Priestley, D. A. 1986. Seed aging: implications for seed storage and persistence in the soil. Comstock, Ithaca, NY. Ramanathan, V. 1988. The greenhouse theory of climate change: a test by an inadvertant global experiment. Science 240:293-299. Reynolds, T. 1. 1992. Strategies for survival in the desiccation-tolerant fern Polypodium virginianum. Thesis, Univ. Guelph, Canada. Reynolds, T. L., and J. D. Bewley. 1993a. Characterization of protein synthetic changes in a desiccation-tolerant fern, Polyp odium virginianum: comparison of the effects of drying, rehydration and abscisic acid. J. Exp. Bot. 44:921-928. Reynolds, T. L., and J. D. Bewley. 1993b. Abscisic acid enhances the ability of the desiccation-tolerant fern Polypodium virginianum to withstand drying. J. Exp. Bot. 44:1771-1779.

212

M.

J. OLIVER AND J. D. BEWLEY

Roberts, J. K., N .A. DeSimone, W. L. Lingle, and L. Dure III. 1993. Cellular concentrations and uniformity of cell-type accumulation of two LEA proteins in cotton embryos. Plant Cell 5:769-780. Sargent, J. A., S. Sen Mandi, and D. J. Osborne. 1981. The loss of desiccation tolerance during germination: an ultrastructural and biochemical approach. Protoplasma 105:225-239. Scherer, S., and M. Potts. 1989. Novel water stress protein from a desiccation-tolerant cyanobacterium. J. BioI. Chem. 264:12546-12553. Schneider, K., B. Wells, E. Schmelzer, F. Salamini, and D. Bartels. 1993. Desiccation leads to the rapid accumulation of both cytosolic and chloroplastic proteins in the resurrection plant Craterostigma plantagineum Hoscht. Planta 189:120-131. Schonbeck, M. W., and J. D. Bewley. 1981a. Responses of the moss Tortula ruralis to desiccation treatments, L: effects of minimum water content and rates of dehydration and rehydration. Can. J. Bot. 59:2698-2706. Schonbeck, M. W., and J. D. Bewley. 1981b. Responses ofthe moss Tortula ruralis to desiccation treatments, II: variations in desiccation tolerance. Can. J. Bot. 59:27072712. Scott, H. B., II, and M. J. Oliver. 1994. Accumulation and polysomal recruitment of transcripts in response to desiccation and rehydration of the moss Tortula ruralis. J. Expt Bot. 45:577-583. Seel, W. K, G. A. F. Hendry, and J. K Lee. 1992a. Effects of desiccation on some activated oxygen processing enzymes and anti-oxidants in mosses. J. Exp. Bot. 43:1031-1037. Seel, W. E., G. A. F. Hendry, and J. K Lee. 1992b. The combined effects of desiccation and irradiance on mosses from xeric and hydric habitats. J. Exp. Bot. 43:10231030. Senaratna, T., B. D. McKersie, and S. R. Bowley. 1990. Artificial seeds of alfalfa: indication of desiccation tolerance in somatic embryos. In Vitro Cell Dev. BioI. 26:85-90. Sgherri, C. L. M., B. Loggini, S. Puliga, and P. Navari-Izzo. 1994. Antioxidant system in Sporobolus staphianus: changes in response to desiccation and rehydration. Phytochemistry. 35:561-565. Siebert, G., J. Loris, B. Zollner, B. Frenzel, and R. K. Zahn. 1976 The conservation of poly(A) containing RNA during the dormant state of the moss Polytrichum commune. Nucleic Acids Res. 3:1997-2003. Simon, K W. 1974. Phospholipids and plant membrane permeability. New Phytol. 73:377-420. Simon, E. W. 1978. Membranes in dry and imbibing seeds. p. 205-224. In: J. H. Crowe and J. S. Clegg (eds.), Dry biological systems. Academic, New York. Simon, K W., and L. K. Mills. 1983. Imbibition, leakage, and membranes. p. 9-27. In: C. Nozzolillo, P. J. Lee, and F. A. Loewus (eds.), Mobilization of reserves in germination. Plenum, New York. Skriver, K., and J. Mundy. 1990 Gene expression in response to abscisic acid and osmotic stress. Plant Cell 2:503-512. Smirnoff, N. 1992. The carbohydrates of bryophytes in relation to desiccation tolerance. J. Bryol. 17:185-191. Smirnoff, N. 1993. The role of active oxygen in the response of plants to water deficit and desiccation. Tansley Review No 52. New Phytol. 125:27-58. Smith, L K., A. Polle, and H. Rennenberg. 1990. Glutathione. p. 201-215. In: R. G. Alscher and J. R. Cummings (eds.), Stress responses in plants: adaptation and acclimation mechanisms. Wiley-Liss New York.

3.

DESICCATION-TOLERANCE OF PLANT TISSUES

213

Stewart, G. R., and J. A. Lee. 1972. Desiccation-injury in mosses, II: the effect of moisture stress on enzyme levels. New Phytol. 71:461-466. Stewart, R. R. C., and J. D. Bewley. 1982. Stability and synthesis of phospholipids during desiccation and rehydration of a desiccation-tolerant and a desiccationintolerant moss. Plant Physiol. 69:724-727. Strauss, G., and H. Hauser. 1986. Stabilization of small uni-lamellar phospholipid vesicles by sucrose during freezing and dehydration. p. 318-326. In: A. C. Leopold (ed.), Membranes, metabolism and dry organisms. Cornell Univ. Press. Ithaca, NY. Thanos, C. A., K. Georghiou, and H. C. Passam. 1989. Osmoconditioning and ageing of pepper seeds during storage. Ann. Bot. 63:65-69. Tuba, Z., H. K. Lichtenthaler, Z. Csintalan, and T. pocs. 1993a. Regreening of desiccated leaves of the poikilochlorophyllous Xerophyta scabrida on rehydration. J. Plant Physiol. 142:103-108. Tuba, Z., H. K. Lichtenthaler, 1. Maroti, and Z. Csintalan. 1993b. Resynthesis of thylakoids and functional chloroplasts in the desiccated leaves of the poikilochlorophyUous plant Xerophyta scabrida on rehydration. J. Plant Physiol. 142:742-748. Vertucci, C. W. 1990. Calorimetric studies on the state of water in seed tissues. Biophys. J. 58:1463-1471. Vertucci, C. W. 1993. Towards a unified hypothesis of seed aging. p. 739-746. In: D Come and F Corbineau (eds.),Vol. 3. Fourth International Workshop on Seeds. Basic and Applied Aspects of Seed Biology. ASFIA, Paris. Vertucci, C. W., and J. M. Farrant. 1995. Aquisition and loss of desiccation-tolerance. p. 237-271. In: J. Kigel, and G. Galili (eds.), Seed development and germination. Dekker, New York. Welbaum, G. E., and K. J. Bradford. 1989. Water relations of seed development and germination in muskmelon (Cucumis melo 1.), II: development of germinability, vigor and desiccation tolerance. J. Exp. Bot. 40:1355-1362. Wellington, P. S. 1956. Studies on the germination of cereals, I: the germination of wheat grains in the ear during development, ripening and after-ripening. Ann. Bot. 20:105-120. Werner, 0., R. M. R. Espin, M. Bopp, and R. Atzorn. 1991. Abscisic-acid-induced drought tolerance in Funaria hygrometrica Hedw. Planta 186:99-103. Whitton, B. A. 1992. Diversity, ecology, and taxonomy of the cyanobacteria. p. 151. In: N.H. Mann and N.G. Carr (eds.), Photosynthetic prokaryotes. Plenum, New York. Willis, A. J. 1964. Investigations on the physiological ecology of Tortula ruraliformis. Trans. Br. Bryol. Soc. 4:668-683. Xu, N., and J. D. Bewley. 1994. Desiccation and the switch from seed development to germination: alfalfa embryos can synthesize storage proteins after germination if maturation drying is prevented. Seed Sci. Res. 4:247-255. Yamaguchi-Shinosaki, K., and K. Shinozaki. 1993. Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol. Gen. Genet. 236:331-340. Yamaguchi-Shinosaki, K., M. Koizumi, S. Urao, and K. Shinozaki. 1992. Molecular cloning and characterization of 9 cDNAs for genes that are responsive to desiccation in Arabidopsis thaliana: sequence analysis of one eDNA clone that encodes a putative transmembrane channel protein. Plant Cell Physiol. 33:217-224.

4 Physiology of Light Tolerance in Plants* Barbara Demmig-Adams, William and Stephen C. Grace Department of Environmental, Population, and Organismic Biology University of Colorado Boulder, Colorado 80309-0334, USA

I.

II.

III.

w: Adams III,

Introduction Processes Involved in Leaf Acclimation A. Photosynthetic Acclimation B. Optimizing Light Interception C. Photoprotective Dissipation of Excess Absorbed Light D. Detoxification of Activated Oxygen Role of the Xanthophyll Cycle in Photoprotective Energy Dissipation A. Diurnal Operation of the Xanthophyll Cycle B. Current Views on Mechanistic Aspects of Xanthophyll Cycle-Dependent Dissipation C. Synthesis, Localization, and Distribution Among Taxa D. Acclimation of the Xanthophyll Cycle 1. From Deep Shade to Sunflecks to Full Sunlight 2. Response to Environmental Stresses 3. The Dynamic Range: From Responses Within Seconds to Seasonal Acclimation E. Photo inhibition and the Xanthophyll Cycle

*Support for our work has been provided by Fellowships from the David and Lucile Packard Foundation to B. Demmig-Adams and from the National Science Foundation to S.C. Grace (Grant BIR-9403968) as well as grants from the United States Department of Agriculture, Competitive Research Grant Office (Grants 90-371305422 and 94-37100-0291) and the National Science Foundation (Grant IBN-9207653). We also thank our colleague Thomas Lemieux for his comments on the manuscript.

Horticultural Reviews, Volume 18, Edited by Jules Janick ISBN 0-471-57334-5 © 1997 John Wiley & Sons, Inc. 215

216

IV.

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

Concluding Remarks Literature Cited

I. INTRODUCTION This review summarizes recent major advances in the understanding of the physiological processes contributing to light tolerance. Current research is characterizing the tremendous phenotypic plasticity of plants to adjust their photosynthetic performance as well as the capacity for photoprotection to stresses in their native environments to which they are genetically well adapted. Future research will have to address the question of whether genetic manipulation of the levels of the enzymes involved in photoprotective processes can enhance the tolerance of certain species to stresses to which they are less well adapted. Photosynthesis is the process responsible for primary productivity on this planet over an extremely wide range of different light environments. For certain portions of each day, the level of solar radiation (PFD = photon flux density) absorbed by terrestrial plants is limiting to their photosynthesis rates, that is, during morning and evening hours for plants growing in open sites, and for major portions of the day for those leaves subject to shading from direct sunlight. In these situations the efficiency of the conversion of solar energy into photochemistry is high. At some time during the day, however, most leaves become exposed to excess PFD levels that necessitate the employment of photoprotective processes to prevent damage to the photosynthetic apparatus. Several key processes contribute to this protection of photosynthesis (for previous reviews see Bjorkman and DemmigAdams [1994] and Demmig-Adams and Adams [1992a]). Leaves can reduce the absorption of light at the chloroplast level by various mechanisms. In addition, excess absorbed light can be dissipated directly in the light-collecting pigment complexes of the chloroplasts (for reviews see Demmig-Adams [1990], Demmig-Adams et al. [1996], and Horton et al. [1994]). Furthermore, antioxidants can detoxify products that may result from excess excitation energy in the chloroplast (for reviews see Foyer and Mullineaux [1994], Alscher and Hess [1993], and Asada and Takahashi [1987]). One of the most fascinating aspects of this response of leaves to changes in the light environment is the rapid flexibility with which the photosynthetic apparatus can alter its functioning to allow a leaf

4.

PHYSIOLOGY OF LIGHT TOLERANCE IN PLANTS

217

growing in a sun-exposed site to switch from efficient collection and processing of light through photosynthesis in the early morning hours to efficient removal of the excess energy during peak exposure. A more gradual adjustment of the photosynthetic apparatus occurs in response to seasonal changes in peak irradiance levels as well as in response to environmental stresses such as drought or unfavorable temperatures. II. PROCESSES INVOLVED IN LEAF ACCLIMATION

A. Photosynthetic Acclimation Leaves that develop under different growth light environments display differences in their capacity for photosynthesis. An increased availability of light often leads to an increased maximal capacity for carbon fixation (reviewed in Bjorkman [1981]; see also Fig. 4.1). This allows the leaf to take advantage of the increased growth PFD by utilizing as much light as possible for photosynthesis. This acclimation involves increased capacities for photosynthetic electron transport, ATP synthesis, and carbon fixation (reviewed in Anderson et al. [1988]). However, no existing plant, including rapidly photosynthesizing crop species, can increase its photosynthetic capacity sufficiently to utilize all of full sunlight in the photosynthetic process (Bjorkman and Demmig-Adams 1994 and; Demmig-Adams et al. 1995).

Species tolerant of full sun differ from species tolerant of deep shade in their ability to undergo changes in photosynthetic capacity in response to extremes in the growth PFD, either deep shade or full sunlight. Species with long-lived and often sclerophytic leaves, such as many rainforest species that are also used as house plants, can typically decrease their overall metabolic rates enough to achieve a light compensation point (the PFD where photosynthesis rates and respiration rates are equal) low enough to maintain a positive carbon balance even in deep shade (Bjorkman 1981). On the other hand, rapidly growing mesophytes, such as many sun-tolerant annual species that grow poorly in the shade, exhibit considerably higher maximal photosynthetic rates than more slowly growing perennials, even when both types of species are growing side-by-side in full sun exposure (see below). When high PFDs occur in combination with other stresses, the maximal capacities of photosynthesis often decline. Stress factors

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

218

Horizontal PFD Changes in light interception Lightharvesting antennae

?

,.- -\\ - -

+

I

/

,.$

--

Excitation energy utilized in photochemistry

Antioxidants )

$

Dissipation of excess excitation energy

J

.-/..--?_' - .-_-.. . . Carbon fixation

Fig. 4.1. Schematic depiction of the key factors affecting the allocation of photons to carbon fixation versus other processes. All circled processes are subject to acclimation to growth PFD such that the activities, concentration, or response of compounds or processes involved can increase with increased growth PFD. The fraction of photons arriving on a horizontal plane that becomes absorbed in the light-harvesting antennae of chloroplasts can be regulated by changes in light interception. The fraction of absorbed photons,that is, excitation energy, that is delivered to the photochemical reactions can be regulated by the harmless dissipation of excess excitation energy in the antennae. This dissipation process counteracts the formation of singlet excited oxygen in the pigment complexes. Products of photosynthetic electron transport are consumed in carbon fixation as well as other processes (not shown). A certain unknown fraction of electrons may be consumed in the reduction of oxygen to potentially toxic products (superoxide, hydrogen peroxide). The antioxidant systems of the chloroplast can detoxify both types of activated oxygen species that may potentially be generated.

such as limiting nitrogen supply in the soil lead to decreases in the capacity for photosynthesis (Field and Mooney 1986; Evans 1989). Water stress can have similar effects (Bjorkman and Demmig-Adams 1994). Under such combinations of stress factors, photoprotective mechanisms are of enhanced importance (reviewed in DemmigAdams and Adams [1992a]) since the utilization of light decreases, thereby making the same incident PFD more excessive (see also Section III.D.2).

4.

PHYSIOLOGY OF LIGHT TOLERANCE IN PLANTS

219

B. Optimizing Light Interception Fully 85 to 95 % of the light striking the leaf surface can be absorbed in the chloroplasts of leaves. Losses in the form of reflected or transmitted light are minimized in shade environments to optimize light utilization in photosynthesis. Leaves in deep shade are also typically oriented horizontally, or facing the solar beam, for maximal light interception. In contrast, when leaves are exposed to excessive PFDs, the fraction of incident light that is reflected can be increased. Plants may also orient their leaves parallel to the solar beam for minimal light interception, thus avoiding absorption of excess light and heat. One can categorize (see Bjorkman and Demmig-Adams [1994]) these various means of regulating light interception into (1) leaf angle changes involving either rapid leaf movements, (heliotropism) (Bjorkman and Powles 1981; Koller 1990), or slow developmental responses resulting in the growth of leaves at an optimal angle, (2) chloroplast movements (Haupt and Scheuerlein 1990), and (3) changes in leaf optical properties. Changes in leaf optical properties occur mostly through increases in the fraction of incident light that is reflected by the leaf surface, by virtue of increased deposition of cuticular waxes or, in some cases, increased pubescence (Ehleringer and Bjorkman 1978) or salt deposition (Mooney et al. 1977). Leaf angle changes are probably the most important means of changing light interception (Bjorkman and Demmig-Adams 1994). While developmental changes in leaf angle are less conspicuous and less flexible, they can occur in a wide variety of species (Ehleringer 1988). The very rapid, active leaf movements to evade excessive light are taxonomically restricted to plant species possessing pulvinar tissues, such as those found in the Leguminosae (Fabaceae) (Koller and Shak 1990) or Oxalidaceae (Bjorkman and Powles 1981). These species are typically capable of performing both light-tracking (diaheliotropic) and light-evasive (paraheliotropic) leaf movements. Mechanisms of regulating light interception can become prominent when high PFDs occur in combination with additional environmental stress factors. These stresses include insufficient water availability (Ehleringer 1982), insufficient nitrogen availability (Kao and Forseth 1992), and high temperatures (Ludlow and Bjorkman 1984). The various means of regulating light interception also regulate the heat load of leaves under high PFDs and have typically been thought of as means of preventing overheating of the leaves as much as regulating the absorption of photosynthetically active radiation. Two biochemical mechanisms are discussed in the following two sections that do not alter the overall heat load of leaves and protect

220

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

only against an excess of absorbed light. These mechanisms are ubiquitous among all higher plants and are employed irrespective oftaxonomic group. C. Photoprotective Dissipation of Excess Absorbed Light When low PFDs limit the rate of photosynthesis, virtually all excitation energy is utilized in photosynthetic electron transport (Bjorkman and Demmig 1987). Under these conditions any dissipation of excitation energy via alternative pathways would be undesirable and does not occur (Fig. 4.1). In contrast, under high PFDs not all of the absorbed excitation energy can be utilized in the Calvin cycle. As a consequence, electrons could become "backlogged" in the electron transport chain and result in the formation of toxic activated oxygen species directly in the light-collecting pigment complexes (singlet oxygen formation) and/or as a result of oxygen reduction by the electron transport chain to superoxide and hydrogen peroxide (As ada and Takahashi 1987). However, a photoprotective dissipation process safely removes excess excitation energy directly where it first arises in the light-harvesting antennae, thereby counteracting the formation of toxic activated oxygen species (Fig. 4.1). This process depends on two factors. One is the presence of the de-epoxidized carotenoids zeaxanthin and antheraxanthin that are invariably formed under excess light via the xanthophyll cycle, which is present in the thylakoid membranes of higher plants (Yamamoto 1979; Demmig-Adams and Adams 1993a,b; Demmig-Adams et al. 1996). The second factor is the magnitude of the pH gradient across the thylakoid membrane, which increases under excess light (Gilmore and Yamamoto 1993a,b; for reviews see Horton et al. 1994; DemmigAdams et al. 1996). Details of the molecular mechanism for energy dissipation, dependent on the xanthophyll cycle as well as L\pH, are discussed in Section lILB. For the many species of higher plants that do not perform lightevasive leaf movements, the level of incident PFD increases and decreases dramatically over the course of a clear day in a sun-exposed location (Fig. 4.2). Leaves of species with high photosynthetic capacities, such as sunflower, can utilize up to 40-50% of the absorbed light at midday (Fig. 4.2A). In contrast, leaves of the evergreen perennial Vinca major utilize only 10-20% of the absorbed light at midday (Fig. 4.2B). The difference between the absorbed PFD and that utilized for photosystem II (PSlI) photochemistry is defined as excess PFD, and is greater for the Vinca leaf compared to the sunflower leaf (Fig. 4.2A,B).

4.

PHYSIOLOGY OF LIGHT TOLERANCE IN PLANTS

221

Analysis of chlorophyll fluorescence characteristics is a commonly employed approach to estimate the activity of photoprotective energy dissipation (Krause and Weis 1991; Schreiber et a1. 1994; Demmig-Adams and Adams 1996a). Since energy dissipation is enhanced under environmental stresses, fluorescence measurements have also become a tool in stress detection. Fluorescence is the reemission of a very small percentage of the light absorbed by chlorophyll. Activation of the photoprotective energy dissipation process leads to a characteristic decrease in fluorescence emission that is termed nonphotochemical fluorescence quenching (NPQ; Fig. 4.2C,D). The sunflower leaf that utilized a greater fraction of the absorbed light in photosynthesis, and thus experienced a lesser degree of excess PFD, also exhibited a smaller increase in NPQ than the Vinca leaf (Fig. 4.2C,D). In contrast, the Vinca leaf reached very high levels of NPQ. This indicates that the Vinca leaf was able to compensate for its lower photosynthetic capacity by a greater dissipation of excitation energy in the antennae. To quantify how complete this compensation is in sun-acclimated leaves, we have recently taken a modified approach to estimate the percentage of absorbed light utilized in photosynthesis versus that dissipated in the antennae (Demmig-Adams and Adams 1996a). Increases in NPQ are accompanied by decreases in the efficiency of PSII. This is an expected consequence of energy dissipation in the antennae: Removal of excitation energy before it reaches the PSII reaction centers will lead to a decrease in the efficiency of the conversion of absorbed light into photochemistry (Kitajima and Butler 1975). The increases in NPQ observed under natural conditions (Demmig-Adams and Adams 1996a; Demmig-Adams et al. 1995) were of a sufficient magnitude to explain all observed decreases in intrinsic PSII efficiency. These changes thus reflect regulatory decreases in the efficiency with which excitation energy is delivered to the photochemical reactions under excess PFD and probably do not limit photosynthetic productivity. The percentage of absorbed light dissipated in the antennae (D) can be estimated from these changes in PSII efficiency for different time points over the course of the day (Fig. 4.2G,H). At midday, about 50% of the absorbed light was dissipated in the antennae in the sunflower leaf, whereas dissipation of about 75% was observed in the Vinca leaf. The sum of the two processes, photosynthetic electron transport and energy dissipation in the antennae, was thus able to account for nearly all of the absorbed light. This indicates that the compensation for light not utilized in photosynthesis by virtue of energy dissipation in the antennae is virtually complete. From these

222

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

percentages, the actual rates of photosynthetic electron flow and energy dissipation in the antennae can also be estimated (Fig. 4.2I,J). The flux of energy being dissipated was again higher for the photosynthetically less active Vinca leaf than for the very active sunflower leaf. D. Detoxification of Activated Oxygen

The chloroplast possesses an array of scavenger systems that can detoxify activated oxygen species (reviewed in Alscher and Hess [1993] and Foyer and Mullineaux [1994]). While some of these scavengers have the potential to deactivate the electronically excited form of oxygen (singlet oxygen) generated in the pigment bed, the main scavenging system is employed in the removal of the reduced forms of activated oxygen. Diversion of electrons from the electron transFig. 4.2. Diurnal changes in incident, absorbed, and excess PFD as well as the rate of photochemistry (A, B) on a clear summer day in sun-exposed leaves of sunflower (Helianthus annuus 1. cv. Taiyo) and Vinca major L. plants growing outdoors in the ground. Based on an assumed absorptance of 0.85, absorbed PFD was estimated as 0.85 x incident PFD. Excess PFD was calculated as absorbed PFD minus the rate of photochemistry (in .umol photons/m 2 s). The rate of photochemistry (PC rate) was estimated from [(Fm'- F}IFJ x absorbed PFD (Genty et al. 1989) under the assumption that the rate of PSI photochemistry matches that of PSII. Changes in NPQ (C, D) are expressed as changes in Stern-Volmer quenching = Fm /F";-l (Bilger and Bjorkman 1990; Demmig-Adams 1990). Changes in the intrinsic efficiency of PSII (E, F) were estimated from Fv'/F"; (Le., the ratio of variable to maximal fluorescence emission at open PSII centers during actual illumination; for nomenclature see van Kooten and Snel [1990]). The allocation of absorbed PFD (G, H) to energy dissipation in the antennae (D; hatched areas) was estimated from D = 1 - F' IF' (see Demmig-Adams and Adams 1996a; Demmig-Adams et al. 1996). Dashellin~s = 1 - (FvFm at predawn). (The remaining fraction can be divided into that utilized in photosynthesis (P; dotted areas) versus that not going into either photosynthesis or energy dissipation in the antennae, which is thus in excess (white areas). The fraction utilized in photosynthetic electron transport was estimated from the expression (Fm' F)/ F"; (Genty et al. [1989], where F is the actual fluorescence emission during illumination), that is obtained by rearrangement of (Fv'/F,:J qp (where qp is the quenching coefficient of photochemical quenching; see Schreiber et al. [1994]). The third fraction of light, that is, the excess absorbed light that is neither utilized through photosynthesis nor dissipated in the antennae, was estimated from (Fv' /Fm') (1 qp). Also shown (1, J) are the rates of energy dissipation in the antennae estimated from (1 Fv'IF";) x absorbed PFD, in analogy to the estimation of the rates of photochemistry shown in A, B, again assuming a matching behavior of PSII and PSI. All fluorescence measurements were performed in situ directly in the field with the portable PAM-2000 chlorophyll fluorometer (Walz, Effeltrich, Germany). (Data from DemmigAdams et al. 1997).

Vinca major, sun

Sunflower, sun _r-

2000

.....00..· O~


~~c

-0 .- 0

cEo 1000 <'O
5

~

°E


°

°

°

4 3

2

0

Z

.......t----J 1.0 r - - - - - - - - - - - - - - , , . . . . - - - - - - - - - - - - . , 0L..J....~c....£...J.~~~.£.."'-c....£...J.w:...:~

~~ ~ E

.
::::»

o.s

.- 0

~.c

PSII efficiency

05~~ -

.;:: 0..

Co

......

..... .c

o

D>

en=

o

F

E I

I

I

~.".".""'

I

I

~".".'.'''

1.00 G·~:.···"·.·.·".,,, . . . . ' .. '. ..:'.'''.• '. 0.75

~ ~ .........~.: .. : H~

C-o

.2
LL..o <'0

c

0.50 0.25 0 1800

J

0


~ 0:

Rate of dissipation in antennae

300 0

4

8

12

16 20 4 8 Time of day, hours

12

16

20

224

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

port chain to oxygen leads to the formation of the reduced oxygen species superoxide and hydrogen peroxide. Superoxide is also converted to hydrogen peroxide by the enzyme superoxide dismutase (SOD), followed by the reductive conversion of hydrogen peroxide to water by the enzyme ascorbate peroxidase (APX) where ascorbate acts as the reductant. Reduced ascorbate is regenerated either enzymatically by a series of reactions in the chloroplast stroma (Asada and Takahashi 1987) or by direct reduction of the ascorbate radical by the photosynthetic electron transport chain (Grace et al. 1995). Figure 4.3 shows that increasing growth PFDs leads to an acclimation in the capacity for this pathway, involving increases in the pool size of ascorbate as well as increases in the activities of SOD and APX. It has also been speculated that differences in stress tolerance between closely related plant species or cultivars may be related to differences in their scavenger levels (Bowler et al. 1992). In addition, there have been reports that genetically altered plant lines with higher levels of, for example, SOD possess a greater tolerance to certain often rather unphysiological stress treatments (for reviews see, e.g. Bowler et al. [1992] and Allen [1995]). Clearly, more work is needed, where genetically altered plant lines that either overexpress or underexpress the enzymes involved in scavenging are grown over an entire growth period in the field to evaluate whether there is any effect on productivity. Such studies also need to consider the effect of changes in the level of one enzyme in the detoxification pathway on other enzymes in this pathway (e.g., Sen Gupta et al. 1993) as well as the interaction between scavenging and energy dissipation in the antennae. Initially, detoxification of superoxide and xanthophyll cycle-dependent energy dissipation in the antennae were thought of as independent processes that each contributes toward the protection of photosynthesis. More recently, several interactions between these two processes have been investigated. First, ascorbate is not only the reductant assisting in the enzymatic detoxification of hydrogen peroxide as well as in the direct reduction of superoxide, but it is also the reductant in the enzymatic and reductive conversions in the xanthophyll cycle (the diepoxide violaxanthin is reduced to the monoepoxide antheraxanthin and the epoxide-free zeaxanthin) (Yamamoto 1979; Neubauer and Yamamoto 1992). Increases in the pool size of ascorbate closely match increases in the pool size of the xanthophyll cycle with increasing growth PFD (Logan et al. 1996). Furthermore, under conditions where Calvin cycle activity is very

4.

PHYSIOLOGY OF LIGHT TOLERANCE IN PLANTS ..-,

3.0...-----------~

su..

2.5

0> 0>

2.0

E

(]) +-'

co

225

A

1.5

..0

1.0

U

0.5

lo-

o

CJ)

~

o 1200 1000

B

800 600 400 200

o (]) CJ)

co..-

:'Q

'lo-

x..c

3000 2500

0"-

Q)~ 2000

Q.u..

20> co

og:::t.E

..0

~

0

1500 1000 500

o

Low

Med.

High

Growth PFD Fig. 4.3. Effect of growth PFD on (A) the levels of reduced ascorbate, (B) the activities of superoxide dismutase (SOD), and (C) ascorbate peroxidase (APX) in leaves of Vinca major 1. growing at low (15/lmol photons/m z s), medium (85 /lmol photons/ m Z s), and high (880 /lmol photons/mz s) growth PFD. These PFDs represent the PFDs incident on the leaves chosen for analysis. Plants were growing in growth chambers under a 12-h lightl12-h dark photoperiod. Air temperatures were regulated to achieve leaf temperatures of approximately 24°C during the day and 18°C at night; relative humidity was maintained at approximately 75%. Duplicate determinations were made on two separate extractions for each growth PFD. Error bars = SE. (S. Grace, unpublished data.)

226

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

low, electron flow to oxygen and the ascorbate radical may generate a minimal rate of electron transport. This could contribute toward generating a sufficiently high pH-gradient to allow xanthophyll cycledependent energy dissipation in the antennae (Neubauer and Yamamoto 1992; Schreiber et al. 1994). III. ROLE OF THE XANTHOPHYLL CYCLE IN PHOTOPROTECTIVE ENERGY DISSIPATION

A. Diurnal Operation of the Xanthophyll Cycle Leaves in their natural environment undergo pronounced changes in the composition of the xanthophyll cycle over the course of a day (Fig. 4.4; see also Adams and Demmig-Adams [1992] and Bjorkman and Demmig-Adams [1994]). In the early morning hours leaves typically possess a large amount of violaxanthin (V). As the day progresses V is converted to antheraxanthin and zeaxanthin (A + Z) and subsequently reconverted to V as the flux of solar radiation in-

Helianthus

N

«+ + G ........ (J)

>.

£ CL

Euonymus

1.0 0.8 0.6 0.4

0

£

C 0.2 a:l

X

0

6:00

12:00

18:00

6:00

12:00

18:00

Time of day, hours Fig. 4.4. Diurnal changes in the xanthophyll cycle composition in leaves of sunflower and Euonymus kiautschovicus Loessner on a clear summer day. The plants were growing outdoors in the ground. A, antheraxanthin; V, violaxanthin; Z, zeaxanthin. Carotenoid determinations were made by HPLC (Gilmore and Yamamoto 1991) as described by Adams and Demmig-Adams (1992). (Data recalculated from Adams et al. (1992) and unpublished data by B. Demmig-Adams, W. Adams, D. Barker, B. Logan, D. Bowling, and A. Verhoeven.)

4.

PHYSIOLOGY OF LIGHT TOLERANCE IN PLANTS

227

creases and decreases over the course of the day. These conversions follow the changes in excess PFD (cf. Fig. 4.2). While Fig. 4.4 illustrates the pattern found in south-facing leaves that receive peak PFDs at midday, east- or west-facing leaves show peak levels of A + Z in the morning or afternoon, respectively (Adams et a1. 1992). The maximal levels of A + Z vary among species and match the levels of NPQ. The highly photosynthetically active sunflower leaf displayed moderately high levels of energy dissipation (Fig. 4.3C) as well as A + Z (Fig. 4.4). A leaf of Euonymus kiautschovicus with similarly low rates of photosynthesis as Vinca showed very high levels of both NPQ (not shown) and A + Z (Fig. 4.4). This trend has been observed among all species examined in their natural habitat. High maximal rates of photosynthesis are associated with a moderately high percentage of absorbed light dissipated in the antennae and a lesser fraction of the xanthophyll cycle pool present as A + Z at peak irradiance (Fig. 4.5). On the other hand, low maximal rates of photosynthesis are matched by high percentages of absorbed light dissipated in the antennae and high maximal conversion of the xanthophyll cycle to A + Z (Fig. 4.6). The same pattern was found in a survey that included 24 different species and varieties of higher plants with widely different maximal photosynthesis rates (Fig. 4.7). This analysis included trees, shrubs, and herbs representing a wide variety of lifeforms, taxa, and leaf anatomical properties. In all species examined, the percentage of absorbed light dissipated in the antennae by virtue of the xanthophyll cycle-dependent dissipation process increased uniformly with the degree of light stress. Furthermore, the response of leaves to excess PFD was the same regardless of whether a greater excess of excitation energy arose as a consequence of lower rates of photosynthetic utilization or as a consequence of increasing incident PFDs (Fig. 4.7). We conclude that these increases in xanthophyll cycle-dependent energy dissipation in response to excess light are species-independent and represent a fundamental and general property of the photosynthetic apparatus. B. Current Views on Mechanistic Aspects of Xanthophyll CycleDependent Dissipation

Early research on the energy dissipation processes demonstrated that an increase in energy dissipation is induced by acidification within the thylakoid membrane (Briantais et a1. 1979; see also Krause and Weis 1991). Subsequently, significant correlations were reported between NPQ and the levels of Z (Demmig et a1. 1987) or A + Z

228

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

Photosynthetically highly active species Allocation of absorbed light

Xanthophyll cycle composition

Helianthus

t

,

45% 62%

Malva 38% 56%

Convolvulus

46%

Fig. 4.5. Allocation of absorbed light to photosynthetic electron flow (P) versus energy dissipation in the antennae (D) and composition of the xanthophyll cycle at peak irradiance at noon (1500-2000 /-lmol photons/m 2 s) in three photosynthetically highly active species (Helianthus annuus, Malva neglecta, and Convolvulus arvensis) growing outdoors in the ground. P and D were determined from chlorophyll fluorescence measurements made in situ directly in the field as described in the legend to Fig. 4.2. For pigment analyses see the legend to Fig. 4.4. The white areas in the pies for allocation of absorbed light represent the excess light that is neither utilized through photosynthesis nor dissipated in the antennae. (Demmig-Adams and Adams 1996b)

4.

PHYSIOLOGY OF LIGHT TOLERANCE IN PLANTS

229

Photosynthetically less active species Allocation of absorbed light

Xanthophyll cycle composition

Mahonia

6%

Parthenocissus

20%

Magnolia

7%

Fig. 4.6. Allocation of absorbed light to photosynthetic electron flow (P) versus energy dissipation in the antennae (D) and composition of the xanthophyll cycle at peak irradiance at noon (1500-2000 !lmol photons/m 2 s) in three photosynthetically less active species (Mahonia rep ens, Parthenocissus quinquefolia, and Magnolia stellata). P and D were determined from chlorophyll fluorescence measurements made in situ directly in the field as described in the legend to Fig. 4.2. For pigment analyses see the legend to Fig. 4.4. The white areas in the pies for allocation of absorbed light represent the excess light that is neither utilized through photosynthesis nor dissipated in the antennae. (Demmig-Adams and Adams 1996b)

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

230

100 (])~

..c .......

-

(])

80

OU>. c ()

0-

:E"5(/)..c

00... 0...0

E:5 o c x

Oco

a 100 (])

co

..c c

.~c

80

(])

-0 .......

(]) c

..0 '-

co

C

0-(/)-0

60

co ....... _co

40

~.~

20

..o(])

0.9-0

10 20

30 40

50

60 70 80

% of absorbed light utilized in PSII photochemistry Fig. 4.7. Relationship between the percentage of absorbed light utilized in PSII photochemistry and the composition of the xanthophyll cycle (top) as well as the percentage of absorbed light dissipated in the antennae (bottom) in 24 different species and varieties of higher plants growing in sun-exposed sites in the ground on the Boulder campus of the University of Colorado during the summer of 1993. Open squares = 24 species or varieties of higher plants monitored at peak irradiance (15002000 /lmol photons/m 2 s) and exhibiting a wide range of different maximal rates of PSII photochemistry. These species included Acer saccharinum 1. [Silver Maple], Catalpa bignonioides Walt., Convolvulus arvensis 1. [Small Bindweed], Cotinus coggyria Scop. cv. Royal Purple [Smoke Tree], Cucurbita pepo L. cv. Autumn Gold [Pumpkin], Euonymus kiautschovicus Loesener, Helianthus annuus 1. cv. Taiyo [Sunflower], Magnolia stellata Maxim. [Star Magnolia]; Mahonia aquifolium (Pursh) Nuttall [Oregon Grape Holly]; Mahonia repens (Lindley) Don [Creeping Holly]; Malus sp. [Crabapple], Malva neglecta Wallr., Parthenocissus quinquefolia (L.) Planch. [Virginia Creeper], Plantago major 1. [Common Plantain], Quercus palustris Muenchh. [Pin Oak], Rosa sp. [Rose], Solanum melongena L. cv. Ichiban [Eggplant], S. melongena L. cv. Black Magic [Eggplant], Sonchus oleraceus 1., Spinacia oleracea

4.

PHYSIOLOGY OF LIGHT TOLERANCE IN PLANTS

231

(Gilmore and Yamamoto 1993a; Adams et al. 1995b; Demmig-Adams and Adams 1995). It has now been clearly established that the key process that dissipates excess energy under physiological conditions requires both A + Z and a low pH within the thylakoid membrane (Gilmore and Yamamoto 1992, 1993a,b, 1994; Bilger and Bjorkman 1994; Gilmore et al. 1995; Mohanty et al. 1995; for a review see Demmig-Adams et al. 1996). This dissipation process leads to the deexcitation of the excited singlet state of chlorophyll in the antennae (Demmig-Adams 1990; Horton et al. 1994; Demmig-Adams et al. 1996). Whether the actual mechanism is a pH-activated direct energy transfer from chlorophyll to A or Z (Frank et al. 1994) or an (A + Z)-activated deexcitation by protonation is currently unresolved (see Horton et al. 1994; Demmig-Adams et al. 1996; Gilmore et al. 1995; Owens 1996). A recent study by Gilmore et al. (1995) suggests a model whereby protonation induces structural changes allowing A + Z to form dissipating centers in specific antenna complexes. C. Synthesis, Localization, and Distribution Among Taxa The carotenoids of the xanthophyll cycle are synthesized from f3carotene, which becomes hydroxylated to zeaxanthin (reviewed in Demmig-Adams and Adams 1993a). Virtually all organisms performing aerobic photosynthesis have the capability to form zeaxanthin or, in some algal groups, an analogous xanthophyll, diatoxanthin (Hager 1980; Demmig-Adams and Adams 1993a; Arsalane et al. 1994). Thylakoid membranes of all higher plants possess a xanthophyll cycle consisting of the enzyme zeaxanthin epoxidase that adds one or two epoxy groups to zeaxanthin, forming antheraxanthin and violaxanthin, respectively, under limiting light conditions, and the enzyme violaxanthin deepoxidase, which removes these groups uncv. Giant Nobel [Spinach], Syringa vulgaris L. [Lilac], TWa cordata L. [Linden], Vinca minor L. [Periwinkle]' and Viburnum lantana L. [Wayfaring Tree]. Filled circles = 9 different leaves of Euonymus kiautschovicus with different leaf angles and exposed to widely different peak incident PFDs at midday. Of the 9 leaves, 8 exhibited similar rates of PSII photochemistry. A 10th leaf was a heavily self-shaded leaf from within the canopy that was exposed to very low PFDs and utilized 80% of the absorbed light in PSII photochemistry. The percentage of absorbed light utilized in PSII photochemistry was estimated from chlorophyll fluorescence measurements as (F: - F)/ (Genty et al. 1989) and the percentage of absorbed light dissipated in the antennae from 1 - F '/ F ' (Demmig-Adams and Adams 1995). For further details see the legend of Fig. 4.2. Fffior carotenoid analyses see legend of Fig. 4.4. (Data from Demmig-Adams and Adams [1996a].)

F:

232

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

der excess light (Yamamoto 1979; Demmig-Adams and Adams 1993a; pfiindel and Bilger 1994). It has been shown that the main pathway for the synthesis of the plant hormone abscisic acid (ABA) is from violaxanthin (Duckham et al. 1991; Rock and Zeevaart 1991). This is an interesting finding considering the fact that stresses such as high salinity and drought lead to an increased emphasis on the xanthophyll cycle carotenoids (Lovelock and Clough 1992; Demmig et al. 1988) as well as on ABA (Davies and Zhang 1991). Furthermore, one of the intermediates in the synthesis of ABA from violaxanthin is xanthoxin (Fransen and Bruinsma 1981), a substance that has been implicated as a hormone involved in slowing growth (a valuable response to stress when environmental factors might not permit continued growth unabated). Thus, this entire pathway seems to be upregulated in response to stress, with various intermediates playing crucial roles in maintaining the functional integrity of the plant. The carotenoids of the xanthophyll cycle are bound to the chlorophyll/carotenoid-binding, light-harvesting antenna complexes of photosystems II and I (Thayer and Bjorkman 1992). While they are present in all of these complexes, including the peripheral complexes (LHCs), they are enriched in the inner minor complexes (CPs) (Yamamoto and Bassi 1996). The CPs are also thought of as a key site for energy dissipation (Walters et al. 1994; Gilmore et al. 1995; Demmig-Adams et al. 1996). Mutants that are chlorophyll b-deficient, and do not possess the peripheral LHCs, typically exhibit increased ratios of the xanthophyll cycle components to chlorophyll or to other carotenoids (Falbel et al. 1994). Many of these "golden" mutants are grown as ornamentals such as 'Golden Privet' (Fig. 4.8). Particularly the younger leaves are quite yellow and contain extremely high levels of the xanthophyll cycle components. The older, greener leaves have a carotenoid composition that is more similar to normal leaves. While the xanthophyll cycle is apparently confined to photosynthetic organisms, the presence of zeaxanthin is not. Zeaxanthin and, in lesser quantities, lutein are also present in the human eye. Recent medical research has suggested a link between the presence of these carotenoids in the human diet and the prevention of age-related macular degeneration that can cause blindness (Seddon et al. 1994). This finding suggests a function of these xanthophylls in photoprotection of the eye as well. Optimization of the levels of these xanthophylls in the human diet may therefore become a target in the production of fruits and vegetables.

4.

PHYSIOLOGY OF LIGHT TOLERANCE IN PLANTS

233

Golden Privet, full sun Young, yellow leaf of top shoot

51%

3%

Mature, greener leaf of older shoot

35% Fig. 4.8. Carotenoid composition of young, yellow leaves as well as mature, greener leaves of 'Golden' privet (Ligustrum x vicaryi) growing in full sunlight during the summer of 1993 in Boulder, Colorado. The total area of the pie reflects the total carotenoid concentration on a chlorophyll basis (1.43 ± 0.30 and 0.62 ± 0.07 mol carotenoid/mol chlorophyll for yellow and greener leaves, repectively; n = 3). V + A + Z , violaxanthin + antheraxanthin + zeaxanthin; N, neoxanthin; L, lutein; j3C, 13carotene. The chlorophyll content was 63 ± 10 and 194 ± 8 I-lmol/m 2 for yellow and green leaves, respectively. The ratio of (A + Z)/(V + A + Z) was extremely high both in the yellow (0.98 ± 0.01) and greener leaves (0.95 ± 0.01). For details on the carotenoid analyses, see the legend to Fig. 4.4. (W. Adams and B. Demmig-Adams, unpublished data.)

234

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

D. Acclimation of the Xanthophyll Cycle 1. From Deep Shade to Sunflecks to Full Sunlight. Within a spe-

cies increasing growth PFD results in an increase in the pool size of the xanthophyll cycle relative to chlorophyll or other carotenoids (Fig. 4.9); that is, a greater demand for energy dissipation in the growth environment leads to a larger size of the pool of V + A + Z (Thayer and Bjorkman 1990; Demmig-Adams and Adams 1992b) and greater maximal levels of A + Z (Brugnoli et al. 1994; Demmig-Adams and Adams 1994; Demmig-Adams et al. 1995). The pool of V + A + Z exhibits by far the most pronounced percent increase among all carotenoids (Fig. 4.9). In an understory environment with multiple sunflecks (Fig. 4.10A), the levels of A + Z measured in Stephania japonica increased during the first minor sunflecks and remained high throughout the course of the day until after the last major sunflecks (Fig. 4.10B). Previous experimental transfers of leaves from low to high to low PFDs indicated that conversion of V to A + Z is very rapid and can be completed in only a few minutes, whereas the reconversion of A + Z to V upon a return to a low PFD takes considerably longer (Yamamoto

Deep shade

34%

Open shade + sunflecks

Full sun

35%

Fig. 4.9. Effect of light environment on the carotenoid composition of Stephania japonica (Thunb.) Miers var. discolor plants growing naturally in deep shade, open shade, and full sunlight at a site on the eastern coast of Australia. V + A + Z, violaxanthin + antheraxanthin + zeaxanthin; N, neoxanthin; L, lutein; I3C, l3-carotene; aC, a-carotene. The total areas of the pie reflect the total carotenoid concentration on a chlorophyll basis (46, 66, and 134 mol carotenoid/mol chlorophyll for deep shade, open shade, and full sun, respectively; means of 2-16 samples from two leaves each). For details on the carotenoid analyses, see the legend to Fig. 4.4. (W. Adams III, B. Demmig-Adams, B. Logan, and D. Barker, unpublished data.)

4.

PHYSIOLOGY OF LIGHT TOLERANCE IN PLANTS

235

1500 r - - - - - - - : - - - - - - - - - - - . . . : . . - - - - - - - .

A

o~

LL

a..

C

CD "0

CJ)

E 0

() E

C

Incident PFD

';J 1000

500

:::t 0'---_+----

N

«++

G

--N

0.8 r - - - - - - - - - - - - - - - - : = - - - - - - - - - , 0.6 0.4

+ 0.2

~

O'------J-----'---------JL.-_ _---I.

---I._---I.

3r------------------------.

c

aa..

2

z

0'------6 8

10

12

14

16

Time of day, hours Fig. 4.10. Diurnal changes in (A) incident PFD, (B) the levels of antheraxanthin and zeaxanthin (A + Z)/(V + A + Z), and (C) energy dissipation activity (NPQ) in leaves of Stephania japonica growing naturally in an understory environment and receiving repeated sunflecks over the course of a clear day. Actual carotenoid concentrations per ChI are reported in Fig. 4.9. NPQ was quantified as Fm/F;' - 1 from measurements of chlorophyll fluorescence with a portable PAM-2000 chlorophyll fluorometer. For further details, see the legend to Fig. 4.2. For details on the carotenoid analyses, see the legend to Fig. 4.4. (W. Adams, B. Demmig-Adams, B. Logan, and D. Barker, unpublished data.)

1979; Bilger and Bjorkman 1990). This pattern is confirmed in this natural environment with multiple transitions between extreme PFDs (Fig. 4.10B). The obvious question is whether the actual level of energy dissipation follows the rapid changes in PFD, as would be desirable, or the slow changes in A + Z. Figure 4.10C illustrates that

236

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

the former is the case, that changes in energy dissipation are extremely rapid and closely match the changes in PFD. This allows the leaf to quickly return to an efficient photosynthetic energy conversion upon return to PFDs that are limiting to photosynthesis while being protected under high PFD. In this scenario rapid changes in the pH gradient across the thylakoid membrane modulate the (A + Z)-dependent energy dissipation activity on a time scale of seconds to allow optimization of carbon gain. 2. Response to Environmental Stresses. When plants are subjected

to environmental stress factors that lower the rates of photosynthetic electron transport, the percentage of absorbed light utilized in photosynthesis decreases, resulting in even greater degrees of excess PFD. Under severe stress even a rather low incident PFD can represent a high degree of excess PFD. Increases in the pool size of the xanthophyll cycle and/or its maximal conversion state (Fig. 4.11) have been reported for nitrogen stress, that is, limiting nitrogen supply in the soil (Khamis et al. 1990; Demmig-Adams et al. 1995), decreased availability of iron (Morales et al. 1994), and low-temperature stress (Bilger and Bjorkman 1991; Adams and Demmig-Adams 1994, 1995; Adams et al. 1995a,b). Earlier studies that did not include xanthophyll cycle analyses nevertheless concluded that increased energy dissipation in the antennae can also occur under high salinity (Bjorkman et al. 1988; see also Lovelock and Clough 1992) or low water availability (Bjorkman 1987; see also Demmig et al. 1988). At present it is unknown whether overexpression of the enzymes involved in the xanthophyll cycle can improve the stress tolerance of certain species (see Section IV). 3. The Dynamic Range: From Responses Within Seconds to Seasonal Acclimation. Changes in the level of (A + Z)-dependent energy dissipation operate on a time scale of minutes to hours (Fig. 4.4) in sun leaves on a clear day without additional environmental stresses. These changes match the diurnal biochemical conversions of the xanthophyll cycle (see also Adams et al. 1992; Adams and Demmig-Adams 1992; Bjorkman and Demmig-Adams 1994). In leaves that have accumulated and retain A + Z throughout periods when they are exposed to limiting PFDs, A + Z can become engaged and disengaged in energy dissipation within seconds. One example for such a pattern was discussed above for leaves exposed to repeated intense sunflecks. Another example can be found under low-temperature stress. Under low-temperature stress during the

4.

PHYSIOLOGY OF LIGHT TOLERANCE IN PLANTS

Control

237

Stress

9%

9~

Spinach, nitrogen stress

82%

•"•~ 25%

-

2{)4%Z A

19%

V

57%

66%

_..

I

......~

Ponderosa pine, 19% low temperature stress

Fig. 4.11. Effect of environmental stresses on the pool size and the midday conversion state of the xanthophyll cycle. Spinach (Spinacia oleracea L.) plants were grown under ample and limiting nitrogen supply in the soil and at an incident PFD of 900 !J.mol photons/m 2 s in a growth chamber. (Data from Demmig-Adams et al. [1995].) Ponderosa pine (Pinus ponderosa Laws. var. scopulorum Engelm.) was growing naturally in Boulder, Colorado, in a fully sun-exposed location during the summer and winter seasons. Data from Adams and Demmig-Adams (1994). The carotenoid concentrations per ChI for controls was normalized and in each case the increase in the total size of the pies from "control" to "stress" reflects the increase in the carotenoid concentration per chlorophyll within each pair.

winter season, not only the pool size and the maximal conversion state increase (see above), but there can also be a dramatic change in the diurnal pattern of xanthophyll cycle operation (Fig. 4.12). On a cold day during the winter season, maximal levels of A + Z were maintained 24 h a day in Yucca glauca. In such a leaf, an abrupt exposure to excess PFDs on a morning following a cold night leads to an instantaneous high level of energy dissipation. In contrast to this response of cold-acclimated and (A + Z)-retaining leaves, which presumably have rather complete photoprotection, leaves not acclimated to cold temperatures were unable to form A + Z and increase energy dissipation levels rapidly at low temperatures (Adams et a1. 1995a).

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

238

Summer 2000

..... I

A

Winter

B

Incident PFD

CJ)

Incident PFD

1500

~

E 0 1000 E ~

0

500

LL

0...

0 0.8

N

0.6

e-+

0.4

::5-

0.2

«+

--N+

C

0

•

•••••• • ••• • A+Z

0 >.

0.8

()

c '0 0.6 Q)

;,;::

Q5

~

(f)

••• ••••• •• •

0.4

0...

() CJ)

c

0.2

C

0

F

E

.;::

6:00

10:00

14:00

18:00 6:00

10:00

14:00

18:00

Time of day Fig. 4.12. Diurnal changes in incident PFD, levels of antheraxanthin and zeaxanthin (A + Z)/(V + A + Z), and intrinsic PSII efficiency in leaves of Yucca glauca Nuttal growing naturally in a short grass prairie on clear days during the summer and winter seasons. Intrinsic PSII efficiency was quantified in situ from fluorescence measurements directly in the field with the PAM-2000 chlorophyll fluorometer as F)Fm (predawn) and Fv'/Fm' during actual illumination. For further details, see the legend to Fig. 4.2. (Modified from Adams et al. [1995b].)

4.

PHYSIOLOGY OF LIGHT TOLERANCE IN PLANTS

239

E. Photoinhibition and the Xanthophyll Cycle

Not only can high A + Z levels be maintained throughout a cold day during the winter, but the PSII efficiency can remain low throughout the entire day and night period as well (Fig. 4.12). A sustained lowering of the efficiency with which excitation energy is delivered to PSII centers is, in fact, the expected consequence of a sustained engagement of A + Z for energy dissipation (see above). The changes in chlorophyll fluorescence characteristics from these leaves indeed suggest that all decreases in PSII efficiency can be accounted for by sustained high levels of xanthophyll cycle-dependent energy dissipation in these leaves (Adams et al. 1995b). In this case, the low PSII efficiency would represent a down-regulation of PSII to match the low rates of carbon fixation. We suggested that this phenomenon may not limit carbon gain by these leaves significantly, since this low PSII efficiency was observed on days when low temperatures inhibited rates of photosynthesis directly (Adams et al. 1995b). In contrast to the above interpretation, the phenomenon of low predawn levels of PSII efficiency in response to environmental stresses has previously often been addressed as "photoinhibition" of photosynthesis, where it is implied that light-induced changes limit photosynthesis rates directly. While it was originally assumed that "photoinhibition" is caused by damage to PSII that can be reversed by repair of PSII (see Melis 1991; Ohad et al. 1994), there is now a new school of thought proposing that a modification of the PSII reaction center can convert these PSII centers into dissipating centers that contribute to photoprotection (reviewed in Critchley 1994; Krause 1994; Long et al. 1994; Osmond 1994). Our recent work on stress-induced sustained changes in PSII efficiency suggests that a large fraction (and perhaps all) of the decreases in PSII efficiency result from sustained high levels of xanthophyll cycle-dependent energy dissipation in the antennae. We are currently exploring a link between the slowed xanthophyll cycle turnover and protein turnover. In addition, Gilmore and Bjorkman (1994a,b) have suggested a mechanism by which a low pH and high levels of A + Z could be maintained in darkness in chilled leaves. IV. CONCLUDING REMARKS

One of the most important findings discussed in this review is the fact that acclimation of leaves to light stress leads to an enhanced employment of xanthophyll cycle-dependent dissipation of excess

240

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

excitation energy in all species of higher plants we have examined. This photoprotective dissipation of energy was sufficient to largely compensate for the inability of photosynthetic electron transport to utilize all of the absorbed light at certain times. The potential for removal of activated oxygen via the chloroplast antioxidant system also increased during this acclimation process. We conclude that leaves acclimated to light stress are well protected from any direct photo damage. In our studies on the interaction of environmental stresses, we have focused on plant species tolerant of these stresses, as is the case for evergreens and low-temperature stress (cf. Figs 4.11 and 4.12). In these cases, even slowly reversible changes in photochemical efficiency are probably entirely of a photo protective nature. In contrast, shade leaves or plants that are suddenly transferred to high PFDs have an insufficient capacity to increase their A + Z levels, resulting in the dissipation of only a part of the excess excitation energy (Demmig-Adams and Adams 1994; Demmig-Adams et al. 1995). When A + Z levels are insufficient, the chloroplast antioxidant system should be able to prevent photooxidative damage for a certain, and presently unknown, period of time. However, these antioxidants are present at much lower levels in shade compared to sun leaves. After that time period, photooxidative damage is likely to occur. Upon sudden shade-sun transfer, appropriate acclimatory increases in the levels of A + Z and thus energy dissipation can apparently occur within less than 1 day (Demmig-Adams et al. 1989; D. Moeller, B. Demmig-Adams, B. Logan, W. Adams, unpublished data). We speculate that any actual leaf damage during sudden shadesun transfers may also involve direct heat damage. These shade leaves are likely to experience an excessive heat load under high PFDs, due to insufficient rates of water uptake by the root system, resulting in insuffient transpiration rates. Future studies should examine to what extent oxidative damage, as a direct consequence of insufficient levels of A + Z and thus insufficient energy dissipation, actually contributes to leaf damage during shade-sun transfers. Genetic differences in the ability of plants to increase the capacity for (A + Z)-dependent energy dissipation have not yet been identified. Rather, our work to date has demonstrated a surprising degree of uniformity in the response of a wide diversity of higher plant species to excess light. Even the rainforest species Monstera deliciosa, when examined 1 day after transfer to a high PFD (Demmig-Adams et al. 1989), and Alocasia brisbanensis, growing naturally in a sunexposed location (B. Logan, D. Barker, W. Adams, B. Demmig-Adams,

4.

PHYSIOLOGY OF LIGHT TOLERANCE IN PLANTS

241

unpublished data), were found to possess large pools of V + A + Z. We speculate that the ability to show high levels of (A + Z)-dependent energy dissipation is a highly conserved trait because (1) it arose early during evolution, (2) it probably does not "involve a large metabolic cost, and (3) excess light is experienced daily by virtually all leaves at certain times, remarkably even in extremely shaded environments such as the rainforest floor (B. Logan, D. Barker, W. Adams, B. Demmig-Adams, unpublished data). It needs to be determined whether crop species such as coffee or cacao, for which shading is a standard horticultural practice, have an inherently lower capacity for photoprotective processes (energy dissipation and/or oxygen scavenging) or whether their limitations reside in an inability to cope directly with other factors such as heat or water stress. Similarly, the response of species to stresses to which they are inherently susceptible, such as the response of tropical species to chilling stress in combination with light, remains to be examined to determine whether this susceptibility involves limitations in the ability to carry out photoprotective processes under the stress conditions. Such additional work will be needed before the discovery of xanthophyll cycle-dependent energy dissipation can be applied directly to horticulture. LITERATURE CITED Adams, W. W., III, and B. Demmig-Adams. 1992. Operation ofthe xanthophyll cycle in higher plants in response to diurnal changes in incident sunlight. Planta 186:390-398. Adams, W. W., III, and B. Demmig-Adams. 1994. Carotenoid composition and down regulation of photosystem II in three conifer species during the winter. Physiol. Plant. 92:451-458. Adams, W. W., III, and B. Demmig-Adams. 1995. The xanthophyll cycle and sustained thermal energy dissipation activity in Vinca minor and Euonymus kiautschovicus in winter. Plant Cell Environ. 18:117-127. Adams, W. W., III, M. Volk, A. Hoehn, and B. Demmig-Adams. 1992. Leaf orientation and the response of the xanthophyll cycle to incident light. Oecologia 90:404410. Adams, W. W., III, A. Hoehn, and B. Demmig-Adams. 1995a. Chilling temperatures and the xanthophyll cycle: a comparison of warm-grown and overwintering spinach. Austral. J. Plant Physiol. 22:75-85. Adams, W. W., III, B. Demmig-Adams, A. S. Verhoeven, and D. H. Barker. 1995b. "Photoinhibition" during winter stress: involvement of sustained xanthophyll cycle-dependent energy dissipation. Austral. J. Plant Physiol. 22:261-276. Allen, R. D. 1995. Dissection of oxidative stress tolerance using transgenic plants. Plant Physiol. 107:1049-1054.

242

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

Alscher, R. G., and J. L. Hess. 1993. Antioxidants in higher plants. CRC, Boca Raton, FL.

Anderson, J. M., W. S. Chow, and D. J. Goodchild. 1988. Thylakoid membrane organization in sun/shade acclimation. Austral. J. Plant Physiol. 15:11-26. Arsalane, W., B. Rousseau, and J.-C. Duval. 1994. Influence of the pool size of the xanthophyll cycle on the effects of light stress in a diatom: competition between photoprotection and photoinhibition. Photochem. Photobiol. 60:237-243. Asada, K., and M. Takahashi. 1987. Production and scavenging of active oxygen in photosynthesis. p. 227-287. In: D. J. Kyle, C. B. Osmond, and C. J. Arntzen (eds.), Photoinhibition: topics in photosynthesis, Vol. 9. Elsevier, Amsterdam. Bilger, W., and a. Bjorkman. 1990. Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes. fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynth. Res. 25:173185. Bilger, W., and a. Bjorkman. 1991. Temperature dependence of violaxanthin deepoxidation and non-photochemical fluorescence quenching in intact leaves of Gossypium hirsutum L. and Malva parviflora L. Planta 184:226-234. Bilger, W., and a. Bjorkman. 1994. Relationship among violaxanthin deep oxidation, thylakoid membrane conformation, and nonphotochemical chlorophyll fluorescence in cotton leaves. Planta 193:238-246. Bjorkman, 0.1981. Responses to different quantum flux densities. p. 57-107. In: a. L. Lange, P. S. Nobel, C. B. Osmond, and H. Ziegler (eds.), Physiological plant ecology, I: responses to the physical environment. Encyclopedia of plant physiology, NS, Vol. 12A. Springer, Berlin. Bjorkman, a. 1987. High-irradiance stress in higher plants and interaction with other stress factors. p. 11-18. In: J. Biggins (ed.), Progress in photosynthesis research, Vol. IV. Martinus Nijhoff, Dordrecht. Bjorkman, 0., and B. Demmig. 1987. Photon yield of O 2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta 170:489-504. Bjorkman, 0., and B. Demmig-Adams. 1994. Regulation of photosynthetic light energy capture, conversion, and dissipation in leaves of higher plants. p. 17-47. In: E.-D. Schulze and M. M. Caldwell (eds.), Ecophysiology of photosynthesis. Springer, Berlin. Bjorkman, 0., and S. B. Powles. 1981. Leaf movement in the shade species Oxalis oregana., I: response to light level and light quality. Carnegie Inst. Wash. Yearb. 80:59-62. Bjorkman, 0., B. Demmig, and T. J. Andrews. 1988. Mangrove photosynthesis: response to high-irradiance stress. Austral. J. Plant Physiol. 15:43-61. Bowler, C., M. Van Montagu, and D. Inze. 1992. Superoxide dismutase and stress tolerance. Annu. Rev. Plant Physiol. Plant Mol. BioI. 43:83-116. Briantais, J.-M., C. Vernotte, M. Picaud, and G.H. Krause. 1979. A quantitative study ofthe slow decline of chlorophyll a fluorescence in isolated chloroplasts. Biochim. Biophys. Acta 548:128-138. Brugnoli, E., A. Cona, and M. Lauteri. 1994. Xanthophyll cycle components and capacity for non-radiative energy dissipation in sun and shade leaves of Ligustrum ovalifolium exposed to conditions limiting photosynthesis. Photosynth. Res. 41:451-463. Critchley, C. 1994. Dl protein turnover: response to photo damage or regulatory mechanism? p. 195-203. In: N. R. Baker and J. R. Bowyer (eds.), Photoinhibition of photosynthesis from molecular mechanisms to the field. Bios Scientific, Oxford, UK.

4.

PHYSIOLOGY OF LIGHT TOLERANCE IN PLANTS

243

Davies, W. J., and J. Zhang. 1991. Root signals and the regulation of growth and development of plants in drying soil. Annu. Rev. Plant Physiol. Plant Mol. BioI. 42:55-76. Demmig-Adams, B. 1990. Carotenoids and photoprotection in plants: a role for the xanthophyll zeaxanthin. Biochim. Biophys. Acta 1020:1-24. Demmig-Adams, B., and W. W. Adams III. 1992a. Photoprotection and other responses of plants to high light stress. Annu. Rev. Plant Physiol. Plant Mol. BioI. 43:599-626. Demmig-Adams, B., and W. W. Adams III. 1992b. Carotenoid composition in sun and shade leaves of plants with different life forms. Plant Cell Environ. 15:411419. Demmig-Adams, B., and W. W. Adams III. 1993a. The xanthophyll cycle. p. 206251 In: A. Young and G. Britton (eds.), Carotenoids in photosynthesis. Chapman & Hall, London. Demmig-Adams, B., and W. W. Adams III. 1993b. The xanthophyll cycle. p. 91110. In: R. G. Alscher and J. L. Hess (eds.), Antioxidants in higher plants. CRC, Boca Raton, FL. Demmig-Adams, B., and W. W. Adams III. 1994. Capacity for energy dissipation in the pigment bed in leaves with different xanthophyll cycle pools. Austral. J. Plant Physiol. 21:575-588. Demmig-Adams, B., and W. W. Adams III. 1996a. Xanthophyll cycle and light stress in nature: uniform response to excess direct sunlight among higher plant species. Planta, 198:460-470. Demmig-Adams, B., and W. W. Adams III. 1996b. Chlorophyll and carotenoid composition in leaves of Euonymus kiautschovicus acclimated to different degrees of light stress in the field. Austral. J. Plant Physiol. 23 (in press). Demmig, B., K. Winter, A. Kruger, and F.-C. Czygan. 1987. Photoinhibition and zeaxanthin formation in intact leaves: a possible role of the xanthophyll cycle in the dissipation of excess light energy. Plant Physiol. 84:218-224. Demmig, B., K. Winter, A. Kruger, and F.-C. Czygan. 1988. Zeaxanthin and the heat dissipation of excess light energy in Nerium oleander exposed to a combination of high light and water stress. Plant Physiol. 87:17-24. Demmig-Adams, B., K. Winter, E. Winkelmann, A. Kruger, and F.-C. Czygan. 1989. Photosynthetic characteristics and the ratios of chlorophyll, f3-carotene, and the components of the xanthophyll cycle upon a sudden increase in growth light regime in several plant species. Bot. Acta 102:319-325. Demmig-Adams, B., W. W. Adams III, B. A. Logan, and A. S. Verhoeven. 1995. Xanthophyll cycle-dependent energy dissipation and flexible PSII efficiency in plants acclimated to light stress. Austral. J. Plant Physiol. 22:249-260. Demmig-Adams, B., A. M. Gilmore, and W. W. Adams III. 1996. In vivo functions of carotenoids in higher plants. FASEB J. 10:403-412. Demmig-Adams, B., W. W. Adams III, D. H. Barker, B. A. Logan, D. R. Bowling, and A. S. Verhoeven. 1997. Using chlorophyll fluorescence to assess the allocation of absorbed light to thermal dissipation of excess excitation. Physiol. Plant. (In press). Duckham, S., R. S. T. Linforth, and I. B. Taylor. 1991. Abscisic-acid-deficient mutants at the aba gene locus of Arabidopsis thaliana are impaired in epoxidation of zeaxanthin. Plant Cell Environ. 14:601-606. Ehleringer, J. R. 1982. The influence of water stress and temperature on leaf pubescence development in Encelia farinosa. Am. J. Bot. 69:670-675. Ehleringer, J. R. 1988. Changes in leaf characteristics of species along elevational gradients in the Wasatch Front, Utah. Am. J. Bot. 75:680-689.

244

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

Ehleringer, J. R., and O. Bjorkman. 1978. Pubescence and leaf spectral characteristics in a desert shrub, Encelia farinosa. Oecologia 36:151-162. Evans, J. R. 1989. Photosynthesis and nitrogen relationships in leaves of C3 plants. Oecologia 78:9-19. Falbel, T. G., A. Staehelin, and W. W. Adams III. 1994. Analysis of xanthophyll cycle carotenoids and chlorophyll fluorescence in light intensity-dependent chlorophyll-deficient mutants of wheat and barley. Photosynth. Res. 42:191-202. Field, c., and H. A. Mooney 1986. The photosynthesis-nitrogen relationship in wild plants. p. 25-55. In: T. J. Givnish (ed.) On the economy of form and function. Cambridge Univ. Press, Cambridge, UK. Foyer, C. H., and P. M. Mullineaux. 1994. Causes of photooxidative stress and amelioration of defense systems in plants. CRC, Boca Raton, FI. Frank, H. A., A. Cua, V. Chynwat, A. Young, D. Gosztola, and M. R. Wasielewski. 1994. Photophysics of the carotenoids associated with the xanthophyll cycle in photosynthesis. Photosynth. Res. 41:389-395. Fransen, J. M., and J. Bruinsma. 1981. Relationships between xanthoxin, phototropism, and elongation growth in sunflower seedling Helianthus annuus L. Planta 151:365-370. Genty, B., Briantais, J.-M., and N. R. Baker. 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta 990:87-92. Gilmore, A. M., and O. Bjorkman. 1994a. Adenine nucleotides and the xanthophyll cycle in leaves, I: effects of CO z- and temperature-limited photosynthesis on adenylate energy charge and violaxanthin deep oxidation. Planta 192:526-536. Gilmore, A. M., and O. Bjorkman. 1994b. Adenine nucleotides and the xanthophyll cycle in leaves, II: comparison of the effects of CO z- and temperature-limited photosynthesis on photo system II fluorescence quenching, the adenylate energy charge and violaxanthin deepoxidation in cotton. Planta 192:537-544. Gilmore, A. M., and H. Y. Yamamoto. 1991. Resolution of lutein and zeaxanthin using a nonendcapped, lightly carbon-loaded C-18 high-performance liquid chromatographic column. J. Chromatogr. 543:137-145. Gilmore, A. M., and H. Y. Yamamoto. 1992. Dark induction of zeaxanthin-dependent nonphotochemical fluorescence quenching mediated by ATP. Proc. Nat. Acad. Sci. (USA) 89:1899-1903. Gilmore, A. M., and H. Y. Yamamoto. 1993a. Linear models relating xanthophylls and lumen acidity to non-photochemical fluorescence quenching: evidence that antheraxanthin explains zeaxanthin-independent quenching. Photosynth. Res. 35:67-78. Gilmore, A. M., and H. Y. Yamamoto. 1993b. Biochemistry of xanthophyll-dependent nonradiative energy dissipation. p. 162-165 In: H. Y. Yamamoto and C. M. Smith (eds.), Photosynthetic responses to the environment. Am. Soc. Plant PhysioI., Rockville, MD. Gilmore, A. M., N. Mohanty, and H. Y. Yamamoto. 1994. Epoxidation of zeaxanthin and antheraxanthin reverses non-photochemical quenching of photosystem II chlorophyll a fluorescence in the presence of a trans-thylakoid L1pH. FEBS Lett. 350: 271-274 Gilmore, A. M., T. L. Hazlett, and Govindjee. 1995. Xanthophyll cycle dependent quenching of photosystem II chlorophyll a fluorescence: formation of a quenching complex with a short fluorescence lifetime. Proc. Nat. Acad. Sci. (USA) 92:2273-2277.

4.

PHYSIOLOGY OF LIGHT TOLERANCE IN PLANTS

245

Grace, S., R. Pace, and T. Wydrzynski. 1995. Formation and decay of monodehydroascorbate radicals in illuminated thylakoids as determined by EPR spectroscopy. Biochim. Biophys. Acta 1229:155-165. Hager, A. 1980. The reversible, light-induced conversions of xanthophylls in the chloroplast. p. 57-79. In: F.-C. Czygan (ed.), Pigments in plants. Gustav Fischer, Stuttgart. Haupt, W., and R. Scheuerlein. 1990. Chloroplast movements. Plant Cell Environ. 13:595-614. Horton, P., A. V. Ruban, and R. G. Walters. 1994. Regulation of light harvesting in green plants. Plant Physiol. 106:415-420. Kao, W. Y., and 1. N. Forseth. 1992. Diurnal leaf movement, chlorophyll fluorescence and carbon assimilation in soybean grown under different nitrogen and water availabilities. Plant Cell Environ. 15:703-710. Khamis, S., T. Lamaze, Y. Lemoine, and C. Foyer. 1990. Adaptation of the photosynthetic apparatus in maize leaves as a result of nitrogen limitation. Plant PhysioI. 94:1436-1443. Kitajima, M., and W. L. Butler. 1975. Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone. Biochim. Biophys. Acta 376:105-115. Koller, D. 1990. Light-driven leaf movements. Plant Cell Environ. 13:615-632. Koller, D., and T. Shako 1990. Light-driven movements in the solar-tracking leaf of Lupinus palaestinus Boiss. Photochem. Photobiol. 52:187-196. Krause, G. H. 1994. Photoinhibition induced by low temperatures. p. 331-348 In: N. R. Baker and J. R. Bowyer (eds.), Photo inhibition of photosynthesis from molecular mechanisms to the field. Bios Scientific, Oxford, UK. Krause, G. H., and E. Weis. 1991. Chlorophyll fluorescence and photosynthesis: the basics. Annu. Rev. Plant Physiol. Plant Mol. BioI. 42:313-349. Logan, B. A., D. H. Barker, B. Demmig-Adams, and W. W. Adams III. 1996. Acclimation of leaf carotenoid composition and ascorbate levels to gradients in the light environment within an Australian rainforest. Plant Cell Environ. 19 (in press). Long, S. P., S. Humphries, and P. G. Falkowski. 1994. Photoinhibition of photosynthesis in nature. Annu. Rev. Plant Physiol. Plant Mol. BioI. 45:633-662. Lovelock, C. E., and B. F. Clough. 1992. Influence of solar radiation and leaf angle on leaf xanthophyll concentrations in mangroves. Oecologia 91:518-525. Ludlow, M. M., and O. Bjorkman. 1984. Paraheliotropic leaf movement in Siratro as a protective mechanism against drought-induced damage to primary photosynthetic reactions: damage by excessive light and heat. Planta 161:505-518. Melis, A. 1991. Dynamics of photosynthetic membrane composition and function. Biochim. Biophys. Acta 1058:87-106. Mohanty, N., A. M. Gilmore, and H. Y. Yamamoto. 1995. Mechanism of non-photochemical chlorophyll fluorescence quenching: II: resolution of rapidly reversible absorbance changes at 530 nm and fluorescence quenching by the effects of antimycin, dibucaine and cation exchanger, A23187. Austral. J. Plant Physiol. 22:239247. Mooney, H. A., J. R. Ehleringer, and O. Bjorkman. 1977. The energy balance ofleaves of the evergreen desert shrub Atriplex hymenelytra. Oecologia 29:301-310. Morales, F., A. Abadfa, R. Belkhodja, and J. Abadfa. 1994. Iron deficiency-induced changes in the photosynthetic pigment composition of field-grown pear (Pyrus communis L.) leaves. Plant Cell Environ. 17:1153-1160.

246

B. DEMMIG-ADAMS, W. W. ADAMS III, AND S. C. GRACE

Neubauer, C., and H. Y. Yamamoto. 1992. Mehler-peroxidase reaction mediates zeaxanthin formation and zeaxanthin-related fluorescence quenching in intact chloroplasts. Plant Physiol. 99:1354-1361. Ohad, 1., N. Keren, H. Zer, H. Gong, T. S. Mor, A. Gal, S. Tal, and Y. Domovich. 1994. Light-induced degradation of the photosystem II reaction centre D1 protein in vivo: an integrative approach. p. 161-177. In: N. R. Baker and J. R. Bowyer (eds.), Photo inhibition of photosynthesis from molecular mechanisms to the field. Bios Scientific, Oxford, UK. Osmond, C. B. 1994. What is photoinhibition? Some insights from comparisons of shade and sun plants. p. 1-24. In: N. R. Baker and J. R. Bowyer (eds.), Photoinhibition of photosynthesis from molecular mechanisms to the field. Bios Scientific, Oxford, UK. Owens, T. G. 1996. Processing of excitation energy by antenna pigments. In: N.R. Baker (ed), Photosynthesis and the environment. Kluwer, Dordrecht, in press. Pfiindel, E., and W. Bilger. 1994. Regulation and possible function ofthe violaxanthin cycle. Photosynth. Res. 42:89-109. Rock, C. D., and J. A. D. Zeevaart. 1991. The aba mutant of Arabidopsis thaliana is impaired in epoxy-carotenoid biosynthesis. Proc. Nat. Acad. Sci. (USA) 88:74967499. Schreiber, U., W. Bilger, and C. Neubauer. 1994. Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis. p. 49-70. In: E.-D. Schulze and M. M. Caldwell (eds.), Ecophysiology of photosynthesis. Springer, Berlin. Seddon, J. M., U. A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gradoudas, J. Haller, D. T. Miller, L. A. Yannuzzi, W. Willett. 1994. Dietary carotenoids, vitamins A, C, and E, and advanced age-related macular degeneration. JAMA 172:1413-1420. Sen Gupta, A., R. P. Webb, A. S. Holaday, and R. D. Allen. 1993. Overexpression of superoxide dismutase protects plants from oxidative stress. Plant Physiol. 103:1067-1073. Thayer, S. S., and O. Bjorkman. 1990. Leaf xanthophyll content and composition in sun and shade determined by HPLC. Photosynth. Res. 23:331-343. Thayer, S. S., and O. Bjorkman 1992. Carotenoid distribution and deep oxidation in thylakoid pigment-protein complexes from cotton leaves and bundle-sheath cells of maize. Photosynth. Res. 33:213-225. van Kooten, 0., and J. F. H. Snel. 1990. The use of fluorescence nomenclature in plant stress physiology. Photosynth. Res. 25:147-150. Walters, R. G., A. V. Ruban, and P. Horton. 1994. Higher plant light-harvesting complexes LHCIIa and LHCIIc are bound by dicyclohexylcarbodiimide during inhibition of energy dissipation. Eur. J. Biochem. 226:1063-1069. Yamamoto, H. Y. 1979. Biochemistry of the violaxanthin cycle. Pure Appl. Chern. 51:639-648. Yamamoto, H. Y., and R. Bassi. 1996. Carotenoids: localization and function. pp. 539-563. In: D. R. Ort and C. F. Yocum (eds), Oxygenic photosynthesis: the light reactions. Advances in Photosynthesis 4, Kluwer, Dordrecht, The Netherlands.

5 Acquired Resistance to Disease in Plants Ray Hammerschmidt Department of Botany and Plant Pathology Michigan State University East Lansing, Michigan 48824, USA Jennifer Smith Becker Department of Plant Pathology University of California Riverside, California 92521, USA

I. II.

III.

Introduction Examples of Acquired Resistance A. Cucumber and Other Cucurbits B. Green Bean C. Tobacco, Potato, and Tomato D. Arabidopsis E. Other Examples F. Systems Acquired Resistance to Arthropod Herbivores Mechanisms of Resistance A. Histology 1. Cucumber 2. Green Bean 3. Tobacco, Potato, and Tomato 4. Arabidopsis B. Biochemical Changes 1. Phytoalexins 2. Peroxidase and Associated Biochemical Changes 3. Lipoxygenase 4. Activated Oxygen 5. Pathogenesis-Related (PR) Proteins

Horticultural Reviews, Volume 18, Edited by Jules Janick ISBN 0-471-57334-5 © 1997 John Wiley & Sons, Inc. 247

248 IV.

V.

VI.

R. HAMMERSCHMIDT AND

J. S. BECKER

C. Summary The Systemic Signal for Resistance A. Evidence for a Systemic Signal B. Salicylic Acid C. How Does Salicylic Acid Induce Resistance? D. Systemic Signal Generation Acquired Resistance and Disease Control A. Microbial Inducers B. Chemical Inducers 1. Amino Acid Analogs 2. Phosphates and Oxalates 3. Fatty Acid Derivatives 4. Phenolic Compounds 5. Silicates 6. 2,6-Dichloroisonicotinic Acid Summary Literature Cited

I. INTRODUCTION

Although many thousands of pathogens are present in nature, individual plant species are susceptible to only a very small number of these pathogens. In many cases, when a pathogen comes into contact with a plant, the potential pathogen attempts to infect the plant. However, only in a few specific cases where the pathogen has the ability to somehow suppress or nullify the host defense mechanism does disease develop. In all other cases, the constitutive or inducible defenses of the plant stop further invasion by the pathogen. Based on this information, it has been concluded that susceptibility to disease is at least partly due to overcoming preformed defenses or to a failure of the plant to induce its defenses rapidly enough to stop a pathogen rather than being solely based on heritable "genes for resistance" (Kuc 1983). In other words, all plants appear to have the necessary mechanisms for defense. In the early part of this century, evidence that plants could be protected against infection by prior infection of the plant with another pathogen began to accumulate. This phenomenon became known as induced or acquired resistance to disease (Chester 1933). Induced resistance can be expressed either locally or systemically. In the former case, plant tissues that respond to an initial infection attempt by a nonpathogen or to an avirulent form of a pathogen become resistance to subsequent infection by a virulent pathogen inoculated at or very near to the site of nonpathogen attack. In systemic induced resistance, the localized infection of one part of a

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

249

plant by a nonpathogen or a necrotic lesion-inducing pathogen results in the expression of an enhanced state of resistance throughout the plant. The induction of systemic and localized acquired resistance has been closely associated with the development of pathogen-induced necrosis (Hammerschmidt 1993). The necrotic tissues can develop from a highly resistant, hypersensitive response to an avirulent or nonpathogen, or the necrosis can be the result of lesion formation by pathogens that are virulent on the host plant. Damage caused by insect feeding, abrasion, or dry ice, even when it appears similar to a necrotic lesion induced by a pathogen, is generally ineffective in the induction of systemic resistance (Hammerschmidt 1993).

Induced resistance is also characterized by nonspecificity (Kuc 1983, 1987). In general, once a plant has been induced, the resis-

tance that is expressed is effective against the full spectrum of pathogen types. In addition to a lack of specificity in the expression of resistance, a number of different pathogen types may be capable of inducing resistance in any given host plant. In this review, we describe several aspects of induced resistance with some emphasis on horticultural crops. We provide a brief assessment of the state of knowledge on the mechanisms and biochemical basis of induced resistance, discuss the nature of the signals generated in the plant that are involved in systemic induced resistance, and provide a description of research that indicates that induced resistance can be part of disease control. Many reviews on the individual topics covered in this review are available and can be consulted for more details (for example, see Hammerschmidt 1993; Hammerschmidt and Kuc 1995; Kuc 1983, 1987; Ryals et al. 1994; Sequira 1983; Wilson et al. 1994). II. EXAMPLES OF ACQUIRED RESISTANCE

Acquired resistance to disease has been observed in many plant families, Several of which have been studied extensively. The biology of acquired resistance in three dicot families has recently been reviewed (Deverall and Dann 1995; Hammerschmidt and Yang-Cashman 1995; Ozeretskovskaya 1995). Graminaceous monocots have been studied with respect to induced resistance, which has also been recently reviewed by Steiner and Schonbeck (1995). In this section, a brief overview of induced resistance in several important families is presented.

250

R. HAMMERSCHMIDT AND

J. S. BECKER

A. Cucumber and Other Cucurbits

Ku6 and coworkers were the first to observe that prior inoculation of cucumber plants with certain pathogens or nonpathogens of cucumber resulted in the expression of local and systemic induced resistance to subsequent infection by a diverse group of pathogens. In the case of local induced resistance, Hammerschmidt et a1. (1976) reported that prior inoculation of cucumber plants with the bean pathogen Colletotrichum lindemuthianum (Sacc. & Magnus) Lams.Scrib. protected the plants against a subsequent challenge infection by the cucumber scab pathogen, Cladosporium cucumerinum Ellis & Arth. In the same study, prior inoculation of scab-resistant seedlings with C. cucumerinum protected them against infection by the anthracnose fungus, Colletotrichum lagenarium (Pass.) Ellis & HaIst. Systemic acquired resistance in cucumber was first reported by Ku6 et a1. (1975) and expanded upon by Ku6 and Richmond (1977). In these reports, the inoculation of one leaf of anthracnose-susceptible cucumber plants with the anthracnose fungus, C. lagenarium, resulted in systemic resistance of the plant against a subsequent challenge inoculation with the same fungus. Further reports from this group demonstrated that the systemic resistance was effective against and induced by a number of different pathogens of cucumber. These pathogens include C. cucumerinum (Staub and Ku6 1980), Fusarium oxysporum (Schlechtend) f. sp. cucumerinum J. H. Owen (Gessler and Ku6 1982), Mycosphaerella melonis (Pass.) Chiu & J. C. Walker (Bergstrom 1981), Pseudoperonospora cubense (Berk & M. A. Curtis) Rostovzes (Okuno et a1. 1991), Sphaerotheca fuliginia (Schlechtend: Fr.) Pollaci (Bashan and Cohen 1983), Pseudomonas syringae pv. lachrymans (Smith and Bryan) Young, Dye & Wilkie (Caruso and Ku6 1979; Doss and Hevisi 1981), Erwinia tracheiphila (Smith) Bergey et a1. (Bergstrom 1981), cucumber mosaic virus (Bergstrom et a1. 1982), and tobacco necrosis virus (Jenns and Ku6 1977).

Inoculation of cucumber with avirulent forms of F. oxysporum has also been reported to induce local (Michail et a1. 1989) and systemic resistance (Mandeel and Baker 1991). Systemic induced resistance was demonstrated in the latter study by inoculation of onehalf of the roots of cucumber (using a "split-root" system). This treatment protected the other half of the root system from infection by F. oxysporum f.sp. cucumerinum. Similarly, infection of the roots also protected the plants against infection of stem tissue through

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

251

wounds. Because the inducing inoculum and the challenge were spatially separated, one conclusion was that systemic resistance was induced. The systemic nature of the resistance was also demonstrated by an increase in resistance to foliar challenge by C. lagenarium. Unlike cucumber, other cucurbits have received much less attention. Caruso and Kuc (1977a,b), modeling their work after that of Kuc et al. (1975) with cucumber, found that prior inoculation of the first leaf of watermelon plants with C. lagenarium resulted in systemic induction of resistance against the same pathogen. Inoculation of the roots of watermelon with the maize pathogen Cochliobolus carbonum Nelson protected the plants against infection with the vascular wilt fungus F. oxysporum (Shimotsuma et al. 1972). Biles and Martyn (1989) later reported that resistance could be induced in watermelon against F. oxysporum Schlechtend f. sp. niveum (E.F. Sm.) W. C. Snyder and Hans. by prior inoculation with either the cucumber wilt pathogen F. oxysporum f.sp. cucumerimum or with avirulent isolates of the watermelon pathogen. Interestingly, the avirulent watermelon strains were more effective than the cucumber pathogen in inducing resistance. Resistance can also be induced in muskmelon. Caruso and Kuc (1977a,b) found that prior infection of one leaf with C. lagenarium elicited systemic protection against the same fungus. Thus, the same systemic phenomenon observed in cucumber and watermelon was effective in muskmelon. Roby et a1. (1987) found that prior treatment of one leaf of muskmelon plants with fungal elicitors would also induce systemic resistance to C. lagenarium. No comparable studies have been carried out with other members of the cucurbit family. This is unfortunate since many of these cucurbits are very important economically. More details on the biology of induced resistance in cucurbits can be found in Hammerschmidt and Yang-Cashman (1995). B. Green Bean Both local and systemic induced resistance has also been demonstrated in legumes. This topic has recently been extensively reviewed by Deverall and Dann (1995), and this section only briefly describes results obtained with green bean. The initial studies on local induced resistance to C. lindemuthianum in bean were carried out by Rahe et al. (1969) and Skipp and Deverall (1973). These investigators used etiolated green bean

252

R. HAMMERSCHMIDT AND

J.

S. BECKER

hypocotyls and green bean plants, respectively. In the report by Rahe et al., whole hypocotyls were sprayed with spores of nonpathogens of bean and then later challenged with spores of a virulent race. In the study by Skipp and Deverall (1973), different parts of green bean plants were induced by placing droplets containing spores of an avirulent race on locations where subsequent challenge inoculations with a virulent race of the pathogen were made. These two studies demonstrated that several different plant parts susceptible to anthracnose could have resistance induced against that disease. Elliston et al. (1971, 1976a) reported that if etiolated bean hypocotyls were inoculated with droplets containing spores of an avirulent race of C. lindemuthianum or a Colletotrichum species pathogenic on another host species, the hypocotyl tissue 0.5 to 1 em away from the initial infection site became resistant to infection by a virulent race of C. lindemuthianum. Systemic resistance in green bean plants to C. lindemuthianum was first reported by Sutton (1979) and later confirmed by Cloud and Deverall (1987). Prior inoculation of lower leaves of green bean plants with C.lindemuthianum conidia resulted in systemic expression of resistance to the same fungus in upper leaves of the plants. Recently, Dann and Deverall (1995) expanded on these previous studies. They inoculated the unifoliate leaves of green bean plants with spores of C.lindemuthianum and subsequently challenge-inoculated the trifoliate leaves with either C. lindemuthianum or Uromyces appendiculatus (Pers.:Pers.) Unger. The prior infection with C. lindemuthianum resulted in significant systemic resistance to both pathogens. Treatment of bean plants with the synthetic resistanceinducing chemical 2,6-dichloroisonicotinic acid (INA) (MMraux et al. 1991) also induced systemic resistance to these pathogens as well as to the bacterial pathogen Pseudomonas syringae pv. phaseolicola (Burkholder) Young, Dye & Wilkie. No induced resistance against Rhizoctonia solani Kuhn or Fusarium solani (MarL) Sacco f.sp. ph,aseoli (Burkholder) Snyder & Hans. was observed in plants treated with INA. Systemic acquired resistance to tobacco necrosis virus following infection of lower leaves of green bean plants with U. appendiculatus (Kutzner et al. 1993) and to infection with U. appendiculatus by prior infection with C. lindemuthianum (Takahashi et al. 1985) has also been reported. These two reports, along with those cited above, indicate that induced resistance in bean, like that in cucumber, can be induced by and is active against a wide range of pathogen types.

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

253

C. Tobacco, Potato, and Tomato Systemic induced resistance has also been documented in solanaceous plants. This topic was recently reviewed in detail (Ozeretskovskaya 1995); thus, the subject is only briefly reviewed in this section. Extensive studies on systemic induced resistance have been carried out in tobacco since Ross (1961a,b) reported that inoculation of a lower leaf of tobacco, carrying the N gene for hypersensitive resistance to tobacco mosaic virus (TMV), with TMV induced local and systemic resistance to TMV. The resistance was characterized by a decrease in the size, although not the number, of TMV lesions. Cruickshank and Mandryk (1960) demonstrated that resistance in tobacco could be systemically induced against the blue mold fungus, Peronospora tabacina Adam, by injecting sporangia of this pathogen into the stem tissues of tobacco. Subsequent research by Kut and coworkers confirmed these results and demonstrated that TMV inoculation could also induce resistance to blue mold. The resistance that developed was characterized by lesion development that stopped by about 5 days after challenge inoculation, the formation of fewer lesions, and much reduced sporulation of the fungus on induced tissues (see Tuzun and Kut [1989] for more details on the induced resistance of tobacco to P. tabacina). Doke et a1. (1987) reported that prior treatment of potato foliage with an elicitor preparation from the late blight fungus Pbytopbtbora infestans (Mont.) de Bary induced resistance to this pathogen in untreated upper leaves of the plant. The resistance was characterized by a decrease in the number of lesions and also in a change in the appearance of the lesions from a susceptible type to a resistant type of reaction. Stromberg and Brisshammar (1991, 1993) also reported that prior infection of lower leaves of potato with either P. infestans or P. cryptogaea Pethybr. and Lafferty, a nonpathogen of potato, would also systemically protect the plants against late blight. Similarly, tomato can also be systemically protected against P. infestans by a prior inoculation with the same fungus (Enkerli et a1. 1993; Heller and Gessler, 1986; Kovats et a1. 1991b).

D. Arabidopsis The small mustard, Arabidopsis tbaliana L., has become widely used as a model for many plant processes (Meyerowitz and Somerville

254

R. HAMMERSCHMIDT AND

J. S. BECKER

1994). This now includes acquired resistance. Uknes et a1. (1992) were the first to demonstrate acquired resistance to disease in Arabidopsis. In this study, they treated plants with INA prior to challenge inoculation with Pseudomonas syringae pv. tomato Young, Dye & Wilkie. Resistance was observed as a decrease in both symptoms and bacterial populations in the leaves. Biological induction of resistance was later reported by Uknes et a1. (1993) and Cameron et a1. (1994), who reported that a prior inoculation of a lower leaf of Arabidopsis with turnip crinkle virus (TCV) or a hypersensitive response-inducing pseudomonad, respectively, induced systemic protection against a number of virulent pathogens. In the study by Uknes et a1. (1993), TCV protected plants against a subsequent challenge inoculation with P. syringae. In addition, pre-treatment of the plants with salicylic acid induced resistance to challenge inoculation with the downy mildew fungus Peronospora parasitica (Pers. :Fr.) Fr. (Uknes et a1. 1993). In the study by Cameron et a1. (1994), prior infection by an avirulent bacterium resulted in the development of acquired resistance to a virulent strain of Pseudomonas. Foliar infection by a noncharacterized, pathogenic isolate of F. oxysporum isolated from Arabidopsis roots induced resistance to infection by P. parasitica (Mauch-Mani and Slusarenko 1994). Systemic resistance was observed if the plants were challenged 4 days after inoculation with Fusarium. No resistance to Peronospora was seen if only 1 day separated the inducing and challenge inoculations. Complete resistance was observed if the plants were inoculated with F. oxysporum 7 days prior to the challenge. This is in line with other studies of induced resistance that reported that a minimum amount of time was needed for full expression of systemic resistance (Kuc 1983).

E. Other Examples A number of other plants have been shown to exhibit induced resistance to disease following treatment with biological or chemical inducers of resistance. Treatment of red cabbage seedlings with INA has also been reported to induce resistance to P. parasitica (Hijwegen and Verhaar 1993). The resistance was characterized by a dramatic decrease in the size of sporulating lesions as a function of INA concentration. Sugar beet (Beta vulgaris 1.) was protected against Cercospora beticola Sacco by prior treatment with INA (Nielsen et a1. 1994), and carnation could be protected from Fusarium wilt by treatment with root-colonizing pseudomonads (Peer et a1. 1991). Woody perennials

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

255

have also been protected against disease by a mechanism thought to involve induced resistance. For example, pear can be protected against Erwinia amylovora (Burrill) Winslow et a1. (McIntyre et a1. 1972), and pear leaves can also have resistance induced against Alternaria alternata (Fr.:Fr.) Keiss1. using spore germination fluids of this fungus (Hayami et a1. 1982). Resistance to the rust pathogen Hemileia vastatrix Berk & Br. has also been induced in coffee (Moraes et a1. 1976). Finally, Brock et a1. (1994) found that inoculation of cotten cotyledons with the pathogen Alternaria macrospora Zimm. or treatment of the cotyledons with the resistance-inducing compound INA induced systemic resistance to subsequent infection by A. macrospora. Taken together with the examples described above, induced resistance to pathogen attack may indeed be widespread, if not universal, in the plant kingdom. However, a much more systematic study of the distribution of acquired resistance among plant families is needed. F. Systemic Acquired Resistance to Arthropod Herbivores Wounding of various plant parts by mechanical means or by insect feeding has been shown to systemically increase resistance to arthropod herbivores in both cucurbits and tobacco (Tallamy 1985; Baldwin 1988a,b). In spite of the similarity of this response to systemic acquired resistance to pathogens, there is only little evidence that supports a reciprocal induction of systemic resistance to a pathogen by an arthropod herbivore or the induction of resistance to arthropod herbivores by pathogens in more than a few cases (Hammerschmidt 1993; Karban and Carey 1984; Karban et a1. 1987). Induction of systemic resistance to pathogens in cucumber (Ajlan and Potter 1991; Apiryanto and Potter 1990) or in tobacco (Ajlan and Potter 1992) did not induce systemic resistance to arthropod herbivores.

III. MECHANISMS OF RESISTANCE The lack of symptom development observed in plants with acquired resistance suggests that some effect on pathogen development must be part of the resistance. Histological studies can provide clues as to the nature of the resistance (particularly with fungal pathogens) because histology can relate the location of the pathogen with anatomical and, sometimes biochemical, responses of the host.

256

R. HAMMERSCHMIDT AND

J.

S. BECKER

A. Histology 1. Cucumber. Histological studies on acquired resistance to several fungal pathogens of cucumber have been reported. In the first of these, Richmond et al. (1979) reported a rather extensive light microscopy study on the induced resistance to C. lagenarium. They found that C. lagenarium spores germinated and formed appressoria equally well on induced and control tissues. The major difference observed between induced and control plants was a lack of penetration by the pathogen into induced host tissues. Thus, the induced resistance response against the cucumber anthracnose fungus appeared to be expressed as a reduction of successful penetrations. This conclusion was reinforced by the observation that genetically resistant cultivars of cucumber that exhibited hypersensitive-like resistance also could exhibit this penetration-related resistance mechanism if these plants were previously induced by infection on another leaf. The inhibition of penetration by C. lagenarium into induced tissues was also observed in cucumber plants that were systemically induced by prior infection by tobacco necrosis virus Oenns and Kuc 1980). The first attempt to explain how the induced plants could block penetration by fungi was reported in a study by Hammerschmidt and Kuc (1982). Using histochemistry, these authors found that there was a direct correlation between a failure of C. lagenarium to penetrate into the host and the deposition of a lignin-like material in the outer epidermal cell wall under appressoria. In general, for every successful penetration observed on control plants, there was a lignin-associated nonpenetration on the induced plant. These results were also consistent with results reported in a subsequent paper by Stumm and Gessler (1986) who found autofluorescent papillae associated with induced resistance in cucumber (autofluorescence is often an indicator of a phenolic material like lignin). Stumm and Gessler (1986) found a higher proportion of appressoria without penetrations associated with fluorescent papilla in induced as compared to control plants. Working with the same system, Kovats et al. (1991a) came to different conclusions. They found fewer appressoria on induced as compared to control leaves. Thus, at least part of the resistance could be the result of a mechanism that blocks appressorium formation. Kovats et al. did find that papilla were more frequently produced under appressoria that did not penetrate, and that these papilla contained callose. Kovats et al. (1991a), however, reported that lignification also occurred in epidermal cells of induced plants, and concluded

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

257

that lignification of epidermal cells after penetration was a major component of the induced resistance response. In a recent study, Kauss and coworkers (Siegrist et al. 1994) reported that prior treatment of etiolated cucumber hypocotyl segments with the resistance-inducing chemicals salicylic acid or INA resulted in an enhanced ability of the tissues to respond to infection by C. lagenarium. Induced plants produced autofluorescent, lignin-positive papillae under nonpenetrating appressoria more frequently than in control plants. These results were very similar to and confirmed earlier reports of Hammerschmidt and Kuc (1982) and of Stumm and Gessler (1986). Two ultrastructural studies on induced resistance to C. lagenarium in cucumber have been published. In the first study, Xuei et al. (1988) found that appressoria on induced leaves were more frequently associated with an electron-dense material in the host epidermal wall and a granular-like material deposited between the outer epidermal cell wall and the plasma membrane than were appressoria on controlleaves. No successful penetration through these wall alterations were reported. In a subsequent report, Stein et al. (1993) confirmed the results of Xuei et al. (1988) and provided some additional information on the possible chemical nature of the wall alterations. Using potassium permanganate as a stain Stein, et al. (1993) found that the wall modifications stained positively for lignin. Energy-dispersive X-ray analysis of the section showed that the deposited manganese (as manganese dioxide) was found to be associated only with the wall modifications. Cell wall associated peroxidase activity was also found in the areas that tested positively for lignin (Stein 1991). X-ray analysis was also used to detect other elements in the cell wall modifications. A very strong signal for silicon was found in the same areas that stained for peroxidase and lignin. The location of the silicon at the same site as the lignin suggests that both lignin and silicon may be functioning to strengthen the cell wall and, therefore, prevent penetration. Epidermal lignification was also reported to be part of the resistance response mounted against the powdery mildew fungus, Sphaerotheca fuliginia. Bashan and Cohen (1983) reported that prior inoculation of the first true leaf of cucumber plants with tobacco necrosis virus resulted in protection against powdery mildew. This protection involved the inhibition of penetration by the pathogen into the host epidermis by production of lignified papillae. In a recent study, Conti et al. (1990) presented a more detailed examination of the induced resistance to S. fuliginea. These authors demon-

258

R. HAMMERSCHMIDT AND

J. S. BECKER

strated that the preinfection behavior of the pathogen on induced plants was the same as on controls. This is similar to what was observed for C. lagenarium (see above). Sites of attempted penetration by S. fuliginea into epidermal cells of induced plants were characterized by localized autofluorescence under appressoria and the presence of papilla. These cytological events were found at a lower frequency in the control plants. In addition, the cell walls and papilla of induced plants exhibited increased peroxidase activity. Increases in cell wall-associated peroxidase activity were not observed in the control plants. These authors also noted that lignification occurred in the epidermal cells of the induced and control plants during the first 3 days after inoculation, but only in induced plants after that time. Interestingly, the epidermal cells of the induced plants exhibited cell wall thickening and lignification under the hyphae as the pathogen grew across the leaf surface. This type of lignin deposition was not seen in the controls. 2. Green Bean. One of the first studies on the histology of induced resistance was reported by Elliston et al. (1976b), working with the bean Phaseolus vulgaris-Colletotrichum lindemuthianum system. As described above, inoculation of etiolated bean hypocotyls with nonpathogenic Colletotrichum species or avirulent races of C. lindemuthianum resulted in the induction of resistance of the bean tissue against virulent races of the pathogen (Elliston et al. 1971, 1976a,b). Elliston and coworkers reported that induced tissues did allow penetration by the pathogen and that the initial phases of infection were the same in induced and control tissues. Primary infection hyphae developed in all tissues (representative of the hemibiotrophic phase of the infection). However, in induced tissues, the pathogen stopped developing at the primary hyphae phase and the host tissue became necrotic. In control tissues, the primary hyphae differentiated into the rapidly growing secondary hyphae that quickly colonized the issue. Thus, the induced resistance state appeared to be associated with failure of the pathogen to differentiate from the hemibiotrophic primary hyphae stage into the necrotrophic secondary hyphae. These authors also showed that this type of hostpathogen behavior was very similar to that seen in the resistance that develops in all cultivars of bean as a result of tissue maturity. The histological events described above were independent of the type of inducing pathogen used. The reason the pathogen is not able to develop beyond the primary infection hyphal stage into the rapidly growing and more destructive secondary hyphae is not known

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

259

but may be key to understanding how acquired resistance operates in bean. The histology of infection into systemically induced trifoliate leaves of green bean plants was reported by Cloud and Deverall (1987). In this study, the primary leaves of bean were inoculated with C. lindemuthianum, and 1 week later, the first set of trifoliate leaves were challenge-inoculated with a compatible race of the pathogen. Unlike the findings of Elliston et al. (1976b) described above, Cloud and Deverall (1987) reported that there was a significant reduction of penetrations from appressoria into leaf epidermal cells of induced as compared to control plants. The numbers of epidermal cell wall appositions under appressoria were more frequently seen in induced as compared to control plants, and these structures may have been at least partly responsible for the decrease in penetration efficiency. The authors reported that the cell wall appositions stained purple with toluidine blue, which suggests that these structures were not lignified. When penetration did occur into the epidermal cells of induced plants, the hyphae were often shorter than in epidermal cells of control plants. By 72 h after infection, the fungal hyphae in control plants had penetrated into the mesophyll tissues, while those hyphae in induced plants had not penetrated beyond epidermal cells, which were becoming necrotic. 3. Tobacco, Potato, and Tomato. Induced resistance in tobacco, potato, and tomato has also been examined microscopically. Peron asp ora tabacina, the cause of blue mold, was studied in tobacco. In both tomato and potato, the challenge pathogen was the late blight fungus Phytophthora infestans. The cytology of infection by P. tabacina into induced tobacco leaf tissue was studied by Stolle et al. (1988). They reported that there was no effect on the penetration of the pathogen into either control or induced leaf tissues. Development of P. tabacina was identical in both induced and control tissues for the first 5 days after inoculation. However, at 5 days after challenge inoculation, the development of the pathogen in the induced plants stopped and the pathogen remained within the lesion area; in control tissues the pathogen continued normal development for up to 9 days. No evidence for a hypersensitive-like reaction to the pathogen was detected. In a later study, Ye et al. (1992) reported that the development of P. tabacina in systemically induced tobacco leaf tissue was restricted within 2 days of inoculation. Host cells became necrotic in induced plants by 5 days after challenge. Lignin deposition, as determined

260

R. HAMMERSCHMIDT AND

J.

S. BECKER

by histochemistry, was observed in the induced-infected leaves at 2 days (the time when pathogen growth was starting to be restricted). No lignification was observed in the inoculated control plants. Material that encased the haustoria of P. tabacina was also seen in the induced plant tissues. Working with potato plants that had been previously inoculated on the lower leaves with a resistance-inducing cell wall elicitor preparation from P. infestans, Doke et al. (1987) and Chai and Doke (1987) reported that leaf-induced plants supported a lower rate of zoospore cyst germination (cystospores). In addition, penetration from the cystospores that did germinate was much lower on induced than on control leaf tissue. Thus, these studies suggested that not only did the induced resistance state influence the ability of the pathogen to penetrate and become established in the host tissue, but that there was also an inhibitory effect on cystospore germination. Stromberg and Brisshammar (1993), however, reported different results from those of Doke and coworkers. After inoculation of potato leaves with zoospores, it was found that a higher proportion of cystospores germinated on induced as compared to control leaves. However, these authors did report fewer penetrations of the pathogen into induced as compared to control, which was in agreement with previous results (Doke et al. 1987; Chai and Doke 1987). Associated with the decreased penetration was the more frequent formation of papillae under the appressoria of cystospores on induced as compared to control plants. More localized host cell necrotization was observed in the induced as compared to the control plant at 40 h after inoculation (Stromberg and Brisshammar 1993). This necrotization of individual cells was similar in appearance to the hypersensitive-like cell death that has been observed in potato after infection with an incompatible race of P. infestans. Heller and Gessler (1986) reported on the histology of induced resistance of tomato to P. infestans. These authors found that cystospores germinated and directly penetrated into noninduced host tissue without any visible host responses. In induced tissues, the cystospores germinated Clnd formed appressoria similar to what was observed on the controls. However, papilla-associated lack of penetration was associated with appressoria on the induced rate. When successful penetrations did occur, the host cells rapidly died and the resultant onset of cellular necrosis was associated with cessation of fungal development. Kovats et al. (1991a) also studied the histology of induced resistance of tomato to P. infestans. These authors reported that the in-

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

261

duced resistance was expressed in three forms. The first was a leaf surface phenomenon that decreased the number of cystospores that germinated. Second (in plants exhibiting a high level of induced resistance based on lesion severity measurements), the infection process was often stopped at the point of penetration into the epidermis. Some evidence for the deposition of phenolics (as autofluorescent materials) and callose under appressoria was noted in this record type of resistance expression, but these events were seen in both control and induced plants. Finally, if the pathogen penetrated the host mesophyll tissue, the induced plants responded with more hypersensitive cell death than was observed in the controls. This third type of response was consistent with what Heller and Gessler (1986) reported for tomato and Stromberg and Brisshammar (1993) reported for induced resistance to P. infestans on potato. 4. Arabidopsis. Using Fusarium oxysporum as an inducing agent, Mauch-Mani and Slusarenko (1994) reported that the induced resistance response of Arabidopsis to P. parasitica was related to a more rapid necrotization of the host tissue after challenge infection. The responses observed ranged from single-cell hypersensitivity to what the authors called a "trailing necrosis" of host cells that was associated with restriction of hyphal growth. The hypersensitive-like response was observed most frequently in plants that were induced 7 days prior to challenge, while the trailing necrosis was seen in plants that were induced only 4 days prior to challenge. In plants induced for 4 days, the authors also noted the encasement of haustoria. The nature of this encasing material was not determined. The same types of histological events were also observed in plants that were induced by pretreatment with INA.

B. Biochemical Changes One of the characteristics of acquired resistance is that it is effective against a broad spectrum of pathogens. This suggests that a number of different mechanisms or the enhanced ability to induce these mechanisms after challenge infection must accompany the development of acquired resistance. In this section, a summary of what is known about these putative mechanisms is presented. The molecular regulation of the following defense responses is currently under investigation for a number of systems. Several reviews of this topic have recently been published, and the reader is

262

R. HAMMERSCHMIDT AND

J.

S. BECKER

referred to these for current details and perspectives on this topic (Ryals et a1. 1994; Stermer 1995). 1. Phytoalexins. One of the classical responses of plants to infection is the synthesis of low molecular weight antibiotics known as phytoalexins. Although the original concept of phytoalexins was derived from studies on local acquired resistance in potato to the late blight fungus (Muller and Borger 1940), the role of these compounds in acquired resistance is not clear. The importance of phytoalexins in the bean-C. lindemuthianum system was studied by Elliston et a1. (1977). All of the avirulent races of C. lindemuthianum as well as many of the Colletotrichum species that were nonpathogens of bean induced a local accumulation of the phytoalexin phaseollin at the site of infection. Since local resistance to virulent races of C. lindemuthianum could be demonstrated by placing the challenge inoculum at the site of the inducing infection (Rahe et a1. 1969; Skipp and Deverall 1973), it was possible that phytoalexins could playa role in the local induced resistance. That phaseollin did not playa direct role in the establishment of systemic induced resistance was demonstrated by Elliston et a1. (1977). They found that noninfected tissues at a distance from the primary inoculation (inducer) site were resistant to infection but did not contain phytoalexins. Thus, the phytoalexins produced at the infection site were not translocated to other parts of the hypocotyl to directly confer resistance. However, phytoalexins were produced more rapidly in the induced tissues after challenge inoculation with a virulent race of C. lindemuthianum. It was therefore concluded that the initial inducing infection conditioned the tissue to respond more quickly to the challenge infection, and that part of this response was the enhanced ability to produce phaseollin. A different situation was found for the systemic acquired resistance of tobacco to P. tabacina. Stolle et a1. (1988) reported that tobacco leaf tissue accumulated several sesquiterpenoid phytoalexins after infection with P. tabacina. There was, however, no correlation in either timing or total quantity of phytoalexins produced with the resistance response. It was thus concluded that phytoalexins do not playa major role in the systemic acquired resistance response to P. tabacina.

2. Peroxidase and Associated Biochemical Changes. The first sys-

temic biochemical change detected in plants expressing acquired resistance was enhanced peroxidase activity in tobacco in response

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

263

to inoculation with TMV (Simons and Ross 1970). Subsequent studies have confirmed the increase in peroxidase in systemically protected tobacco (Ye et al. 1990a,b) and have reported systemic peroxidase increases in cucurbits after infection of lower leaves (Hammerschmidt et al. 1982; Smith and Hammerschmidt 1988). In both tobacco and cucurbits, the increases in peroxidase activity appeared to be associated with enhanced activity of a specific group of peroxidase isoforms. One of the earliest detectable biochemical changes that is observed in tobacco and cucumber that are developing acquired resistance is the accumulation of apoplastic and cell wall-associated peroxidase (Hammerschmidt et al. 1982; Ye et al. 1990a). One possible function for the production of these enzymes is to cross-link additional hydroxyproline-rich glycoproteins (HRGP) into the cell wall. In tobacco, inoculation of lower leaves with TMV resulted in a systemic increase in the amount of cell wall bound hydroxyproline (Ye et al. 1992). Since cell walls that have additional HRGP cross-linked into them are more resistant to enzymatic attack (Stermer and Hammerschmidt 1987), it is possible that the induction-associated increases in HRGPs may increase the resistance of the host cell walls to pathogen attack. This potential role for HRGP was strengthened by the observation that challenge inoculation of the induced tobacco with P. tabacina increased the amount of HRGP over time (Ye et al. 1992). There was no increase in control tobacco cell walls even after infection with P. tabacina. Although local increases in HRGP have been documented as resistance responses in cucumber (Hammerschmidt et al. 1984), no systemic changes in cell wall bound HRGPs have been documented, even though there is a systemic increase in peroxidase. Peroxidases are also thought to be involved in lignin polymerization. In systemically induced tobacco, the host responded to infection with P. tabacina by depositing lignin in its cell walls within a few days of challenge (Ye et al. 1992). No comparable cell wall changes were observed in the control. Additional evidence for a role for peroxidase in systemic resistance-associated lignification comes from studies with cucumber. Inoculation of one leaf of cucumber plants with any number of necrotic lesion-inducing pathogens results in the systemic increase in the amount of three 30- to 33-kD, acidic, apoplastic peroxidases (Hammerschmidt et al. 1982; Smith and Hammerschmidt 1988). Associated with the appearance of these peroxidases is an enhanced ability of the host tissue to deposit lignin after challenge inoculation or even wounding (Dean and KulS 1985; Hammerschmidt and KulS 1982). Although it is tempting to

264

R. HAMMERSCHMIDT AND

J.

S. BECKER

speculate that the increased peroxidase activity in cucumber is directly related to the enhanced resistance and lignification capacity, the evidence linking these phenomena is only correlative. In addition, the total activity of peroxidase cannot be correlated to the level of resistance because apoplastic peroxidases accumulate in all leaf tissues as they mature regardless of whether or not the plant was induced (R. Hammerschmidt, unpublished observations). The systemic induction of these peroxidases, however, is correlated very well with the onset of systemic resistance in cucumber. The peroxidases appear just prior to when the plants can be shown to have resistance induced (Hammerschmidt et a1. 1982; Smith et a1. 1991). Thus, when proper controls are present, these enzymes can provide a marker for the ind uction of resistance. Further understanding of the role of peroxidase in systemic resistance induction and expression requires understanding the regulation of the peroxidase genes. Recently, Rasmussen et a1. (1995) reported the cloning of the 33-kD acidic peroxidase that is associated with the expression of systemic resistance. RNA gel blot analysis indicated that the message for this peroxidase was systemically induced by 16 h after inoculation of the first leaf of cucumber plants with the bacterium P. syringae pv. syringae. This bacterium was previously shown to induce the appearance of the peroxidase protein by 20 h after inoculation and systemic resistance to anthracnose within 24 h of the inducing inoculation (Smith et a1. 1991). 3. Lipoxygenase. Lipoxygenase activity has been reported to in-

crease systemically in cucumber after infection with C. lagenarium (Avdiushko et a1. 1993). Similarly, induction of systemic resistance in rice (Smith and Metraux 1991) has also been shown to be correlated with enhanced levels of lip oxygenase (cited in Kessmann, et a1. [1994]). How the increase in this enzyme is related to the expression of acquired resistance is not known, but there are two possibilities. First, the enhanced lip oxygenase activity may result in the production of lipid peroxidation products that are toxic to the pathogen (Croft et a1. 1993). Thus, the enzyme may have a direct effect on resistance through the generation of antimicrobial lipid peroxidation products. Alternatively, since lip oxygenase is known to have a role in the production of lipid-derived signal molecules, such as jasmonic acid, the induction of lip oxygenase may be needed for the synthesis of these signals (Farmer 1994). The role of jasmonic acid in systemic acquired disease resistance, however, has not been established.

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

265

4. Activated Oxygen. Activated oxygen species have recently been given attention as an integral part of the active defense response to pathogens (Mehdy 1994; Sutherland 1991). It is now well documented that one of the early events in the hypersensitive response is the generation of hydrogen peroxide (Levine et a1. 1994). These reactive oxygen species are thought to be involved in the rapid cross-linking of cell wall polymers that may result in increased resistance to pathogen-produced cell wall-degrading enzymes (Stermer and Hammerschmidt 1987). Alternatively, they may function as antimicrobial factors (Peng and KUG 1992). The systemic induction of peroxidase and lipoxygenase that was described above suggests that activated oxygen species may also play a role in systemic resistance expression. This is further supported by the observations of Chai and Doke (1987), who found that local and systemic induction of resistance in potato foliage to P. infestans was accompanied by an increase in the activity of a superoxide generating activity. This was accompanied by increased superoxide dismutase, which could be involved in the conversion of superoxide into hydrogen peroxide. Although not addressed in the report by Chai and Doke, the systemic increase in cross-linked hydroxyproline-rich glycoproteins in tobacco cell walls (Ye et a1. 1992) indicates that the increase in activated oxygen may function in the strengthening of cell walls. Hydrogen peroxide has also been reported to function as a local signal in the induction of systemic induced resistance (Chen et a1. 1993). This is discussed later in this review.

5. Pathogenesis-Related (PR) Proteins. In addition to peroxidase, many plants also systemically accumulate a group of proteins collectively known as the pathogenesis-related proteins. These proteins have been classified into several major groups that have been given the names PR1 through PR5. Several lengthy reviews on these proteins and their regulation have been recently published, and the reader is directed to these for more details (Cutt and Klessig 1992; Linthorst 1991; Ryals et a1. 1994; Stermer 1995). Only a brief description of the proteins and the evidence supporting their role in systemic acquired resistance is described in this section. The role that PR proteins play in acquired resistance is based on two lines of evidence. First, these proteins are systemically induced by pathogens or resistance-inducing chemicals such as salicylic acid (Ye et a1. 1990b; Ward et a1. 1991). Other evidence comes from use of

266

R. HAMMERSCHMIDT AND

J.

S. BECKER

transgenic plants expressing these proteins or from the biological activity of these proteins. Expression of PRl in transgeneic tobacco increases resistance to oomycete pathogens (Alexander et al. 1993). This may be due to the antifungal properties of PR1. The [3-1,3glucanases and chitinases (the PR-2 and PR-3 groups, respectively) have the ability to degrade fungal cell walls (Linthorst 1991), which suggests that they may be involved in active defense. Overexpression of chitinase in transgenic plants has been shown to enhance resistance to some pathogens (Broglie et al. 1991). However, the observation that the cucumber chitinase is developmentally regulated as well as induced by pathogens suggests that these proteins may play other roles in plant growth and development (Lawton et al. 1992). Expression of the antifungal protein osmotin (a member of the PR-5 family) in potato resulted in increased resistance of the plants to the late blight fungus, P. infestans (Liu et al. 1994). C. Summary Each of the defense-related phenomena reported above have characteristics and correlations that strongly suggest that each is associated with the resistant state. However, when examined individually (e.g., use of transformation to overexpress one specific gene), the contribution of each to resistance appears to be small. This strongly suggests that the expression of full resistance requires the expression of several mechanisms, many of which are known to be coordinately regulated (Ward et al. 1991). Further studies evaluating each putative mechanism alone or in combination with other defense responses will eventually help to determine the contribution of each mechanism to the resistant state.

IV. THE SYSTEMIC SIGNAL FOR RESISTANCE A. Evidence for a Systemic Signal The observations that infection of one part of a plant with a pathogen results in the systemic expression of disease resistance strongly suggest that there is a systemically mobile signal that moves from the inducing inoculation (or treatment) site. In this section, the evidence for the presence of the systemic signal for resistance will be presented along with an evaluation of some possible candidates.

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

267

In cucumber, Dean and Kuc (1986a,b) showed that the source of the translocated signal or signals that are involved in systemic expression of resistance is the inoculated leaf used for induction. Removing the inoculated leaf prior to the appearance of necrotic lesions prevented systemic resistance from developing in leaves above the inoculated leaf. These experiments, therefore, provided some evidence that the systemic signal was generated and/or released at a specific time during lesion development. Because systemic resistance develops in leaf tissue both above and below the induction site and in roots, it was postulated that the signal is phloem-translocated. This theory was supported by experiments in which signal movement was inhibited by heat killing of the phloem of petioles (Guedes et al. 1980). However, until the actual chemical or physical nature of the signal or signals is known, the precise route of transport will not be known for certain. The signal for systemic resistance in cucumber also can move through graft unions, and this further supports the intraplant transport of the signal. Grafting experiments in which cucumber, muskmelon, or watermelon scions were grafted onto cucumber rootstocks with one true leaf that was used as the signal source (i.e., the leaf used for induction inoculation) demonstrated that the signal generated in cucumber was also effective in inducing resistance in other cucurbits (Jenns and Kuc 1979). This suggested that the cucumber signal is identical or very similar to the signals used in other cucurbits. Therefore, if one signal molecule can be identified from one cucurbit, its role in induced resistance should be strengthened by identifying the same compound in another cucurbit and by showing biological activity in all cucurbits. Systemic acquired resistance in tobacco also appears to require a systemically translocated signal that is produced in an infected leaf and then transported throughout the plant (Tuzun and Kuc 1985). Similar to cucumber and other cucurbits, the tobacco signal is also graft transmissible (Tuzun and Kuc 1985; Vernooij, et al. 1994). B. Salicylic Acid There have been several proposals for what the local and systemic signals for resistance might be, including abscisic acid, oligosaccharides, jasmonic acid, peptides, and salicylic acid (Farmer 1994; Hammerschmidt 1993; Pierpont 1994). Several of these have been proposed as wound-induced signals that increase plant resistance

268

R. HAMMERSCHMIDT AND

J.

S. BECKER

to arthropod herbivores (Farmer 1994; Hammerschmidt 1993). Of these putative signals, the molecule that has received the most attention is salicylic acid, and the role that this phenol plays in various plant processes has been extensively reviewed (Klessig and Malamy 1994; Pierpont 1994; Raskin 1992). Thus, the discussion in this section provides only a brief overview of the role salicylic acid plays in acquired resistance. Treatment of tobacco (White, 1979) and cucumber (Mills and Wood 1984) with acetylsalicylic acid (aspirin) induced resistance to TMV in tobacco and in cucumber to C. lagenarium, respectively. Exogenous application of salicylic acid is also a well-known inducer of PR proteins (see reviews by Cutt and Klessig 1992; Linthorst 1991; Pierpont 1994) Such evidence, along with the wide-spread occurrence of salicylic acid in the plant kingdom (Pierpont 1994; Raskin 1992) suggested that this compound might function as an endogenous signal for induced resistance. A possible role for salicylic acid in systemic induced resistance was reported for both cucumber (Metraux et a1. 1990) and tobacco (Malamy et a1. 1990). In both cases, the amount of salicylic acid in noninfected tissues rose prior to the onset of resistance. This was supported by the observations of Mills and Wood (1984) and Rasmussen et a1. (1991), who demonstrated that cucumber could be protected against infection by c. lagenarium by prior treatment with salicylic acid. Exogenous application of salicylic acid has also been shown to induce resistance in tobacco to Peron asp ora tabacina (Ye et a1. 1990b) and to Erwinia carotovora (Jones) Bergey et a1. (PaIva et a1. 1994). An extensive review by Pierpont (1994) provides further information on the ability of salicylic acid to induce as well as not induce resistance in a number of plant species. Treatment of cucumber plants with exogenous salicylic acid was also shown to induce two markers for systemic acquired resistance in cucumber: an apoplastic acidic chitinase and three apoplastic acidic peroxidase isoforms. Metraux et a1. (1989) and Lawton et a1. (1992) have shown that chitinase transcripts are strongly induced by salicylic acid at concentrations that induce resistance. Similarly, Rasmussen et a1. (1991,1995) have demonstrated that salicylic acid will induce both peroxidase activity and peroxidase transcript accumulation. In addition, Rasmussen et a1. (1991) have shown that the increase in salicylic acid in the phloem of cucumber precedes the increase in acidic peroxidase activity. In N-gene tobacco, salicylic acid systemically increased in TMVinfected plants prior to the expression of the acquired resistance-

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

269

associated PR protein, PR-1 (Malamy et al. 1990; Malamy and Klessig 1992). Furthermore, exogenus feeding of salicylate to leaves demonstrated that the levels of salicylic acid found in plants after TMV infection are sufficient to induce PR-1 expression (Yalpani et al. 1991). Salicylic acid is a benzoic acid derivative, and is thought to be produced by O-hydroxylation of benzoic acid. (Leon 1993; Yalpani et al. 1993). Since phenols are synthesized at wound or infection sites (Nicholson and Hammerschmidt 1992), production of salicylic acid at these sites is a possibility. This suggests that the local production of salicylic acid after infection may be the source of the translocated material. Some of the most convincing evidence for a role for salicylic acid in the induction of resistance was reported in a series of papers by Ryals and his coworkers. Gaffney et al. (1993) transformed tobacco with a bacterial gene that converts salicylic acid to catechol (salicylic acid hydroxylase encoded by the nahG gene). Gaffney et a1. (1993) reported that transgenic tobacco constitutively expressing nahG did not accumulate salicylic acid after infection with TMV and did not express systemic resistance. In a subsequent paper, (Delaney et al. 1994) nahG transformed tobacco and Arabidopsis plants were found to be more susceptible to a number of pathogens. The observation of Delaney et al. (1994), along with the previous report from this group, supported the role for salicylic acid in both systemic acquired resistance and local resistance to infection. The fact that the signal for systemic resistance appears to be phloem mobile and the observation that enhanced amounts of salicylic acid are found in phloem exudates during the induction of resistance (M8iraux et al. 1990; Yalpani et al. 1991) suggested that salicylic acid might be the translocated signal. Rasmussen et al. (1991), addressed this question and proposed that salicylic acid accumulates in the phloem of cucumber in response to another signal that is translocated throughout the plant. These experiments were based on previous observations by Smith et al. (1991). These authors reported that systemic acquired resistance developed in cucumber within 24 h of inoculation with the nonpathogen P. syringae pv. syringae and that systemic acquired resistance developed to a small degree if the inoculated first leaf was left on the plant for as little as 6 h. Using P. syringae pv. syringae to induce systemic resistance, Rasmussen et al. (1991) reported that detaching the inoculated the leaf at 4 h after inoculation resulted in the systemic accumulation of salicylic acid in the phloem, even though no increases in salicylic acid were detected in the phloem exudate of inoculated leaf at the

270

R. HAMMERSCHMIDT AND

J. S. BECKER

time it was detached. Allowing the P. syringae pv. syringae-inoculated leaf to remain on the plant for as little as 6 h allowed the accumulation of over 200 I-lM salicylate in the petiole of the leaf above the inoculated leaf when sampled at 24 h postinoculation. Salicylate levels in phloem exudates of the detached P. syringae pv. syringae-inoculated leaves were no different than the controls at 4 and 6 h and did not increase in the petioles of inoculated leaves until 8 h after inoculation (Rasmussen et a1. 1991). These results suggest that salicylic acid is induced throughout the plant by another signal generated at the infection site. Evidence that salicylic acid is not the translocated signal was supported in a recent study by Vernooij et a1. (1994). These authors grafted wild-type tobacco scions onto nahG transformed tobacco rootstock. The hypothesis behind the experiment was that if salicylic acid was the translocated signal, then inoculation of the nahG rootstocks would not systemically protect the wild-type scions. TMV infection of the nahG root stocks did, however, result in the development of resistance in the wild-type scion. These results indicated that salicylic acid was not the translocated signal, because the rootstocks could not accumulate salicylic acid. Thus, the translocated systemic signal that results in the onset of acquired disease resistance may function by inducing the accumulation of salicylic acid. C. How Does Salicylic Acid Induce Resistance? Although there is now very good evidence to support a role for salicylic acid as an endogenous signal in the induction of resistance in cucumber, tobacco, and Arabidopsis, less is known about how this compound acts to induce resistance. Klessig and coworkers have addressed this question by searching for a receptor for salicylic acid. They have recently reported on the presence of a soluble protein that is capable of binding salicylic acid and analogs of salicylic acid that are capable of inducing resistance in tobacco (Chen and Klessig 1991). Analysis of this protein has revealed that it is a catalase (Chen et a1. 1993). Interestingly, the binding of salicylic acid to this catalase inhibits the enzymatic activity of this protein, which has led to the hypothesis that salicylic acid induces resistance by blocking catalase activity. This, in turn, would result in the accumulation of hydrogen peroxide, which then activates resistance (Chen et a1. 1993). Because of the observation that exogenous hydrogen peroxide can induce PR gene expression in tobacco (Chen et a1. 1993) as well as

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

271

the expression of various other defense processes (Mehdy 1994), this hypothesis is worth confirmation (see Klessig and Malamy 1994; Dempsey and Klessig 1995). Genetic analysis may also provide an approach to understanding how salicylic acid regulates resistance. Two types of acquired resistance mutants in Arabidopsis have recently been described. One of these constitutively expresses resistance that is biochemically similar to the state of acquired resistance (Bowling et al. 1994). These plants contain elevated levels of salicylic acid and PR proteins, and studies suggest that this phenotype is the result in a change in the regulation of salicylic acid in these plants. A second class of mutants are noninducible by exogenous salicylic acid (Cao et al. 1994). These may prove to be very useful in understanding the mechanism of salicylic acid induction of resistance, but may not yield information that is specific for salicylic acid action, because these mutants are also not induced by the synthetic inducer of resistance, INA. Selection of more mutants that are insensitive to salicylic acid may provide the best approach to understanding how this simple phenol can induce resistance. D. Systemic Signal Generation

A prerequisite for the induction of systemic induced resistance by pathogens is the development of a necrotic lesion. Whether or not cell death itself or the events leading to cell death are necessary for the generation of the signal is not known. Some results have been presented that may help to clarify the role of plant cell death in the generation of the signal. Dean and Kuc (1986b) demonstrated, as described above, that detaching the inoculated first leaf of cucumber prior to the onset of visible symptoms resulted in no systemic resistance. These experiments were carried out using C. lagenarium as the inducing agent, and the time intervals used were in days. Thus, this work could only approximate the relationship between host cell death and the generation of the signal. Smith et al. (1991) provided more precise information on the timing of host cell death and the generation of the signal. These authors reported that inoculating cucumber with the hypersensitive responseinducing bacterium P. syringae pv. syringae would result in the systemic expression of resistance within 24 h. Detaching the Pseudomonas-infected leaf at 2-h intervals after inoculation revealed that the signal for systemic resistance was generated by 6 h after

272

R. HAMMERSCHMIDT AND

J.

S. BECKER

inoculation. This was before there were any visible signs of host cell necrosis. However, based on other studies on the induction of the bacterial hypersensitive response, the host cells were starting to undergo the early metabolic changes that are associated with the hypersensitive response. These events, such as membrane permeability changes, active oxygen production, and lipid peroxidation, have been recently reviewed in relation to the time of induction of the systemic signal (Hammerschmidt 1993). With the large amount of information that is now available on the hypersensitive response at the molecular and physiological level, a full understanding of the hypersensitive response may be of great value in determining the nature of the signal.

V. ACQUIRED RESISTANCE AND DISEASE CONTROL

The fact that acquired resistance occurs has suggested that this type of resistance might be useful in the control of disease in agricultural systems. Trials by Kuc and coworkers (Caruso and Kuc 1977b; Tuzun and Kuc 1989) have demonstrated that both tobacco and several cucurbits can be successfully protected from disease by acquired resistance under field conditions. However, one of the potential problems in the use of acquired resistance in disease control has been finding means for inducing resistance without having to inoculate individual plants. This section describes some of the recent advances that may allow the successful use of acquired resistance in agricultural systems. Several other approaches, have been recently discussed by Kessmann et al. (1994) and Tuzun and Kloepper (1995). We concentrate on two different types of inducing agents-nonpathogenic microbes and chemicals that mimic the biological acquired resistance response-because both can be readily used to treat planting materials or as foliar sprays. A. Microbial Inducers Although effective in inducing resistance, the use of virulent, necrosis-inducing pathogens has some obvious limitations. However, a number of avirulent strains of pathogens have been used to induce some degree of protection. For example, resistance to Fusarium wilt in tomato can be induced by a F. oxysporum forma specialis that is not pathogenic on tomato (Kroon et al. 1991). Sweet potato wilt,

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

273

caused by F. oxysporum Schlechtend f.sp. batatas (Wollenweb.) Snyder and Hans. (cited in Cook and Baker [1983]), has been successfully controlled under field conditions by treatment of the planting material with spores of the avirulent strain. Although effective, this type of approach is probably limited to root diseases of crops that are transplanted. An approach to practical use of acquired resistance that has received recent attention is to use bacteria known as PGPR (plant growth promoting rhizobacteria). These bacteria have the ability to enhance growth of plants as well as reduce the amount of disease seen on the plants colonized by these bacteria (Cook and Baker 1983). One possible means for disease suppression by these bacteria is through induced resistance, which has been demonstrated in several systems (Tuzun and Kloepper 1995). Several examples of PGPR-induced resistance have been recently reviewed by Tuzun and Kloepper (1995). Wei et a1. (1991) reported systemic induction of resistance to C. lagenarium infection as a result of inoculating roots with various PGPR strains. Tuzun and Kloepper (1995) provided more evidence for the resistance-inducing activity of the PGPR strains by showing that these bacteria protected cucumber against E. tracheiphila and P. syringae pv. lachrymans. These authors have also demonstrated that the PGPRmediated resistance was effective in controlling bacterial wilt of cucumber, caused by E. tracheiphila, under field conditions. In a recent paper, Zhou and Paulitz (1994) provided further evidence for systemic resistance induction in cucumber by root colonizing pseudomonads. Using a split root system, they were able to demonstrate enhanced systemic resistance to root infection by Pythium as well as the growth-promoting effects typically associated with these bacteria. Similarly, Maurhofer et a1. (1994) described the systemic induction of resistance in leaves of tobacco to tobacco necrosis virus by a Pseudomenas fluorescens Migula strain. Not only did these authors report an increase in resistance, but they also found an increase in PR proteins that was similar to the amounts induced by a resistanceinducing treatment with tobacco necrosis virus. A strain of the bacterium that did not produce antibiotics was also effective in systemic induction of resistance, thus further supporting the hypothesis that the mode of protection was induced resistance. Further understanding of the way that these bacteria protect plants against disease either through induced resistance or other means could lead to a novel means of plant protection.

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

275

2. Phosphates and Oxalates. Extraction and fractionation of spinach leaf tissue resulted in the isolation of oxalic acid as a resistanceinducing compound (Doubrava et al.19SS). Spraying lower cucumber leaves with oxalate resulted in the systemic expression of resistance to C. lagenarium. Treatment of the leaf resulted in a slight chlorotic stippling that was associated with the induction of resistance. Because of the ability of oxalate to chelate calcium, di- and tribasic potassium phosphate salts were also tested as inducers of resistance in cucumber (Gottstein and Kuc 19S9; Mucharromah and Kuc/ 19S9). Both of these compounds were very effective in inducing both systemic resistance and in inducing systemic accumulation of chitinase and peroxidase, two biochemical markers for the induction of resistance (Irving and Kuc 1990). Phosphates have also been reported as effective inducers of systemic resistance in potato against late blight (Stromberg and Brisshammar 1991). Walters and Murray (1992) reported that phosphates would also induce systemic resistance in broad bean to a subsequent challenge by the rust fungus Uromyces vignae. Based on the observations that these chemical inducers cause a chlorotic/necrotic stippling of leaf tissue that is similar to certain pathogen attacks (Gottstein and Kuc 19S9), it is likely that oxalates and phosphates act by inducing the production of an endogenous, translocated resistance-inducing signal or signals.

3. Fatty Acid Derivatives. The observations that a cell wall elicitor (Doke et al. 19S7) and a lipoglycoprotein (Ozeretskovskaya 1995) from P. infestans are capable of inducing systemic resistance in potato provided evidence for fatty acids as inducers because the active material in these preparations is arachidonic acid, a polyunsaturated fatty acid elicitor (Bostock et al. 19S1). Treatment of lower leaves with pure arachidonic acid or other polyunsaturated fatty acids like linolenic acid also resulted in the systemic induction of resistance to late blight (Cohen et al. 1991). The effective concentration of these pure fatty acids was, however, very high. Although not demonstrated, it is possible that these fatty acids induce resistance not directly, but via the generation of another translocated signal. The fatty acid derivative jasmonic acid has also been shown to protect potato plants against P. infestans (Cohen et al. 1993). The mechanism of protection was not elucidated, and the amount of material needed for protection against this pathogen was very high. However, since jasmonic acid is known to be an inducer of systemic wound responses (Farmer 1994), it is possible that lipid derived sig-

276

R. HAMMERSCHMIDT AND]. S. BECKER

nals like jasmonate may be involved as natural signals for resistance. However, it should be noted that jasmonates did not induce resistance in cucumber seedlings to infection by C. lagenarium (Siegrist et al. 1994). Although the fatty acids and jasmonate do have a protective effect, it is unlikely that these would be useful inducers because of the very high concentrations (1000-2000 ppm) needed to protect the plants. 4. Phenolic Compounds. The discovery that salicylic acid plays a

role in the expression of acquired resistance in several plants has suggested that this compound or other phenols may be useful in induction of resistance. Use of salicylic acid as an inducer is probably not practical because of the narrow range between efficacy and toxicity to the plants (Kessmann et al. 1994). Field trials using salicylic acid as an inducer have not been encouraging (Hammerschmidt and Widders, unpublished results). Some of this may have been the result of failure of the salicylate to penetrate into the tissues. Siegrist et al. (1994) recently reported that both salicylic acid and the 5-chloro derivative of this compound induced resistance to C. lagenarium in hypocotyl segments but not intact hypocotyls. 5. Silicates. Treatment of hydroponically grown cucumber plants with soluble silicates was reported to induce resistance to infection by Pythium (Cherif et al. 1992, 1994). In the latter study, the treated plants reacted to infection by the pathogen by an enhanced accumulation and deposition of phenolic materials. Unlike the report of biologically induced resistance in which silicon was detected in papillae under nonpenetrating appressoria of C. lagenarium, these authors reported no increase in the silicon content of the cucumber root cell walls after infection with Pythium. Thus, they concluded that the silicon was acting as an inducer of resistance. Schneider and Ullrich (1994) reported that foliar application of powdered quartz or silicic acid (80 ppm) to cucumber or tobacco leaves enhanced resistance to Sphaerotheca fuliginea or Pseudomonas syringae pv. tabaci (Wolf and Foster) Young, Dye and Wilkie, respectively. This treatment also enhanced activity of chitinase and peroxidase in these plants and f31,3-glucanase in tobacco. The results of this work also suggested that the silicates were acting by inducing resistance in these plants.

6. 2,6-Dichloroisonicotinic Acid. A recently developed synthetic chemical inducer for disease resistance is the nicotinic acid deriva-

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

277

tive 2,6-dichloroisonicotinic acid (INA, CGA-41396) produced by Ciba-Geigy. Application of this experimental material to a wide variety of crops was shown to induce resistance to disease (Metraux et al. 1991). In tobacco, INA has been shown to induce resistance to a wide variety of pathogens and to also induce the full complement of genes that are associated with biologically induced resistance in this plant (Kessmann et al. 1994). INA also induces resistance to several diseases and activates the expression of the PR1 gene in Arabidopsis. As mentioned earlier, INA also induces resistance in bean to anthracnose, rust, and halo blight (Dann and Deverall 1995) and in cucumber to anthracnose (Siegrist et al. 1994). In the latter case, the histological phenotype of the INA induced resistance in cucumber was identical to that seen in biologically induced resistance (Hammerschmidt and Kuc 1982). Nielsen et al. (1994) also reported that INA was effective in protecting sugar beet against infection by Cercospora beticola. Interestingly, no evidence for PR protein induction by INA was found. However, nothing is known about the characteristics of biological induced resistance in this plant. The lack of PR protein induction may indicate that induced resistance in this plant operates in a manner different from tobacco, cucumber, or Arabidopsis. Field testing of INA as the only control measure has also been carried out on a number of crops (Metraux et al. 1991). We have successfully used INA in field studies on cucumber (R. Hammerschmidt, 1. Widders, and L. Newall, unpublished results) and soybean (R. Hammerschmidt and B. Diers, unpublished results). In 3 years of field tests, treatment of several cultivars of cucumber with a range of concentrations and numbers of applications of INA prior to anthesis resulted in the expression of resistance to a challenge inoculation with the angular leaf spot bacterium P. syringae pv. lachrymans. The resistance was quantified as a reduction of the number of angular leaf spot lesions. Since the experiments were carried out over a 3-year period and under somewhat different environmental conditions each year, the efficacy of this compound appears to be very good. Dann and Deverall have also studied the efficacy of INA in the control of green bean rust disease under field conditions (Dann and Deverall, personal communication). In 2 years of field testing, they found that one treatment with INA when the plants were 16-20 days old reduced the severity of rust as shown by a two- to tenfold reduction in the number of uredinia produced on the inoculated leaves. They also found a reduction of spread of the disease to upper leaves that were not inoculated.

278

R. HAMMERSCHMIDT AND

J.

S. BECKER

VI. SUMMARY

The ability to induce resistance in plants that have no apparent genes for resistance has led to a number of new avenues for research in disease control. The observations that induced resistance utilizes the same mechanisms for restricting pathogen attack as are induced in plants containing major gene for resistance has suggested that this type of resistance is environmentally safe (Kuc: 1983,1987). Because of the apparent safety and the broad-spectrum nature of the induced resistance, research is underway to identify and develop microbes or nontoxic chemicals that can be used to induce resistance in plants and thus make this type of resistance directly applicable to disease control in the field (Kessman et al. 1994; Kuc: 1995; Tuzun and Kloepper 1995). Studies on induced resistance have also led to a new understanding of which genes may be involved in the expression of the resistant state (Ryals et al. 1994; Stermer 1995). Knowing which genes are involved may lead to novel control by engineering plants with these genes. There is, however, a need for more research in several areas. In the first part of this paper, examples of acquired resistance from a number of plant families were described. Unfortunately, the number of plant families that have been studied are few. Thus, we have only a limited amount of information on the distribution of acquired resistance in the plant kingdom and these examples. Only a few plant families (cucurbits, legumes, solanaceous plants, crucifers, and graminaceous monocots) have been studied in any detail, and within each family only one or two plants have received any extensive study. Having a better understanding of the induced resistance response in more than one species within a family is important in developing any generalizations about this type of resistance within a family. Despite many studies, information on the relative importance of putative defense mechanisms utilized in acquired resistance is also very limited. We know that in many cases, the induction of acquired resistance is accompanied by the systemic accumulation of PR proteins and other putative defenses. In addition, a few studies have also demonstrated that the induced plants can respond to subsequent challenge infection by expression of additional defenses. These latter studies have shown that acquired resistance appears to use the same types of defenses as major gene or nonhost resistance. However, it is not known with certainty which of the defenses that are expressed as a result of an inducing treatment or are elected after a challenge infection are required for the expression of resistance. Knowing which defenses are most critical in the expression of in-

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

279

duced resistance to a specific pathogen will be helpful in determining how the resistance may be improved or how pathogens may overcome the resistance. There is also a lack of understanding about the nature of the systemically translocated signal that is generated in the inoculated leaf and conditions resistance throughout the plant. As described earlier in this manuscript, salicylic acid appears to be an important signal molecule in the expression of systemic induced resistance in several plants. However, salicylic acid does not appear to be the translocated signal, but rather the accumulation of this compound is induced by a more primary, translocated signal. Finally, induced resistance may be an important part of disease control strategies of the future. Having more concrete information on the biological spectrum of induced resistance throughout the plant kingdom will be of use in determining which plant families may be good "targets" for this type of control. Furthermore, understanding the defenses and the signals that regulate these defenses will also provide new avenues for implementation of induced resistance via genetic engineering or manipulation of signaling pathways. However, we still need more experience in the management and implementation of induced resistance. The future availability of chemical inducers of resistance will prove one approach that should be compatible with current agricultural practices. Promising studies with biocontrol agents that may control disease via induced resistance may provide another means of "delivering" induced resistance. Thus, inducing plants in agricultural settings does not seem as difficult as it did a few years ago. In addition to having efficient means of inducing resistance, studies on the overall efficacy of induced resistance during a growing season and the effects of the environment on the expression and efficacy of induced resistance need study to determine if these factors can enhance or suppress this type of resistance. The incorporation of induced resistance into a managed, integrated pest management disease control strategy should provide one of the best ways of delivering this type of control to growers. The interfacing of induced resistance with cultural, genetic, and even traditional chemical controls should enhance these other controls and provide a new and effective means of controlling plant diseases. LITERATURE CITED Ajlan, A. M. and D. A. Potter. 1991. Does immunization of cucumber against anthracnose by Colletotrichum lagenarium affect host suitability for arthropods? Ent. Exp. App!. 58:83-91

280

R. HAMMERSCHMIDT AND J. S. BECKER

Ajlan, A. M., and D. A. Potter. 1992. Lack of effect of tobacco mosaic virus-induced systemic acquired resistance on arthropod herbivores in tobacco. Phytopathology 82:647-651. Alexander, D., RM. Goodman, M. Gut-Rella, C. Glascock, K. Weyman, L. Friedrich, D. Maddox, P. Ahl-Goy, T. Luntz, E. Ward, and J. Ryals. 1993. Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein la, Proc. Nat. Acad. Sci (USA) 90:7327-7331. Apriyanto, D., and D. A. Potter. 1990. Pathogen-activated induced resistance of cucumber: response of arthropod herbivores to systemically protected leaves. Oecologia 85:25-31. Avdiushko, S. A., X. S. Ye, D. F. Hildebrand, and J. Kuc. 1993. Induction of lipoxygenase activity in immunized cucumber plants. Physiol. Mol. Plant Pathol. 42:83-95. Baldwin, 1. T. 1988a. Mechanism of damage-induced alkaloidproduction in wild tobacco. J. Chern. Ecol. 15:1661-1680. Baldwin, 1. T. 1988b. Short-term damage-induced increases in tobacco alkaloids protects plants. Oecologia 75:367-370. Baldwin, 1. T. 1988b. The alkaloidal responses of wild tobacco toreal and stimulated herbivory. Oecologia 77:378-381. Bashan, B., and Y. Cohen. 1983. Tobacco necrosis virus induces systemic resistance in cucumbers against Spaerotheca fuliginea. Physiol. Plant Pathol. 23:137-144. Bergstrom, G. C. 1981. Biological induction of physiological resistance in cucurbits to Erwinia tracheiphila, Mycospherellamelonis, and cucumber mosaic virus. Thesis, Univ. of Kentucky, Lexington. Bergstrom, G. C., M. C. Johnson, and J. Kuc. 1982. Effects of local infection of cucumber by Colletotrichum lagenarium, Pseudomonas lachrymans, or tobacco necrosis virus on systemic resistance tocucumber mosaic virus, Phytopathology 72:922-926. Biles, C. L. and R Martyn. 1989. Local and systemic resistanceinduced in watermelons by formae specialis of Fusarium oxysporum, Phytopathology 79: 856860. Boller, T., and J. P. Metraux. 1988. Extracellular localization of chitinase in cucumber, Physiol. Mol. Plant Pathol. 33:11-16. Bostock, R. M., R. Laine and J. Kuc. 1981. Eicospentaenoic and arachidonic acids from Phytophthora infestans elicit fungitoxic sesquiterpenes in the potato. Science 212:67-69. Bowling, S. A., A. Guo, A. S. Gordon, D. F. Klessig, and X. Dong. 1994. A mutation in Arabidopsis that leads to constitutive expression of systemic resistance. Plant Cell 6:1845-1857. Brock, P. M., J. R. B. Inwood, and B. K. Deverall. 1994. Systemic induced resistance to Alternaria macrospora in cotton (Gossypium hirsutum). Austral. Plant Pathol. 23:81-85. Broglie, K., 1. Chet, M. Holliday, R. Cressman, P. Riddle, S. Knowlton, C. J. Mauvias, and R Broglie. 1991. Transgenic plants with enhanced resistance to fungal pathogens. Science 254:1194-1197. Cameron, R K., R. A. Dixon, and C. J. Lamb. 1994. Biologically induced systemic acquired resistance in Arabidopsis thaliana. Plant J. 5:715-725. Cao, H., S. A. Bowling, A .S. Gordon, and X. Dong. 1994. Characterization of an Arabidopsis mutant that is non-responsive to inducers of systemic acquired resistance. Plant Cell 6:1583-1592.

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

281

Caruso, F. L., and J. KuG. 1977a. Field protection of cucumber, watermelon and muskmelon against Colletotrichum lagenarium by Colletotrichum lagenarium. Phytopathology 67:1290-1292. Caruso, F. 1., and J. KuG. 1977b. Protection of watermelon and muskmelon against Colletotrichum lagenarium by Colletotrichum lagenarium, Phytopathology 67:1285-1289. Caruso, F. 1., and J. KuG. 1979. Induced systemic resistance of cucumber to anthracnose and angular leaf spot by Pseudomonas lachrymans and Colletotrichum lagenarium, Physiol. Plant Pathol. 14:191-201. Chai, H. B., and N. Doke 1987. Systemic activation of O 2 - generating reaction, superoxide dismutase, and peroxidase in potato plants inrelation to induction of systemic resistance to Phytophthora infestans, Ann. Phytopathol. Soc. Jpn. 53:645-649. Chen, Z., and D. F. Klessig. 1991. Identification of a solubles alicylic-acid binding protein that may function in <signal transduction in the plant disease resistance response. Proc.Nat. Acad. Sci. (USA) 88:8179-8183. Chen, Z., H. Silva, and D. F. Klessig. 1993. Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science 262:1883-1885. Cherif, M., N. Benhamou, J. G. Menzies, and R. R. Belanger. 1992. Silicon induced resistance in cucumber plants against Pythium ultimum. Physiol. Mol. Plant Pathol. 41:411-425. Cherif, M., A. Asselin, and R. R. Belanger. 1994. Defense responses induced by soluble silicon in cucumber roots infected by Pythium species. Phytopathology 84:236-242. Chester, K. S. 1933. The problem of acquired physiological immunity in plants. Quart. Rev. BioI. 8:128-154, 275-324. < Cloud, A. M. K, and B. J. Deverall. 1987. Induction and expression of systemic resistance to the anthracnose disease in bean, Plant Pathol. 36:551-557. Cohen, Y. 1994a. 3-Aminobutyric acid induces resistance against Peronospora tabacina. Physiol. Mol. Plant Pathol. 44:273-288. Cohen, Y. 1994b. Local and systemic control of Phytophthora infestans in tomato plants by DL-3-amino-n-butanoic acids. Phytopthology 84:55-59. Cohen, Y., and U. Gisi. 1994. Systemic translocation of 14C-DL-3-aminobutyric acid in tomato plants in relation to induced resistance against Phytophthora infestans. Physiol. Mol. Plant Pathol. 45:441-456. Cohen, Y., U. Gisi, and K Mosinger. 1991. Systemic resistance ofpotato plants against Phytophthora infestans induced by unsaturated fatty acids, Physiol. Mol. Plant Pathol. 88:255-263. Cohen, Y., U. Gisi, and T. Niderman. 1993. Local and systemic protection against Phytophthora infestans induced in potato and tomato plants by jasmonic acid and jasmonic-methyl ester. Phytopathology 83:1054-1062. Cohen, Y., T. Niderman, K Mosinger, and R. Fluhr. 1994. (3-Aminobutyric acid induces the accumulation of pathogenesis related proteins in tomato (Lycopersicon esculentum L.) and resistance to late blight infection by Phytophthora infestans. Plant Physiol. 104:59-66. Conti, G., M. Bassi, D. Carminucci, 1. Gatti, and A. M. Bocci. 1990. Preinoculation with tobacco necrosis virus enahnces peroxidase activity and lignification in cucumber as a resistance response to Sphaerotheca fuliginea, J. Phytopathol. 128:191-202. Cook, R. J., and K. F. Baker. 1983. The nature and practice of biological control of plant pathogens. APS, St. Paul, MN.

282

R HAMMERSCHMIDT AND J. S. BECKER

Croft, K. P. C., F. Juttner, and A. J. Slusarenko. 1993. Volatile products of the liopxygenase pathway evolved from Phaseolus vulgaris 1. leaves inoculated with Pseudomonas syringae pc. phaseolicola. Plant Physiol. 101:13-24. Cruickshank, 1. A. M. and M. Mandryk. 1960. The effect of stem infection of tobacco with Peronospora tabacina on foliage reactionto blue mold, J. Austral. Inst. Agr. Res. 26:369-372. Cutt, J. R, and D. F. Klessig. 1992. Pathogenesis-related proteins. p. 181- 216. In: F. Meins and T. Boller (eds.), Plant gene research. Springer, New York. Dann, K K, and B. J. Deverall. 1995. Effectiveness of systemic resistance in bean against foliar and soil-borne pathogens as induced by biological and chemical means. Plant Pathol. 44:458-466. Dean, R A., and J. KuG. 1985. Rapid lignification in response to wounding and infection as a mechanism for induced systemic protection in cucumber. Physiol. Mol. Plant Pathol. 31:69-81. Dean, R. A., and J. KUG 1986a. Induced systemic protection in cucumber: The source of the "signaL" Physiol. Mol. Plant Pathol. 28:227-233. Dean, R A., and J. KUG 1986b. Induced systemic protection in cucumber: time of production and movement of the signal. Phytopathology 76:966-970. Delaney, T. P., S. Uknes, B. Vernooij, L. Friedrich, K. Weymann, D. Negrotto, T. Gaffney, M. Gut-Rella, H. Kessmann, K Ward, and J. Ryals. 1994. A central role of salicylic acid in plant disease resistance. Science 266:1247-1250. Dempsey, D. A. and D. F. Klessig. 1995. Signals in plant disease resistance. Bull. Inst. Pasteur. 93:167-186. Deverall, B. J., and E. K. Dann. 1995. Induced resistance in legumes, p. 1-30. In: R Hammerschmidt and J. KuG. (eds.), Induced resistance to disease in plants. Kluwer Academic, Amsterdam. Doke, N., A. V. Ramirez, and K. Tomiyama. 1987. Systemic induction of resistance in potato plants against Phytophthora infestans bylocal treatment with hyphal wall components of the fungus. J. Phytopathol. 119:232-239. Doss, M., and M. Hevisi. 1981. Systemic acquired resistance of cucumber to Pseudomonas lachrymans as expressed in suppression ofsymptoms, but not in multiplication of bacteria. Acta Phytopathol. Sci. Hung. 16:269-272. Doubrava, N. S., R S. Dean, and J. KuG. 1988. Induction of systemic resistance to anthracnose caused by Colletotrichum lagenarium in cucumber by oxalate and extracts from spinach and rhubarb leaves. Physiol. Mol. Plant Pathol. 33:69-79. Elliston, J., J. KUG, and K B. Williams. 1971. Induced resistance to bean anthracnose at a distance from the site of the inducing interaction. Phytopathology 61:11101112. Elliston, J., J. KUG, and K B. Williams. 1976a. Protection ofPhaseolus vulgaris against anthracnose by Colletotrichum species nonpathogenic to bean. Phytopathol. Z. 86:117-126. Elliston, J., J. KUG, and KB. Williams. 1976b. A comparative study of the development of compatible, incompatible, and induced incompatible interactions between Colletotrichum spp. and Phaseolus vulgaris. Phytopathol. Z. 87:289-303. Elliston, J., J. KUG, K B. Williams, and J. K Rahe. 1977. Relationship of phytoalexin accumulation to local and systemic protection of bean against anthracnose. Phytopathol. Z. 88:114-130. Enkerli, J., U. Gisi, and K Mosinger. 1993. Systemic acquired resistance to Phytophthora infestans in tomato and the role of pathogenesis-related proteins. Physiol. Mol. Plant Pathol. 43:161-171.

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

283

Enyedi, A .J., N. Yalpani, P. Silverman, I. Raskin. 1992. Localization, conjugation and function of salicylic acid during thehypersensitive response to tobacco mosaic virus. Proc. Nat. Acad. Sci. (USA) 89:2480-2484. Farmer, K E. 1994. Fatty acid signalling in plants and thier associated microorganisms. Plant Mol. BioI. 26:1423-1437. Gaffney, T., 1. Friedrich, B. Vernooij, D. Negrotto, G. Nye, S.Uknes, K Ward, H. Kessmann, and J. Ryals. 1993. Requirement of salicylic acid for the induction of systemic acquired resistance. Science 261:754-766. Gessler, C., and J. KuG. 1982. Induction of resistance to Fusarium wilt in cucumber by root and foliar pathogens. Phytopathology 72:1439-1441. Gottstein, H., and J. KuG. 1989. Induction of systemic resistance to anthracnose in cucumber by phosphates. Phytopathology 79:176-179. Guedes, M. K M., S. Richmond, and J. KuG. 1980. Induced systemic resistance to anthracnose in cucumber as influenced by the location of the inducer inoculation with Colletotrichum lagenarium and the onset of lowering and fruiting. Physiol. Plant Pathol. 17:229-233. Hammerschmidt, R. 1993. The nature and generation of systemic signals induced by pathogens, arthropod herbivores and wounds. Adv. Plant Pathol. 10:307-337. Hammerschmidt, R., and J. KuG. 1982. Lignification as a mechanism for induced systemic resistance in cucumber. Physiol. Plant Pathol. 20:61-71. Hammerschmidt, R., and J. KUG (eds.). 1995. Induced resistance to disease in plants. Kluwer Academic, Amsterdam. Hammerschmidt, R., and P. Yang-Cashman. 1995. Induced resistance in cucuurbits. p. 63-85.In: R. Hammerschmidt and J. KUG, (eds.), Induced resistance to disease in plants. Kluwer Academic, Amsterdam. Hammerschmidt, R., S. Acres, and J. KuG. 1976. Protection of cucumber against Colletotrichum lagenarium and Cladosporium cucumerinum. Phytopathology 66:790-793. Hammerschmidt, R., E. M. Nuckles, and J. KuG. 1982. Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiol. Plant Pathol. 20:73-82. Hayami, C., H. Otani, S. Nishimura, K. and Kohmoto 1982. Induced resistance in pear leaves by spore germination fluids of nonpathogens to Alternaria alternata, Japanese pear pathotype and suppression of the induction by AK-toxin. J. Fac. Agr., Tottori Univ. 17:9-18. Heller, W. I., and Gessler, C. 1986. Induced systemic resistance intomato plants against Phytophthora infestans, Phytopathol. Z. 116:323-328. Hijwegen, T. 1963. Lignification, a possible mechanism of active disease resistance against pathogens. Netherlands J. Plant Pathol. 69:314-317. Hijwegen, T., and M. A. Verhaar 1993. Induced resistance to Peronospora parasitica in red cabbage. Netherlands J. Plant Pathol. 99 (Suppl. 3):103-107. Irving, H. R., and J. Kuc. 1990. Local and systemic induction of peroxidase, chitinase and resistance in cucumber plants by potassium phosphate monobasic. Physiol. Mol. Plant Pathol. 37:355- 366. Jenns, A. K, and J. KuG. 1977. Localized infection with tobacco necrosis virus protects cucumber against Colletotrichum lagenarium, Physiol. Plant Pathol. 11 :207212. Jenns, A. K, and J. Kuc. 1979. Graft transmission of systemic resistance of cucumbers to anthracnose induced by Colletotrichum lagenarium and tobacco necrosis virus. Phytopathology 69:753-756.

284

R HAMMERSCHMIDT AND

J. S. BECKER

Jenns, A. E., and J. KuG. 1980. Characteristics of anthracnose resistance induced by localized infection of cucumber with tobacco necrosis virus. Physiol. Plant Pathol. 17:81-91. Karban, R, and J. R. Carey. 1984. Induced resistance of cotton seedlings to mites. Science 225:53-54. Karban, R, R. Adamchak, and W. C. Schnathorst. 1987. Induced resistance and interspecific competition between spider mites and a vascular wilt fungus in cotton plants. Science 235:678-680. Kessmann, H., T. Staub, C. Hofmann, T. Maetzke, J. Herzog, E. Ward, S. Uknes, and J. Ryals. 1994. Induction of systemic acquired disease resistance in plants by chemicals. Annu. Rev. Phytopathol. 32:439- 459. Klessig, D. A, and J. Malamy. 1994. The salicylic acid signal in plants. Plant Mol. BioI. 26:1439-1458. Kovats, K., A. Binder, and H. R Hohl. 1991a. Cytology of induced systemic resistance of tomato to Phytophthora infestans. Planta 183:491-496. Kovats, K., A. Binder, and H. R Hohl. 1991b. Cytology of induced systemic resistance of cucumber to Colletotrichum lagenarium. Planta 183:484-490. Krischik, V. A., R W. Goth, and P. Barbosa. 1991. Generalized plantdefense: effects on multiple species. Oecologia 85:562-571. Kroon, B. A. M., R J. Scheffer, and D. M. Elgersma 1991. Induced resistance in tomato plants against fusarium wilt invoked by Fusarium oxysporum f.sp. dianthi. Netherlands J. Plant Pathol. 97:401-408. KUG, J. 1983. Induced systemic resistance in plants to diseases caused by fungi and bacteria. p. 191-221. In: J. Bailey and B. Deverall (eds), The dynamics of host defense. Academic, Sydney. KUG, J. 1987. Plant immunization and its applicability for disease control. p. 255274. In: 1. Chet (ed.) Innovative approaches to plant disease control. Wiley, New York. KUG, J. 1995. Induced systemic resistance: an overview. p. 169-175 In: R. Hammerschmidt and J. KUG (eds.), Induced resistance to disease in plants. Kluwer Academic, Amsterdam. KUG, J., and S. Richmond. 1977. Aspects of the protection of cucumber against Colletotrichum lagenarium by Colletotrichum lagenarium. Phytopathology 67:533536. KUG, J., E. Barnes, A. Daftsios, and E. B. Williams. 1959. The effect of amino acids on susceptibility of apple varieties to scab. Phytopathology 49:313-315. KUG, J., G. Shockley, and K. Kearney. 1975. Protection of cucumberagainst Colletotrichum lagenarium by Colletotrichum lagenarium, Physiol. Plant Pathol. 7:195-199. Kutzner, B., K.-H. Hellwald, and H. Buchenauer. 1993. Systemic induction of resistance in Phaseolus vulgaris L. to tobacco necrosis virus by Uromyces appendiculatis. J. Phytopathol. 138:9-20. Lawton, K., E. Ward, G. Payne, M. Moyer, and J. Ryals. 1992. Acidic and basic class III chitinase mRNA accumulation in response to TMV infection of tobacco. Plant Mol. BioI. 19:735-743. Leon, J., N. Yalpani, 1. Raskin, and M. A. Lawton. 1993. Induction of benzoic acid 2hydroxylase in virus-inoculated tobacco. Plant Physiol. 103:323-328. Levine, A., R Tenhaken, R. A. Dixon, and C. Lamb. 1994. HzO z from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79:583-593.

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

285

Linthorst, H. J. M. 1991. Pathogenesis-related proteins in plants. Crit. Rev. Plant Sci. 10:123-150 Liu, D., K. G. Ragothama, P. M. Hasegawa, and R. A. Bressan. 1994. Osmotin overexpression in potato delays development of disease symptoms. Proc. Nat. Acad. Sci. (USA) 91:1888-1892. Malamy, J. and D. F. Klessig. 1992. Salicylic acid and plant disease resistance. Plant J. 2:643-654. Malamy, J., J. P. Carr, D. F. Klessig, and 1. Raskin. 1990. Salicylic acid: a likely endogenous signal in the resistance response oftobacco to tobacco mosaic virus. Science 250:1002-1004. Mandeel, Q., and R. Baker. 1991. Mechanisms involved in biological control of Fusarium wilt of cucumber with strains of nonpathogenic Fusarium oxysporum. Phytopathology 81:462-469. Martyn, R. D., C. L. Biles, and E. A. Dillard. 1991. Induced resistance to fusarium wilt of watermelon under simulated field conditions. Plant Dis. 9:874-877. Mauch-Mani, B., and A. Slusarenko. 1994. Systemic acquired resistance in Arabidopsis thaliana induced by a predisposing infection with a pathogenic isolate of Fusarium oxysporum. Mol. Plant-Microbe Interactions 7:378-383. Maurhofer, M. C. Hase, P. Meuwly, J. P. Metraux, and G. Defage. 1994. Induction of systemic resistance of tobacco to tobacco necrosis virus by root-colonizing Pseudomonas fluorescens strain CHAO: influence of the gacA gene and of pyoverdine production. Phytopathology 84:139-146. McIntyre, J. L., J. KUG, and E. B. Williams. 1972. Protection of pear against fire blight by bacteria and bacterial sonicates. Phytopathology 62:872-877. Mehdy, M. C. 1994. Active oxygen species in plant defense against pathogens. Plant Physiol. 105:467-472. Metraux, J. P., L. Streit, and T. Staub. 1988. A pathogenesis related protein in cucumber is chitinase, Physiol. Mol. Plant Pathol. 33:1-10. Metraux, J. P., W. Burkhart, M. Moyer, S. Dincher, W. Middlestadt, S. Williams, G. Payne, G., M. Carnes, and J. Ryals. 1989. Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctionallysozynme/chitinase. Proc. Nat. Acad. Sci. (USA) 86:896-900. Metraux, J. P., H. Signer, J. Ryals, E. Ward, M. Wyss-Benz, J. Gaudin, K. Raschdorf, E. Schmid, W. Blum, and B. Inverardi. 1990. Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 250:1004-1006. Metraux, J. P., P. Ahl Goy, Th. Staub, J. Speich, A. Steinemann, J. Ryals, and E. Ward. 1991. Induced systemic resistance in cucumber in response to 2,6dichloroisonicotinic acid and pathogens. p. 432-439. In: H. Hennecke and D. P. S. Verma (eds.), Advances in the molecular genetics of plant microbe interactions, Vol. 1. Kluwer Academic, Amsterdam. Meyerowitz, E. M., and C. R. Somerville 1994. Arabidopsis. Cold Spring Harbor Laboratory, Plainview, NY. Michail, S. H., H. M. Sheir, and M. R. Rasmy. 1989. Cross protection of watermelon and cucumber plants against wilt by prior inoculation with an irrespective form a specialis of Fusarium oxysporum. Acta Phytopathol. Entomol. Hung. 24:301309. Mills, P. R., and R. K. S. Wood. 1984. The effects of polyacrylic acid, acetyl salicylic acid and salicylic acid on resistance of cucumber to Colletotrichum lagenarium. Phytopathol. Z. 111:209-216.

286

R. HAMMERSCHMIDT AND]. S. BECKER

Moraes, K M. F., D. S. Roveratti, and M. ]. G. Beretta. 1976. Induced protection to Hemileia vastatrix in coffee plants. Summa Phytopathol. 2:30-43. Mucharromah, K, and]. KuG. 1989. Oxalic acid, K2 HP0 4 and K3 HP0 4 as inducers of systematic resistance against diseases caused by fungi, bacteria and viruses in cucumber. Phytopathology 79:1144. Miiller, K. 0., and H. Borger. 1940. Experimentelle Untersuchungenuber die Phytophthora-Resistanz der Kartoffel, Arb. Biolog. Anst. Reichsanstalt. Berlin 23:189-231. Nicholson, R. 1., and R. Hammerschmidt. 1992. Phenolic compounds and their role disease resistance. Annu. Rev. Phytopathol. 30:369-389. Nielsen, K. K., K. Bojsen, D. B. Colinge, and ]. D. Mikkelsen. 1994. Induced resistance in sugar beet against Cercospora beticola: induction by dichloroisonicotinic acid in independent of chitinase and 13-1,3-glucanase transcript accumulation. Physiol. Mol. Plant Pathol. 45:89-99. Okuno, T., M. Nakayama, N. Okajima, and 1. Furusawa. 1991. Systemic resistance to downy mildew and appearance of acid soluble proteins in cucumber leaves treated with abiotic and biotic elicitors, Ann. Phytopathol. Soc. ]pn. 57:203-211. Ozeretskovskaya, O. 1995. Induced resistance in the Solanaceae. p. 31- 62. In: R. Hammerschmidt and ]. KUG (eds.), Induced resistance to disease in plants. Kluwer Academic, Amsterdam. PaIva, T. K., M. Hurtig, P. Saindrenan, and E. Tapio PaIva. 1994. Salicylic acid induced resistance to Erwinia caortovora subsp. carotovora in tobacco. Mol. PlantMicrobe Interactions 7:356-363. Peer, R. G. ]. van Niemann, and B. Schippers. 1991. Induced resistance and phytoalexin accumulation in biological control of fusarium wilt of carnation. Phytopathology 81:728-734. Peng, M., and ]. KuG. 1992. Peroxidase-generated hydrogen peroxide as a source of antifungal activity in vitro and on tobacco leaf disks. Phytopathology 82:696699. Pierpont, W. S. 1994. Salicylic acid and its derivatives in plants: Medicines, metabolism and messenger molecules. Adv. Bot. Res. 20:164-235. Rahe, ]. K, ]. KUG, C.-M. Chuang, and E. B. Williams. 1969. Induced resistance in Phaseolus vulgaris to bean anthracnose. Phytopathology 59:1641-1645. Raskin, 1. 1992. Role of salicylic acid in plants. Annu. Rev. Plant Physiol. Plant Mol. BioI. 43:439-463. Rasmussen,]. B., R. Hammerschmidt, and M. N. Zook. 1991. Systemic induction of salicylic acid accumulation in cucumber after inoculation with Pseudomonas syringae pv. syringae. Plant Physiol. 97:1342-1347. Rasmussen, ]. B., ]. A. Smith, S. Williams, W. Burkhardt, E. Ward, S. Somerville, ]. Ryals, and R. Hammerschmidt. 1995. cDNA cloning and systemic expression of an acidic peroxidase associated with systemic acquired resistance in cucumber. Physiol. Mol. Plant Pathol. 46:389-400. Richmond, S., ] .E. Elliston, and ]. KuG. 1979. Penetration of cucumber leaves by Colletotrichum lagenarium is reduced in plants systemically protected by previous infection with the pathogen. Physiol. Plant Pathol. 14:329-338. Roby, D., A. Toppan, and M.-T. Esquerre-Tugaye. 1987. Cell surfaces in plant-microorganism interactions, VII: increased proet einase inhibitor activity in melon in response to infection by Colletotrichum lagenarium or to treatment with an elicitor fromthis fungus. Physiol. Mol. Plant Pathol. 30:453-460. Ross, A. F. 1961a. Localized acquired resistance to plant virusinfect ion in hypersensitive hosts. Virology 14:329-339.

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

287

Ross, A. F., 1961b. Systemic acquired resistance induced by localized virus infections in plants. Virology 14:340-358. Ryals, J., S. Uknes, and E. Ward. 1994. Systemic acquired resistance. Plant Physiol. 104:1109-1112. Schneider, S. and W. R. Ullrich. 1994. Differential induction of resistance and enhanced enzyme activities in cucumber and tobacco caused by treatment with various abiotic and biotic inducers. Physiol. Mol Plant Path. 45:291-304. Sequira, L. 1983. Mechanisms of induced resistance in plants. Annu. Rev. Microbiol. 37:51-79. Shimotsuma, M., J. KUG, and C. M. Jones. 1972. The effect of prior inoculation with non-pathogenic fungi on Fusarium wilt of watermelon. HortScience 7:72-73. Siegrist, J., W. Jeblick, and H. Kauss. 1994. Defense responses in infected and elicited cucumber (Cucumis sativus L.) hypocotyl segments exhibiting acquired resistance. Plant Physiol. 105:1365-1374. Simons, T. J., and A .F. Ross. 1970. Enhanced peroxidase activity associated with induction of resistance to tobacco mosaic virus in hypersenstive tobacco. Phytopathology 60:383-384. Skipp, R. A., and B. J. Deverall. 1973. Studies on cross-protection in the anthracnose disease of bean. Physiol. Plant Pathol. 3:299-314. Smith, J. A., and R. Hammerschmidt. 1988. Comparative study of acidic peroxidases associated with induced resistance in cucumber, muskmelon and watermelon. Physiol. Mol. Plant Pathol. 33:255-261. Smith, J. A., and J. P. Metraux. 1991. Pseudomonas syringae pv. syringae induces systemic resistance to Pyricularia oryzae in rice. Physiol. Mol. Plant Pathol. 39:451-460. Smith, J. A., D. W. Fulbright, and R. Hammerschmidt. 1991. Rapid induction of systemic resistance in cucumber by Pseudomonas syringae pv. syringae. Physiol. Mol. Plant Pathol. 38:223-235. Staub, T., and J. KuG. 1980. Systemic protection of cucumber plants against disease caused by Cladosporium cucumerinum and Colletotrichum lagenarium by prior localized infection with either fungus. Physiol. Plant Pathol. 17:389-393. Stein, B. D. 1991. The ultrastructure and histochemistry of infection by Colletotrichum lagenarium in cucumbers induced for resistance. Ph.D. Dissertation. Michigan State University, East Lansing. Stein, B. D., K. Klomparens, and R. Hammerschmidt. 1993. Histochemistry and ultrastructure of the induced resistance response of cucumber plants to Colletotrichum lagenarium. J. Phytopathol. 137:177-188. Steiner, U., and F. Schonbeck. 1995. Induced resistance to disease in monocots. p. 86-110. In: R. Hammerschmidt and J. KUG (eds.), Induced resistance to disease in plants. Kluwer Academic, Amsterdam. Stermer, B. A. 1995. Molecular regulation of induced systemic resistance. p. 111140 In: R. Hammerschmidt and J. Kuc (eds.), Induced resistance to disease in plants. Kluwer Academic, Amsterdam. Stermer, B. A., and R. Hammerschmidt 1987. Association of heat shock induced resistance to disease with increased accumulation of insoluble extensin and ethylene synthesis. Physiol. Mol. Plant Pathol. 31:453-461. Stolle, K, M. Zook, L. Shain, F. Heberd, and J. KuG. 1988. Restricted colonization by Peronospora tabacina and phytoalexin accumulation in immunized tobacco leaves. Phytopathology 78:1193-1197.

288

R. HAMMERSCHMIDT AND J. S. BECKER

Stromberg, A., and S. Brishammar. 1991. Induction of systemic resistance in potato (Solanum tuberosum 1.) to late blight caused by local treatment with Phytophtora infestans (Mont.) de Bary, Phytophtora cryptogea Pethyb & Laff., or potassium diphosphate. Potato Res. 34:219-225. Stromberg, A., and S. Brishammar. 1993. A histological evalution of induced resistance to Phytophtora infestans (Mont.) de Bary in potato leaves. J. Phytopathol. 137:15-25.

Stumm, D., and C. Gessler. 1986. Role of papillae in the induced systemic resistance of cucumber against Colletotrichum lagenarium Physiol. Mol. Plant Pathol. 29:405-410.

Sutherland, M. W. 1991. The generation of oxygen radicals during host responses to infection. Physiol. Mol. Plant Pathol. 39:79-94. Sutton, D. C. 1979. Systemic cross protection of bean against Colletotrichum lindemuthianum by Colletotrichum lindemuthianum. Austral. Plant Pathol. 8:45.

Takahashi, K., T. Inaba, and T. Morinaka. 1985. Systemic induction to bean rust induced in Phaseolus vulgaris by the preinoculation with Colletotrichum lindemuthianum. Ann. Phytopathol. Soc. Jpn. 51:399-404. Tallamy, D. W. 1985. Squash beetle (Epilachna borealis) feeding behavior: an adaptation against induced cucurbit defenses. Ecology 66:1545-1579. Tuzun, S., and J. Kloepper. 1995. Practical application and implementation of induced resistance. p. 152-168. In: R. Hammerschmidt and J. Kuc (eds.), Induced resistance to disease in plants. Kluwer Academic, Amsterdam. Tuzun, S.,and J. KuG. 1985. Movement of a factor in tobacco infected with Peronospora tabacina which systemically protects against blue mold. Physiol. Mol. Plant Pathol. 26:321-330.

Tuzun, S., and J. Kuc. 1989. Induced systemic resistance to blue mold of tobacco. p. 177-200 In: W. K McKeen (ed.), Blue mold of tobacco, Am. Phytol. Soc., St. Paul, MN. Uknes, S., B. Mauch-Mani, M. Moyer, S. Potter, S. Williams, S. Dincher, D. Chandler, A. Slusarenko, K Wards, and J. Ryals. 1992. Acquired resistance in Arabidopsis. Plant Cell 4:645-656. Uknes, S., A. M. Winter, T. Delany, B. Vernooij, A. Morse, L. Friedrich, G. Nye, S. Potter, K Ward, and J. Ryals. 1993. Biological induction of systemic acquired resistance in Arabidopsis. Mol. Plant-Microbe Interactions 6:692-698. van Andel, O. M. 1966. Amino acids and plant disease. Annu. Rev. Phytopathol. 4:349-368.

Vernooij, B., L. Friedrich, A. Morse, R. Reist, R. Kolditz-Jawhar, K Ward, S. Uknes, H. Kessmann, and J. Ryals. 1994. Salicylic acid is not the translocated signal responsible for inducing systemic acquired resistance but is required in signal transduction. Plant Cell 6:959-965. Walters, D. R, and D. C. Murray, 1992. Induction of systemic resistance to rust in Vida fabae by phophate and EDTA. Plant Pathol. 41:444-448. Ward, K, S. J. Uknes, S. C. Williams, S. S. Dincher, D. L. Wiederhold, D. C. Alexander, P. Ahl-Goy, J. P. Metraux, and J. A. Ryals. 1991. Coordinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell 3:10851094.

Wei, G., J .W. Kloepper, and S. Tuzun. 1991. Induction of systemic resistance of cucumber to Colletotrichum lagenarium by select strains of plant growth-promoting rhizobacteria. Phytopathology 81:1508-1512.

274

R. HAMMERSCHMIDT AND

J. S. BECKER

B. Chemical Inducers One potential means of utilizing acquired resistance is through the chemical activation of the acquired resistance state. Over the last 30 years, a number of compounds that do not have direct antimicrobial activity have been shown to increase resistance or at least to decrease symptoms in some host-pathogen interactions. In this section, we review some of the compounds that have been reported to induce resistance to disease. A more extensive review of this topic can be found in Kessmann et al. (1994). 1. Amino Acid Analogs. One of the first reports on the use of nonantimicrobial compounds to protect plants against infection used analogs of amino acids. Hijwegen (1963) reported that the treatment of cucumber seedlings with phenylserine protected these plants against infection by C. cucumerinum. Amino acid derivatives were also reported to protect scab susceptible apple leaves from infection by Venturia inaequalis (Cke.) Wint. (Kuc et al. 1959). In 1966, van Andel summarized a number of studies showing the effects of amino acids on the development of disease. In several recent publications, Cohen and coworkers have demonstrated that certain derivatives of aminobutyric acid protect tomato against Phytophthora infestans and tobacco against Peronospora tabacina. In tomato, applications of 250 to 2000 ppm of DL-3-amino-n-butanoic acid resulted in 76.5 to 98.4% protection against infection by P. infestans (Cohen 1994a,b). Applications of this compound at 24 h after inoculation demonstrated that the material also had curative properties. A subsequent study (Cohen et al. 1994) demonstrated that the resistance-inducing aminobutyric acid would also induce the production ofPR proteins. Using radiolabeled aminobutyric acid, Cohen and Gisi (1994) demonstrated that the compound was translocated in tomato plants, and that tissues to which the material was translocated were protected against late blight infection. Cohen (1994a) also reported that the same aminobutyric acid compounds that induced resistance in tomato also protected tobacco against infection by the blue mold pathogen P. tabacina. Unlike the case with tomato, however, PR protein synthesis was not induced in tobacco plants after treatment. The protective effect of the aminobutyric acids needs to be further evaluated in other crops that exhibit biologically induced acquired resistance. In addition, further study on how the protection is expressed is needed to determine if the enhanced resistance is similar to or different from the resistance that is pathogen induced.

5.

ACQUIRED RESISTANCE TO DISEASE IN PLANTS

289

White, R. F. 1979. Acetylsalicylic acid (aspirin) induced resistance to tobacco maosaic virus in tobacco. Virology 99:410-412. Wilson, C. L., A. EI Ghaouth, E. Chalutz, S. Droby, C. Stevens, J.Y. Lu, V. Khan, and J. Arul. 1994. Potential of induced resistance to control postharvest diseases of fruits and vegetables. Plant Dis. 78:837-844. Xuei, X. L., U. Jarlfors, and J. Kuc. 1988. Ultrastructural changes associated with induced systemic resistance of cucumber to disease: host response and development of Colletotrichum lagenarium in systemically protected tissue. Can. J. Bot. 66:1028-1033. Yalpani, N., P. Silverman, T. M. A. Wilson, D. A. Kleier, and 1. Raskin. 1991. Salicylic acid is a systemic signal and inducer of pathogenesis-related proteins in virus infected tobacco. Plant Cell. 3:809-818. Yalpani, N., J. Leon, M. A. Lawton, and 1. Raskin. 1993. Pathway of salicylic acid biosynthesis in healthy and virus-inoculated tobacco. Plant Physiol. 103:315321. Ye, X. S., S. Q. Pan, and J. Kuc. 1990a. Activity, isozyme pattern, and cellular localization of peroxidase as related to systemic resistance of tobacco to blue mold (Peronospora tabacina) and tobacco mosaic virus. Phytopathology 80:1295-1299. Ye, X. S., S. Q. Pan, and J. Kuc. 1990b. Association of pathogenesis related proteins and activities of peroxidase, 13-1,3-glucanse, and chitinase with systemic induced resistance to blue mold or tobacco but not to tobacco mosiac virus. Physiol. Mol. Plant Pathol. 36:523-531. Ye, X .S., J. Jarlfors, S. Tuzun, S. Q. Pan, and J. Kuc. 1992. Biochemical changes in cell walls and cellular responses of tobacco leaves related to systemic resistance to blue mold (Peronospora tabacina) induced by tobacco mosaic virus. Can. J. Bot. 70:49-57. Zhou, T., and T. C. Paulitz. 1994. Induced resistance in the biocontrol of Pythium aphanidermatum by Pseudomonas. spp. on cucumber. J. Phytopathol. 142:51-63.

6 Cacti as Crops* Yosef Mizrahi and Avinoam Nerd Department of Life Sciences Institutes for Applied Research Ben-Gurian University of the Negev Beer-Sheva, Israel 84105 Park S. Nobel Department of Biology University of California Los Angeles, California 90024-1606, USA

I. II. III. IV. V.

VI.

VII.

Introduction Biological Characteristics of Cacti A. Shoots, Crassulacean Acid Metabolism B. Roots, Salinity Tolerance Cacti as Animal Feed Cacti as Vegetables Cacti as Fruit Crops A. Cactus Pears B. Columnar Cacti C. Climbing Cacti Cacti as Industrial Crops A. Cochineal B. Processed Foods C. Mucilage and Medicinal Products Future Prospects A. Low-Input Systems B. High-Input Systems Literature Cited

* The authors thank Sol Leshin for his generous financial support through the UCLABen Gurian Program for Cooperation, which has enabled an ongoing collaboration on cactus research between our two universities.

Horticultural Reviews, Volume 18, Edited by Jules Janick ISBN 0-471-57334-5 © 1997 John Wiley & Sons, Inc. 291

292

Y. MIZRAHI, A. NERD, AND P. S. NOBEL

I. INTRODUCTION In a stimulating article, Vietmeyer (1990) pointed out that relatively few plant species, most of which were domesticated thousands of years ago, serve as food for humans and animals, as medicinal plants, and as industrial crops. Other species may be the new crops that will tolerate the changing climatic conditions on earth-global warming and locally dryer conditions as a result of atmospheric CO 2 increases-serving in marginal, infertile, dry lands where common crops fail. Such new crops can provide diversification to enable sustainable agricultural systems and can offer commercial opportunities. Cacti may have the proper characteristics to fulfill such roles. Cacti are native to North and South America and the West Indies (Gibson and Nobel 1986). They are known around the world as unusual looking plants coming from hot, dry, and hostile desert areas. The dicotyledonous family Cactaceae has 122 genera with approximately 1600 species, nearly all of which characteristically have spines and exhibit stem succulence (Gibson and Nobel 1986; Nobel 1988). Of the three subfamilies, the smallest (Pereskioideae) contains about 20 species, all of which have prominent leaves. About 250 species occur in the Opuntioideae, about half of which are platyopuntias with flattened stem segments known as pads or cladodes, including the widely cultivated Opuntia ficus-indica (L.) Miller. The remaining cacti, which are in the Cactoideae, are diverse in morphology and include small collectable species (e.g., in genus Mammillaria), barrel-shaped species, tall columnar species, and epiphytic vine-like species that climb on other plants (Nobel 1994). Cacti appear in various habitats, from harsh hot deserts through tropical rain forests to cold areas with freezing temperatures. Most cacti grow in arid and semiarid zones with high summer temperatures; indeed, they are among the most tolerant of high temperatures of all plant species, tolerating 50 to 55°C when properly acclimated. Unfortunately, many species of cacti with agricultural potential are damaged by freezing temperatures, such as epiphytic cacti in the genera Hylocereus and Selenicereus that are native to tropical forests. Cacti show great adaptability to various soil conditions, as they can grow in poor, infertile desert soil and have tolerance to wide range of soil pH (Nobel 1988). Their remarkable adaptability together with the their unique shapes, sizes, and appearances have spread cacti around the world. Cacti were introduced into the Mediterranean region as early as the sixteenth century, when ships returned from the newly "discov-

6. CACTI AS CROPS

293

ered" America carrying cladodes of Opuntia. Today, O. ficus-indica and other species of this genus grow so well around the Mediterranean Sea that many consider them to be natives. Yet special care sho'uld be taken when introducing plants from one area into another, especially plants as adaptive as cacti. Introduced cacti have become dangerous weeds, as in the famous case of Australia. In 1832 platyopuntias were used as hedges north of Sydney, and in 1839 a single specimen of Opuntia stricta was introduced into Sydney as an ornamental. They became naturalized and could not be removed by plowing, since cutting the cladodes increased the number of propagation units and enhanced the spreading. Birds also spread the seeds. To make things worse, in 1914, Burbank's collection of various opuntias was introduced into Australia as forage. By 1925 prickly pear cacti such as O. ficus-indica, O. stricta, and O. vulgaris were infesting range lands in a rate of 100 ha/hour and 10 million hectares were infested in Queensland (Nobel 1994). Not until the natural insect enemy of these cacti, Cactoblastis cactorum, was introduced from Mexico were they controlled. Similar occurrences happened in South Africa, indicating the strong adaptation of the Cactaceae to various conditions. Today, cacti are grown in most countries around the world. There are many national societies of cactus enthusiasts. Germplasm exchange is becoming increasingly common, and journals on cacti are published in many countries. Because of the beauty of their flowers and the uniqueness of their shoots, they are important indoor and outdoor ornamentals and gardening plants, as seen in the Huntington Botanical Garden in Pasadena, California. There is also a whole industry of cacti as ornamentals, mainly as potted flowering plants. For example, the Christmas cactus Schlumbergera truncata and the Easter cactus Rhipsalidopsis gaertneri are widely enjoyed, and manuals exist for their cultivation. Ornamental cacti deserve an entire chapter and are not be covered in this review. Cacti increasingly serve as agricultural and industrial crops, including as animal feed, vegetables, and fruits. Cacti are most widely used in Mexico, which has a cactus as part of its national emblem, as depicted on its flag and coins. Much research on cacti has been done in Mexico, although most of the resulting publications are not widely disseminated or translated into other languages. In 1992, the Food and Agricultural Organization of the United Nations (FAO) established the "cactus pear network" with the aim of promoting the cactus pear as an important fruit crop worldwide, and a comprehensive book on cactus pear has recently been published by FAO

294

Y. MIZRAHI, A. NERD, AND P. S. NOBEL

(Barbera e1. aI1995). This review describes the biology of this interesting family of plants and discusses the potential of various species of cacti that may serve as important crops. II. BIOLOGICAL CHARACTERISTICS OF CACTI A number of books on the biology of cacti are available (Gibson and Nobel 1986; Nobel 1988, 1994; Pimienta-Barrios 1990). In this review, only certain aspects of cactus biology relevant to horticulture are emphasized, including the special structures and physiological processes; reproductive biology is discussed elsewhere in this volume (Nerd and Mizrahi 1996). A. Shoots, Crassulacean Acid Metabolism The most distinguishing vegetative feature of the Cactaceae is the areole, which occurs on a raised portion of the stem referred to as a tubercle or on a rib, which is a linear array of fused tubercles (Gibson and Nobel 1986). Areoles have meristematic activity and produce spines, including the nasty thin deciduous spines termed glochids that are a characteristic of opuntias (nasty because they contain barbs that cause glochids to become implanted in the skin of anyone who carelessly touches a cladode or unbrushed platyopuntia fruit). An areole can also produce another organ, such as a new cladode or a fruit (or even an entire plant). Cactus fruits also have areoles on their surfaces; an areole of the fruits of O. ficus-indica can even produce an entire new plant, underscoring the many possibilities for vegetative reproduction among cacti. The massiveness of the stems of cacti indicates that considerable amounts of water can be stored in their shoots. Indeed, such water storage can keep certain opuntias and barrel cacti alive for up to 3 years in the absence of water uptake from the soil (Nobel 1988). Perhaps more importantly, stomatal opening and net CO 2 uptake can occur for 2 to 7 weeks for such cacti without uptake of soil water, because metabolic activity then relies on water stored in the stems. Succulence also occurs at a cellular level for cacti, because the cells typically contain a large, water-filled, central vacuole that can represent 85 to 900/0 of the cell volume. The large central vacuoles suggest the most unusual feature of cacti among cultivated plants-they utilize a pathway for CO 2 fixation known as Crassulacean acid metabolism (CAM). CAM was origi-

6. CACTI AS CROPS

295

nally discovered in the Crassulaceae about 200 years ago based on nocturnal increases in tissue acidity detectable by human taste buds; CAM occurs in about 7% of the approximately 300,000 species of vascular plants (Nobel 1988, 1994). Essentially all CAM plants evolved in water-limited environments, such as in deserts or within tree canopies without access to large soil volumes. Water loss rates for plants depend on the difference in water vapor concentration (or partial pressure) between inside the leaves or stems and the ambient air, differences that average 5- to 10-fold less at night than during the daytime because of lower plant temperatures at night. Stomatal opening at night thus leads to less water loss by CAM plants than for stomatal opening during the daytime by C3 and C4 plants (Fig. 6.1A). C3 plants have the 3-carbon phosphoglycerate as their first photosynthetic product and comprise about 92 % of vascular plant species, and C4 plants have a 4-carbon acid such as malate or aspartate as their first photosynthetic product and comprise about 1% of vascular plants, including many agriculturally important species (e.g., maize and sorghum). A widely held view is that CAM species grow slowly. This is certainly true for various species of Mammillaria, which may be only 15 cm tall after growing for 100 years. However, the CAM pathway is only slightly more costly in terms of utilization of light energy for net CO 2 uptake than is the C4 pathway and actually is less expensive than the C3 pathway, for which 20 to 40% of the fixed CO 2 is generally released by photorespiration at a high energetic cost (Nobel 1991, 1994). CAM plants therefore can take up a relatively large amount of CO 2 (Fig. 6.1B) with respect to the water lost by transpiration (Fig. 6.1A), so their water-use efficiency (ratio of CO 2 uptake to water loss) is high. Averaged over a season, the water-use efficiency in mmol CO 2 per mol H 2 0 typically is 1.0 to 1.5 for C3 plants, 2 to 3 for C4 plants, and 4 to 10 for CAM plants (Nobel 1988). Moreover, certain irrigated CAM plants can have an annual productivity that exceeds that of nearly all cultivated C3 and C4 species. In particular, Opuntia amyc1aea Tenore and O. ficus-indica can produce an aboveground dry weight of at least 45 t ha- 1 yr- 1 (two species of agave, Agave mapisaga and A. salmiana, and pineapple, Ananas comosus, are other CAM plants that are nearly as productive), whereas such high productivities have been recorded for only a few C3 and C4 species (Nobel 1991, 1994). Thus certain CAM species are inherently extremely productive, which can have a major impact on future cultivation of cacti.

Y. MIZRAHI, A. NERD, AND P. S. NOBEL

296 Iv>

20

:.:-:.:.:.:.:.:.:.:.:.:.:.

C\l

IE 0

E E c

.2

15 10

"§

"0.. v> c

5

0

~ Iv> C\l

IE

0 50 40

0

E 30 ::::1..

(1) ~

20

0

0.. :::J

0

U

Q)

z

10

C\l

0

o

6

12

18

24

Time of day (hour) Fig. 6.1. Transpiration (A) and net CO 2 uptake (B) for C3 ( - ) , C4 ( - - - ) , and CAM (•••• ) plants. Data are for species exhibiting the highest rates of net CO 2 uptake and are averaged from many sources (Nobel 1991, 1994). Stippled bars indicate nighttime.

B. Roots, Salinity Tolerance The roots of cacti, unlike the shoots, are nonsucculent. The roots are typically shallow (5-15 em deep) and even for a large arborescent cactus occur chiefly in the upper 30 em of the soil (Gibson and Nobel 1986). Irrigation water is generally applied only to the usual rooting depth, but excessive irrigation can force roots to lower soil layers. As the soil dries, fine lateral roots generally die, while larger roots become covered with a corky layer (periderm). The root water conductivity decreases about 10-fold during soil drying, which reduces water loss from the plant tissues to the soil (North and Nobel 1992; Huang and Nobel 1994). However, water loss to a drying soil is prevented mainly by the large decreases in soil hydraulic conductivity, which can decrease 106-fold as the soil dries and water continuity among soil particles is lost (Nobel and Cui 1992).

6. CACTI AS CROPS

297

The many preformed root primordia (sites for new lateral roots) located beneath the periderm of the main roots grow rapidly when the soil is remoistened, increasing the water and mineral absorption capacity in a matter of days. For platyopuntias, roots generate easily from areoles in contact with the ground, so vegetative propagation can occur for cladodes laid on the soil surface as well as those placed vertically in the soil. Original plant orientation should be retained for stem cuttings of columnar cacti, whose roots develop from meristems located in the vascular cambium of the stem. For epiphytic cacti, such adventitious roots can be formed all along the stem and emerge through its epidermis (Gibson and Nobel 1986). Although root generation in cacti is easy, the time varies from weeks to months, is species and temperature dependent, and can be accelerated by externally applied hormones. Salinity stress has two major components: water stress and ion toxicity (Mass 1986). Cacti are relatively tolerant of water stress but are sensitive to salinity (Nobel et al. 1984; Berry and Nobel 1985; Silverman et al. 1988; Nerd et al. 1991a, 1993b). When salinity occurs in the natural habitats of certain species such as Cereus validus Haworth, the roots die back and uptake of Na+ is avoided (Nobel et al. 1984; Nobel 1988). Ca z+ can often negate the toxic effect of Na+ (Rengel 1992). Indeed, when cacti are irrigated with water in which the ratios ofNa+to Ca z+ + Mgz+and ofCl-to S04Z- are low, they do not suffer from salinity stress (Nerd et al. 1993b). When Ca z+is not abundant in the soil or the irrigation water, gypsum can be added to reduce salinity stress. Despite their drought tolerance, most cacti are commercially unsuitable to saline areas, unless special precautions regarding water and soil are taken, such as can be best learned from local field trials. The few genotypes of Opuntia that exhibit salt tolerance should be used in breeding programs. III. CACTI AS ANIMAL FEED Wild and domestic animals eat cactus stems, especially species of the subgenus Opuntia (Russell and Felker 1987a; Nobel 1994). Their high water-use efficiency makes certain opuntias ideal feed crops for semiarid regions where drought is common and animal food is scarce. For high-density plantings (24 plants m- Z) and under proper management including irrigation, yields of 40 to 50 t aboveground dry weight ha- 1 yr 1 can be obtained for platyopuntias, as already indicated (Nobel 1991, 1994). Similar biomass yields occur only among

298

Y. MIZRAHI, A. NERD, AND P. S. NOBEL

the highest producing C3 and C4 plants, which have a much lower water-use efficiency. Besides opuntias grown for harvesting to provide fodder for cattle and other animals, cacti can be used as forage by free-ranging cattle. In cases where animals are allowed to graze opuntias, the spines can be burned off before the cladodes are eaten. This requires specialized burners with attendant costs of the propane fuel (Maltsberger 1991). In southern Texas this option is 30 to 40% cheaper than the cost of available relief food provided during drought (Russell and Felker 1987a). Although spineless genotypes are available, they are generally less resistant to drought. Indeed, the ranchers prefer the spiny types, because livestock do not generally consume the unburned cladodes, which remain untouched until needed. If range cattle had access to spineless genotypes, they would be consumed in preference to the native grasses and thus eliminated. Although spines can also be burned off for cladodes used as fodder, mechanically chopping the cladodes, sometimes followed by fermentation, also makes them palatable to livestock (Fuentes-Rodriguez 1992). Although confusion exists in the taxonomy of opuntias used as animal feed, Opuntia ficus-indica is the most common species worldwide (Russell and Felker 1987a), O. lindheimeri Engelm. is used in southern Texas (Maltsberger 1991; Nobel 1994), and O. rastera Weber, O. robusta Wendland in Pfeiff., O. engelmanii Salm-Dyck, O. megacantha Salm-Dyck, and O. phaeacantha Engelm. are used in Mexico (Fuentes-Rodriguez 1991). When these plants are not cultivated, yields tend to be much lower than the maximal possible but are still higher than yields for C3 or C4 plants grown under drought and otherwise harsh conditions (Nobel 1994). Growth conditions affect the quality of cladodes as an animal feed, so nutritional analysis should be made on such feed before use (Maltsberger 1991). Cladodes consist mainly of water, usually about 85 to 95% by fresh weight (Fuentes-Rodriguez 1991), but during water stress the water content may drop to 60% (Nobel 1994). In nutritional value, cladodes are similar to immature maize silage on a dry matter basis (Maltsberger 1991); they are relatively high in fiber (average of 18%) and minerals (19%), low in fats (1-4%), and medium in proteins (generally 4-8% total, with 1-2% digestible). Digestibility of cladodes is high (72 %), and carbohydrates can account for up to 71 % of their dry weight (Fuentes-Rodriguez 1991, 1992; Maltsberger 1991). Like many other green plant tissues, cladodes contain f)-carotene (a precursor to vitamin A) and vitamin C. Protein supplementation should always be considered (e.g., cotton seed, which is available in southern Texas and elsewhere, is a good

6. CACTI AS CROPS

299

source). Micro- and macronutrient supplementation should also be considered (Maltsberger 1991). The content of nitrogen and other minerals in cladodes can be increased by fertilization (Nerd and Mizrahi 1992; Nerd et al. 1993a). A clone of Opuntia stricta Haworth has been identified with high nitrogen and phosphorus contents that satisfy the feed requirements of cattle (Nobel 1994). The laxative properties of cladodes can be avoided by gradually increasing the cladode portion in the feed (Maltsberger 1991). Because consumption of cladodes can improve the flavor of milk and the color of the butter produced from it, milk from cladode-fed cows commands higher prices in Mexican markets (Russell and Felker 1987a). Numerous countries (Mexico, Brazil, United States [mainly Texas], Peru, Chile, South Africa, and Tunisia) are already producing significant amounts of animal feed from opuntia cladodes (Nobel 1994), reflecting their low maintenance cost, efficient production, and sustainability (Hamilton 1992). Selected cultivars of opuntias with high yields and high-quality cladodes should play an increasingly important role in the livestock industry on a regular basis, not just as a drought relief source of feed. With the proper management, these cacti can be a competitive source of feed, especially where water shortage is a problem. However, a major problem for the expansion of the cultivation of cacti in the United States and other countries is the plants' sensitivity to freezing temperatures (Russell and Felker 1987b). Most members of subgenus Opuntia growing in the wild or cultivated are heavily damaged by nighttime temperatures of -10 C , although certain wild opuntia species growing in northern latitudes can tolerate temperatures below -20 C when properly acclimated, such as Opuntia fragilis (Nutt.) Haworth and O. humifusa (Rafinesque) Rafinesque, both of which are native to Canada (Nobel and Loik 1990; Loik and Nobel 1993). Indeed, breeding efforts between cold-tolerant native species and highly productive but coldsensitive commercial species should be a major objective of programs to expand the cultivation of cacti (Nobel et al. 1995; Nobel 1996). c

C

IV. CACTI AS VEGETABLES

A traditional vegetable of Mexico is the nopalito, the name for the young cladodes of various species of platyopuntia, such as Opuntia ficus-indica, O. streptacantha Lem., O. amyc1aea, O. robusta, O. inermis De Candolle, and Nopalea cochenillifera (L.) Salm-Dyck (Cantwell et al. 1992; Flores 1992; Pimienta-Barrios 1993). This unique vegetable, which usually is roasted, blanched, or cooked af-

300

Y. MIZRAHI, A. NERD, AND P. S. NOBEL

ter the spines and the young leaves are removed, has great potential for other countries as well. Nopalitos are sold in the Mexican markets in several forms, the simplest being the freshly harvested cladodes. Often they are sold after the spines, young leaves, and edges are sliced off and sometimes after being cut into strips or small cubes. This vegetable is utilized in many forms, including salads and cooked dishes with meat. Even a delicious cactus pie can be prepared, which tastes apple-like, perhaps reflecting the high malic acid levels in both apples and nopalitos (Master 1959). The nutritional value of nopalitos is similar to that of many other vegetables; they contain mostly water (88-95%), some carbohydrates (3-7%), and minerals (about 1.3 %, mainly Ca 2 +). Like most leafy vegetables, nopalitos are low in proteins (about 1 %) and fiber (about 1 %, which is still more than twice that of lettuce; Rodriguez-Felix and Cantwell 1988). They are a typical source of two important vitamins, p-carotene (18-38 mg per 100 g fresh weight) and ascorbic acid as vitamin C (10-18 mg per 100 g fresh weight). Nopalitos are less nutritious than spinach but more nutritious than lettuce (Cantwell 1991). Mucilage, which is secreted from the cut ends of the cladodes, deters some potential consumers but can be minimized by boiling in water for a few minutes with some sodium bicarbonate or salt. Consumption of nopalitos can reduce the blood sugar levels in diabetics who are not insulin dependent (Ibanez-Camacho and Roman-Ramos 1979; Meckes-Lozoya and Roman-Ramos 1986; Frati et al. 1983, 1988, 1989, 1990) and also can reduce fats and cholesterol, especially the low-density cholesterol (Frati et al. 1983; Fernandez et al. 1990, 1992), underscoring the great potential of nopalitos as a crop. Nopalitos corne to Mexican markets from wild as well as backyard plants of various species, from established orchards where the plants are grown as for fruits (Rodriguez-Felix and Cantwell 1988), and from special plantations with dense plantings to maximize vegetative production. "The Nopalitos Capital" is Milpa Alta, where high-quality cultivars, mainly of O. ficus-indica, are grown. The clado des are traditionally bailed in round bundles (Color Plate 1) before being sent to the markets. Over 5000 ha of nopalitos are cultivated in Mexico (Pimienta-Barrios 1993); the limited plantings in the United States are mostly in southern California and Texas, where the main cultivated species is Nopalea cochenil1ifera. Cladodes of the specific cultivar of N. cochenil1ifera are spineless and less mucilaginous, greener, and more tender than those of the various Opuntia species grown for nopalitos. Young cladodes of N. cochenil1ifera

6. CACTI AS CROPS

301

are often sold in U.S. supermarkets under a botanically incorrect term, "cactus leaves" (cladodes are stems). Although more research is needed on its growth and management (Mick 1991, 1992),N. cochenillifera is planted as cuttings with about 20 em between individual cladodes along rows in soil beds with 8 rows about 20 em apart. Fifty cladodes can be harvested in one year from a single cladode planted in the spring (the first harvest is obtained in 3-4 months). High fresh weight yields of 400 to 600 t ha- 1 yr 1 are possible under the severe pruning that stimulates vegetative growth. Cultivar 1308 is sensitive to freezing temperatures, so it is grown under plastic tunnels filled with straw during the wintertime risk period, when ambient outside temperatures can be -10°C. These tunnels are also used to lengthen the cladode production period in the spring and autumn, when outside temperatures are too low (Mick 1991) (Color Plate 2). Opuntia ficus-indica, O. inermis, and O. amyc1aea can have young cladodes that are low in spines. These nopalitos reach their market size of about 20 em in length within 20 to 30 days, depending on the weather. The shelf life of this vegetable is usually a few weeks, provided that proper postharvest techniques are applied (Cantwell et al. 1992). Diurnal variation in acidity because of CAM metabolism can impose a unique postharvest problem, because morning-harvested cladodes are very acidic, whereas afternoon-harvested clado des contain only 10 to 20% as much acidity (Rodriguez-Feliz and Cantwell 1988). The acidity of harvested cladodes decreases with time and after 1 week of storage at 20°C reaches a plateau (Cantwell et al. 1992), so packages should be marked with the earliest day for consumption to guarantee consistent acidity levels in the cladodes. Proper marketing and consumer information about their quality and many uses are essential before the tremendous potential of nopalitos will be realized worldwide (Flores 1991, 1992). Several books and leaflets have been published describing the many dishes that can be prepared from nopalitos, such as, Cookin With Cactus (Haggerton 1992). The bases of the flower buds of N. cochenillifera (Color Plate 3) can also be used as a tasty vegetable (L. Scheinvar, Autonomous University of Mexico, Mexico City, personal communication). The flower base contains glochids that must be removed before use, suggesting that the absence of spines and glochids from the cladodes of N. cochenillifera is controlled by other genes. When this species is grown for nopalito production with intensive cladode removal, flower buds are generally not formed, although much aboutN. cochenillifera

302

Y. MIZRAHI, A. NERD, AND P. S. NOBEL

remains to be determined by future research. Another cactus used as a vegetable in Mexico is Acanthocereus tetragonus (L.) Humlk. (1. Scheinvar, personal communication), which is an epiphyte that may require a trellising system and shade. This cactus lacks mucilage, which may make it more attractive to potential consumers than nopalitos. Basic agricultural research is needed before exploiting A. tetragonus as a crop, which is also true for the leafy nonsucculent cactus Pereskia grandiflora, whose young stems are cooked as a vegetable in Vietnam (N. T. Nguyen, University of Hanoi, personal communication). V. CACTI AS FRUIT CROPS The greatest potential for cacti as horticultural crops, whose reproductive biology has recently been reviewed (Nerd and Mizrahi 1996), lies in the attractive and unique fruits produced by many species in various genera. Already an established crop in some countries is the "cactus pear" (Opuntia sp), formerly known as the "prickly pear" (Color Plate 4). The term "cactus pear," which overcomes the negative connotation for the fruit of "prickly pear," was recommended by Frieda Caplan, a California marketer of exotic fruits and vegetables, and others and was adopted by representatives of 10 countries at the Second International Conference on Tuna and Cochineal in Santiago, Chile, in 1992 (Pimienta-Barrios et a1. 1993). In the country of origin, Mexico, O. ficus-indica, O. amyc1aea, O. streptacantha, O. megacantha, and O. inermis are cultivated for fruits (Pimienta-Barrios 1991, 1994), whereas in other countries the most important species is O. ficus-indica. Cactus pear is already an established commercial crop in Mexico, Italy, United States, Israel, Peru, South Africa, Chile, Argentina, Colombia, and many other Latin American countries. The common name of the fruit is tuna in the Latin American countries, ficodindia (fig of India) in Italy, tzabbar in Israel, and sabar in Arab countries around the Mediterranean. The fruit is spiny and has a thick peel that must be removed before reaching the tasty flesh. Because various books on cactus pear are available (Wessels 1988; Pimienta-Barrios 1990; Barbera and Inglese 1993; Barbera et a1. 1995), this review focuses on some limiting factors and possible solutions to allow this fruit crop to become more widely accepted, as well as on other types of cacti producing edible fruits. Problems include spines, seeds, and the short period of production. Yet even with current limitations, the profitability of cactus pears in

6. CACTI AS CROPS

303

South Africa, Israel, and other countries equals or exceeds that of common orchard crops (such as apples, peaches, and oranges), which have higher cultivation expenditures with comparable fruit prices (Brutsch and Zimmermann 1993). A. Cactus Pears

Cactus pears have glochids (Gibson and Nobel 1986), which generally are removed before the fruit is peeled. A simple solution would be to select spineless cultivars, which so far do not exist-the so called "spineless" fruits are really only low in spines. Of 50 cultivars from around the world tested under Israeli conditions, 'Direktor' from South Africa has the lowest number of spines but produces only a few fruits. Although a genetic approach should not be ignored, opuntias are facultative apomictic plants, because they produce both sexual and asexual (nucellar) embryos in the same ovule; this leads to sexual and apomictic seeds (Perez-Reyes and Pimienta-Barrios 1995), which somewhat decreases the success of hybridization experiments. Farmers wear aprons and gloves to harvest the fruits, which are mechanically brushed to remove the glochids and then often washed, dried, and waxed. In Israel, the cost of harvesting, brushing, waxing, sorting, and packing is about US$ 0.30/kg, so from the marketing and consumer point of view the problem can be solved. Nevertheless, glochids can remain on the fruits in some commercial shipments, which together with variations in fruit size are matters of quality control. People who have consumed cactus pears from an early age are not bothered by the seeds, which readily pass through the digestive tract. In Australia where commercial activity with cactus pears was banned for many years due to their devastating effect as introduced weeds (Nobel 1994), immigrants from Mediterranean countries would pay high prices to obtain this fruit in spite of the seeds. Ten kilograms of fruits in unmarked wooden boxes were sold in the late 1980s "under the table" in Sydney's wholesale market for Aus$ 60-80 CUSS 4256) according to John Discusso Southern Cross Produce, Flemington Market, Sydney (personal communication). Cactus pear is presently legal in Australia and the 10-kg boxes sell for Aus$ 20-30 (US$ 1522). However, first-time tasters are often repelled by the numerous hard seeds, up to several hundred, embedded in the tasty pulp. Although emasculation of flowers and treatment with gibberellins can induce seedless fruit (Gil et al. 1977; Gil and Espinosa 1980), these solutions are not really feasible in horticultural practice. Emascula-

304

Y. MIZRAHI, A. NERD, AND P. S. NOBEL

tion of the flowers by hand is costly and application of gibberellins over a wide range of flower ages does not guarantee 100% seedlessness. A genotype of cactus pear exhibits vegetative parthenocarpy, so seedless cultivars may be obtained by breeding (Weiss et a1. 1993b), although other evidence indicates that ovule fertilization is necessary for the development of the funicular envelope that makes the fruit pulp (Pimienta-Barrios and Engelman 1985; Pimienta-Barrios 1991). In any case, because their seeds are a major problem in the acceptance of cactus pears, more research should be conducted on this aspect to allow a greater adoption of this crop worldwide. Another problem of cactus pear is that the flower buds in a particular region emerge during about 1 month in the spring. As a result, ripening occurs in the summer, also during about 1 month (Nerd et a1. 1989, 1993a; Barbera et a1. 1991; Brutsch and Zimmermann 1993). In Israel, 80% of fruits normally ripen during a 2-week period, which can drive prices below harvesting expenses (the cool wet winters and hot dry summers may synchronize flowering in Israel). In Mexico with its various climates, the wide range of commercial cultivars produce fruits in June through October, but backyard plantings have fruits from May through December (Pimienta-Barrios 1991), indicating a genetic diversity that can be exploited in future breeding programs. Actually, in some locations, fruits of O. ficus-indica can be produced year-round, for example, in the Huonta Valley, Peru, and Njoro, Kenya. A technique called scozzolatura was developed over 100 years ago in Italy by which the first flush of flowers was removed, forcing a second and delayed flush of flower buds, resulting in later ripening of the fruits (Barbera et a1. 1991; Barbera and Inglese 1993). These fruits are marketed during the autumn, avoiding competition with other summer fruits and increasing profitability, a technique also adopted in South Africa (Brutsch and Scott 1991) and Israel (Nerd and Mizrahi 1993, 1994). Out-of-season fruiting can be induced in the Negev Desert by nitrogen fertilization, which causes the plants to flower after the normal period, leading to a winter crop that is more profitable than the summer crop (Nerd et a1. 1991b). In Israel, cactus pears can be harvested 10 months during the year (Fig. 6.2). The lowest wholesale prices (equivalent to US$ O.aO/kg) are obtained during the natural ripening period, which is mid-July to mid-August, and the highest prices (US$ 3.50/kg) are obtained from the out-of-season winter crop. For one particular farmer, the first fruits in June obtained a 7.3-fold higher price than those in midJuly (Fig. 6.2). This farm, which is located in the Arava valley and

6. CACTI AS CROPS

305

experiences arid desert conditions with average maximal daytime temperatures in July and August of 40°C (extremes of 47°C), 30 mm annual rainfall, and 3500 mm annual pan evaporation, produces mango with 2400 mm per year irrigation but cactus pear with only 400 mm, illustrating its high water-use efficiency. Thus fields in Israel can produce a winter crop of cactus pears in addition to the normal summer crop (Nerd et al. 1991b) or a single autumn crop with higher prices (Nerd and Mizrahi 1994). Similarly, winter cactus fruits are produced in orchards that are heavily manured (in Chile with chicken manure) or fertilized (in California). Future yearround availability of cactus pears in international markets can also be enhanced by integrating northern and southern hemisphere producers. B. Columnar Cacti Fruits of many species of columnar cacti are consumed in Mexico as well as other Latin American countries. These fruits are often termed pitayas or sometimes pitahayas, which can be further distinguished by adding a second name; such as pitaya dulce (the sweet pitaya), pitaya agria (sour pitaya), pitaya de Mayo (pitaya that ripens in May), or pitaya amarilla (yellow pitaya). To avoid confusion in dealing with U> 12 0)

.:s:O)

.I:: (f)

0)

2 (f)

3 0)

EO"

.:s:~

0)

00) (J

a=

0

J

F M A M J

J A SON Month

D

Fig. 6.2. Prices of cactus pears 1994 in the Israeli market. Data for solid line are average monthly wholesale prices for fruits of various sizes and qualities; from late April to late June, fruits are from storage (Nerd and Mizrahi 1993, 1994). Data for dashed line represent the farm-gate price received by one grower for fresh fruits and were graciously provided by Yair Fine of Moshav Iddan.

306

Y. MIZRAHI, A. NERD, AND P. S. NOBEL

the many species and genera that differ tremendously from each other, and awaiting the adoption of uniform common names worldwide, each species is here designated by its scientific name. The genus containing the most species with edible fruits is Stenocereus (Pimienta-Barrios and Nobel 1994). The three most important species in Mexico are Stenocereus griseus (Haworth) Buxb., known as pitaya de Mayo; S. queretaroensis (Weber) Buxb., known as pitaya de Queretaro; and S. stellatus (Pfeiff.) Riccob. These are cultivated in various regions with a total area of about 2000 ha; sometimes known cultivars are intermixed with cuttings from wild plants that bear tasty and colorful fruits. The stem cuttings are typically about 1 m in length and can produce fruits in about 4 years, eventually producing about 20 t fresh weight of fruits ha- 1 yr- 1 • Clones differ in their pulp colors, including white, orange, pink, and various hues of red; most skins are green but may vary to red (Color Plate 5). The fruit's spines abscise upon ripening and can be removed with bare hands. The fruit shelf life of the most commercial species, S. queretaroensis, is currently only a few days (Pimienta-Barrios and Nobel 1994), but much remains to be learned about its postharvest physiology, including harvesting at various stages of fruit development and the effects of reduced temperatures or controlled atmospheres. Fruits of Stenocereus thurberi (Engelm.) Buxb. (pitaya dulce, also known as the organ pipe cactus) are collected from the wild in Arizona and northwestern Mexico. Stenocereus gummosus (Engelm.) Gibs. & Horak (pitaya agria) grows in Sonora and Baja California, Mexico. The fruits of the famous saguaro, Carnegiea gigantea (Engelm.) Britt. & Rose, are also edible and are collected from wild plants; this species grows slowly and has little agricultural potential (Crosswhite 1980). Other species that are highly appreciated in Mexico include Escontria chiotilla (Weber) Rose (jiotilla or geotilla) , with small but tasty fruits. The cactus Myrtillocactus geometrizans (Martius) Cons. (garambullo) produces a berry-like, tasty, dark blue to black, small fruit of possible commercial value that can be found in local markets in the central part of Mexico. Two species of Pachycereus also produce edible fruits: P. pecten-aboriginum (Engelm.) Britt. & Rose (cardon barbon) and P. pringlii (Berger) Britt. & Rose (cardon pelon; Felger and Moser 1974; 1976). Cereus peruvianus (1.) Miller (apple cactus) is a very promising columnar cactus (Morton 1987; Nerd et al. 1993b; Weiss et al. 1993a), which probably originated along the subtropical southeastern coast of South America (Backeberg 1984). It has a high growth rate and

6. CACTI AS CROPS

307

can produce fruit 3 to 4 years after propagation from seed. The fruit is smooth and spineless and varies from yellow to deep red (Weiss et al. 1994a). Its pulp is white, juicy, and sweet and sour in taste, and contains soft, black, edible seeds. A 7-year-old plant can annually bear 60 to 80 kg of fruits, mainly in the summer. The stems generally have spines, but spineless genotypes exist and could be selected for. Attempts to domesticate various columnar cacti have recently been made in Israel with respect to fruit production (Nerd et al. 1990, 1993b). The closely related Cereus jamacaru D.C. (Scheinvar 1985) has recently been planted for fruit in western Australia. C. Climbing Cacti An interesting group of cacti bearing edible fruit are the climbing epiphytic species that are native to the forests of northern South America, Central America, and Mexico. They grow on the shaded stems of trees and climb within the canopy. When disconnected from the ground, these cacti can take up water and minerals via their adventitious roots (Gibson and Nobel 1986). Their growth can be inhibited by high radiation, such as direct sunlight (Nerd et al. 1990; Raveh et al. 1993). Although confusion has existed about their identification, climbing cacti apparently belong to two genera: Hylocereus, which has many species with edible fruits, and Selenicereus, with the most important fruit species, S. megalanthus (Schum.) Britt. & Rose (Weiss et al. 1995). Selenicereus megalanthus (pitaya amarilla) grows wild in Colombia, Ecuador, and Nicaragua at elevations between 800 and 1200 m; it has been referred to as Hylocereus triangularis (L.) Britt. & Rose (Arcadio 1986; Barbeau 1990; Morton 1987) and Hylocereus sp. Katom (Nerd et al. 1990; Raveh et al. 1993). After tasting this yellow pitaya in Bogota in the mid 1980s, a Japanese businessman imported several tonnes, stimulating planting of S. megalanthus in Colombia (Cacioppo 1990). Today the fruits are gaining popularity in the European market (Barbeau 1990; Cacioppo 1990). Before the fruits of S. megalanthus are ripe, their areoles contain several 1- to 2-cm-Iong spines that abscise upon ripening. Because the fruits are harvested before full ripening, the spines are removed with a brush (spine degradation in the soil is very slow, so spines are generally removed from the orchard). When ripe, the attractive fruit is yellow with raised tubercles (podaria; Color Plate 6). The flesh, which is white, juicy, sweet, with an appealing touch of acid-

308

Y. MIZRAHI, A. NERD, AND P. S. NOBEL

ity, is scooped from halved fruits, leaving the peel untouched. The black seeds embedded throughout the flesh are soft and edible. Like other climbing cacti, S. megalanthus is grown on a trellis system. In the main country of commercial production, Colombia, many shapes and materials of trellis systems are used, including large boulders. Although costs, productivities, and efficiency of labor under various trellis systems have not been compared in detail, in Israel the trellis system is the major expense in establishing an orchard and has a major impact on the economic feasibility of growing S. megalanthus. In addition, the effects of pruning on yields and on ease of handling this crop need to be investigated. In regions where the photon flux (wavelengths of 400 to 700 nm absorbed by photosynthetic pigments such as chlorophyll) can reach 2000 to 2200 mol photons m-2 S-l, S. megalanthus must be grown in screened houses to prevent stem bleaching and death. The optimal shading is not yet known and its effects on vegetative growth as well as fruit and yield quality must be evaluated (Raveh et a1. 1993). Also, protection from freezing temperatures is needed (Nerd et a1. 1990). The postharvest physiology of this fruit is not well studied, suggesting that the quality found in the international markets can be improved. The other important climbing species is Hylocereus undatus (Haworth) Britt. & Rose (Color Plate 7), known in Mexico as pitahaya and in Central America and northern South America as pitaya roja (red pitaya). Surprisingly, this species is the most profitable crop in Vietnam, where it is known as "the dragon fruit" (thang loy in Vietnamese) or "dragon pearl fruit" in Vietnam's export markets of Hong Kong, Singapore, and Taiwan. In season from July through October, the fruits are offered as a special treat on Vietnam Airlines. Hylocereus undatus was apparently introduced to Vietnam by the French about 100 years ago, and many Vietnamese now regard this species, which has spread along the eastern coast as a backyard plant, as indigenous. Commercial plantations estimated to be several thousand hectares are mainly grown among trees that serve as inexpensive trellises; farmers prune these trees to allow more light, which they believe increases the fruit production of H. undatus (Color Plate 8). Fruits of H. undatus are well known in the local markets (Color Plate 9). The main production area is along the coast from Nha Trang in the north to Ho Chi Minh City in the south. The orchards, which yield 30 t fruit ha- 1 yr\ are fertilized by organic manure and apparently contain a single self-compatible cultivar. The fruit of H. undatus, which has a red peel and large green scales, is larger, with a thicker parenchyma, than that of S. megalanth us.

6. CACTI AS CROPS

309

Upon ripening, the scales turn yellow (and other shades) and the fruit looks like an attractive spineless ornament. Fruits of H. undatus need about 50 days to complete development, and their pulp varies from white to various hues of red; fruits of S. megalanthus need 150 days to complete fruit development, their pulp is white with higher sugar levels, and this species is more sensitive to subfreezing temperatures. Other species of Hylocereus, such as H. costaricensis (Weber) Britt. & Rose and H. polyrhizus (Weber) Britt. & Rose, are also candidates for domestication because they produce large attractive fruits (Raveh et a1. 1993; Weiss et a1. 1994b). Crosses between these species occur and can result in hybrids with good horticultural characteristics (Color Plate 10). VI. CACTI AS INDUSTRIAL CROPS

A. Cochineal

The most established and important industrial crop of cactus origin is the red dye known as cochineal, the carminic acid used for over two centuries as a biological stain for light microscopy as well as a dye for fabrics and foods (Nobel 1994). The dye is produced by female insects of Dactylopius coccus Costa or D. opuntiae Cockerell, which use Opuntia ficus-indica as their host plant (Color Plate 11). The cladodes can be inoculated with fertile oviparous female insects in cloth bags whose mesh allows the larvae to infect the cladode. The insects, which proliferate on healthy cladodes by extracting nutrients from the phloem (Wang and Nobel 1995), are collected when they are full of caraminic acid and have reached maximum infestation. Several hundred insects, about 2 to 3 mm in diameter, can be collected by hand from a single cladode. After air drying, the raw material, called "grana" (meaning "seeds," because the Spanish mistook the small insect bodies for seeds) is used to make carmine, which in turn is used to dye various products with vivid red colors of different hues as well as orange and yellow produced by varying the pH. The grana quality depends on the ease of extraction of the dye (10 to 26% of the insects' dry weight), which is relatively easy for the cultivated cochineal insects, whereas noncultivated insects produce low-quality grana that obtain lower prices in the world markets (Borrego-Escalante 1992). A peak world production of dry grana of 700 t was recorded in the eighteenth century. Demand decreased in the nineteenth century, as cheaper aniline dyes were produced. How-

310

Y. MIZRAHI, A. NERD, AND P. S. NOBEL

ever, aniline dyes are now considered unsafe (carcinogenic), so a resurgence of demand for carminic acid has occurred in the last decade, especially as dyes for the food and cosmetic industries. Current annual world production is about 300 t, with approximately 90% from Peru; the Canary Islands is the second largest producer and Mexico the third. Conditions in the deserts of Peru are suitable for production: freezing temperatures do not occur and prevailing temperatures are 20 to 35°C; growers obtain US$ 45/kg of grana. Because labor is the major expense for cochineal production, establishing an industry in countries where labor is expensive will be difficult. Cochineal can be produced by growing opuntias in plantations similar to those for fruit production or by removing the cladodes to sheds in which the infested cladodes are placed close together on hooks. The latter system was developed in South Africa where approximately 8000 ha of infested opuntias are available to provide cladodes for cochineal inoculation; 90 m 2 of sheds with cladodes from 0.5 ha can yield 75 kg of grana per year with a value of US$ 3000 (Brutsch and Zimmermann 1993). Because good yields of grana in the first system means death of most of the cladodes on the plants, heavily yielding plantations generally deteriorate rapidly and must be uprooted or pruned to the plant bases to renew vegetative growth, so use of sheds may be more efficient (Borrego-Escalante 1992). B. Processed Foods Nopalitos are sold not only as a fresh vegetable in Mexico but also as processed food, including pickled nopalitos, various salads, and cooked dishes. Fifteen firms export products, such as pickled strips or cubes of nopalitos in vinegar and a variety of prepared salads, mainly to the United States (Flores 1991). These processing industries absorb the seasonal overproduction of nopalitos for local markets and also use cladodes from wild plants, such as from O. robusta in San Luis Potosi. Nopalitos are commercially dried only on a limited scale, probably because of the high amount of water in the fresh cladodes. In any case, when the food industries seriously consider this relatively inexpensive, high-quality vegetable, more processed nopalito products should become available in the future and not only in Mexico and the United States. Because the cactus pear is the most common cactus fruit on the market, most of the processed fruit products are of cactus pear origin. Fruit juices, concentrates, jams, and jellies are common in Mexico

6. CACTI AS CROPS

311

and other Latin American countries. A popular product in Mexico is queso de tuna (literally, cheese of cactus pear), which is similar to cheese in consistency but is sweet. Miel de tuna (honey of cactus pear) is another popular fruit product (Inglese et a1. 1993). An alcoholic drink called coloncheis produced in Mexico from cactus pears, (Flores 1991). Even in the Negev Desert of Israel, cactus pear ice cream is sold. Seeds of cactus pear contain 6 to 20% oil, with 82 to 90% linoleic and oleic acids, 9 to 16% palmitic acid, and 1 to 2 % stearic acid. Thus, cactus pear seeds left after processing the fruit can be a source of high-quality culinary oil (Pimienta-Barrios 1991; Inglese et al. 1993). Similar products are made from fruits of other cacti, mainly in Mexico. Pitayas have been used in Mexico for many years as coloring agents and additives to ice cream (Pimienta-Barrios and Nobel 1994). Ice cream is produced from the variously colored fruits of Hylocereus species in Israel. Although fresh fruits yield the highest income to the producers, many fruits from columnar cacti tend to dehisce upon ripening and so are not suitable for the fresh market. Overripe fruits or those with other defects can go to the processing industries and supplement the farmers' income (Pimienta-Barrios and Nobel 1994).

c.

Mucilage and Medicinal Products

The sticky, jelly-like, water-absorbing substance found in certain plants is known as mucilage and can occupy about 3% of the stem volume of Opuntia ficus-indica. It is produced and secreted by specific cells that can be located at the inner side of the chlorophyllcontaining tissue (chlorenchyma), and large droplets are exuded when a cladode is cut (Gibson and Nobel 1986). Mucilage is considered a nuisance by many when cladodes are used as a vegetable, but as a complex polysaccharide it possesses biophysical characteristics that are desired by the cosmetic and food industries, which continually look for such chemicals from a range of plant and algal sources (Arad [Malis] and Cohen 1991). Opuntias and other cacti with large amounts of mucilage may be a competitive source for such biochemicals, as already recognized in Mexico (Flores 1991). Medicinal effects and uses have been found for stems, flowers, and fruits of cacti. As already indicated, consumption of nopalitos can improve glucose control in humans with non-insulin-dependent diabetes mellitus, can reduce glucose levels and increase insulin activity under hyperglycemic conditions, and can reduce the blood

312

Y. MIZRAHI, A. NERD, AND P. S. NOBEL

levels of triglycerides, total cholesterol, and low-density lipoprotein-cholesterol (Fernandez et al. 1990, 1992; Frati et al. 1983,1988, 1989, 1990; Inglese et al. 1993). Such a "health food" does not require any licensing, and dry nopalito capsules are already sold in Mexico as a remedy to the above-mentioned health problems. The mode of action can be partly explained based on fiber content, but changes in cellular sensitivity to insulin may also occur (Frati 1992). Two companies in Israel sell capsules of dried flower corollas of O. ficus-indica to prostate sufferers for diuretic regulation (Inglese et al. 1993). Even after fruit ripening, flowers of the Israeli cultivar 'Ofer' maintain their dry corollas, which are easily removed and sell for US$ 13/kg (harvesting costs half as much), providing an important additional income to cactus pear growers. Hallucinogenic compounds are also found in cacti. Peyote, Lophophora williamsii (Lem.) Coulter, contains the alkaloid mescaline (Gibson and Nobel 1986). Mescaline also occurs in certain columnar cacti including Trichocereus pachanoi (up to 1.3 g mescaline/kg fresh weight), which is sold on the street as an intoxicant in various countries (Mann 1992). If pharmaceutical industries become interested in such biochemicals from cacti, research into the active compounds and development programs of domestication need to be applied to tap what could be a very large market. VII. FUTURE PROSPECTS This review covers 34 species of cacti that can be crops (Table 6.1). Great potential exists for cacti as low-input systems for developing regions and as high-input systems for developed regions. A. Low-Input Systems Cacti can be grown in marginal lands of semiarid zones. In Kenya, bush clearing for conventional crops can lead to desertification, such as near Lake Baringo. Opuntia ficus-indica as well as other opuntias can serve as fruits and products therefrom, vegetables, animal feed, and as a drought-tolerant perennial for reforestation, which can provide firewood, although of low quality. In addition, the cladodes can be used for cochineal insects for dyes and as a medicinal plant to combat diabetes mellitus, high blood pressure, and high cholesterol, and the corollas can serve as a prostate remedy. Planting of several genera and species can increase the success of such projects

6. CACTI AS CROPS

313

Table 6.1. Some cacti crops and their uses. Botanical name

Acanthocereus tetragonus (L.) Humlk Carnegiea gigantea (Engelm.) Britt & Rose Cereus jamacaru D.C. Cereus peruvianus (L.) Miller Escontria chiotilla (Weber) Rose Hylocereus costaricensis (Weber) Britt. & Rose H. polyrhizus (Weber) Britt. & Rose H. undatus (Haworth) Britt. & Rose Lophophora williamsii (Lem.) Myrtillocactus geometrizans (Martius) Cons. Nopalea cochenillifera (1.) Salm-Dyck Pachycereus pecten-aboriginum (Engelm.) Britt. & Rose P. pringlii (Berger) Britt. & Rose Opuntia amyciaea Tenore O. engelmanii Salm-Dyck O. ficus-indica (L.) Miller O. fragilis (Nutt.) Haworth O. humifusa (Rafinesque) Rafinesque O. inermis De Candolle O. lindheimeri Engelm. O. megacantha Salm-Dyck O. phaeacantha Engelm. O. rastera Weber O. robusta Wendland in Pfeiff. O. streptacantha Lem. O. stricta Haworth Pereskia grandiflora Haworth Selenicereus megalanthus (Schum.) Britt. & Rose [=Hylocereus triangularis (1.) Britt & Rosel Stenocereus griseus (Haworth) Buxb. S. gummosus (Engelm.) Gibs. & Horak S. queretaroensis (Weber) Buxb. S. stellatus (Pfeiff.) Riccob. S. thurberi (Engelm.) Buxb. Tricocereus pachanoi Britt. & Rose

Use V F; F; F; F; F;

IC IC IC IC IC F; IC F; IC IC; MP F; IC AF;V F; IC F; IC F; MP; V AF AF; F; IC; MP; V AF AF F; MP; V AF AF; F AF AF AF; MP; V F;MP;V AF V

IC IC IC IC IC IC IC; MP F; F; F; F; F; F;

Note. AF, animal feed; F, fruit; IC, industrial crop; MP, medicinal plant; V, vegetable.

and provide the diversity required for sustainability. The main input is the price of the plants. Although the productivity of some cacti is not high, the productivity of certain cacti can surpass other crops, many of which are relatively recently introduced, such as mango, avocado, banana, maize, potato, and tomato. These crops have been moved from con-

314

Y. MIZRAHI, A. NERD, AND P. S. NOBEL

tinent to continent, from the tropics and temperate zones, but little movement has occurred for semiarid and arid crops such as cacti. For instance, Hylocereus undatus was introduced as a fruit crop to Vietnam and has become naturalized there, often climbing on backyard fences and existing trees; it is now an important export. Selenicereus megalanthus is exported from Colombia, initially based on the realization of its potential by a foreigner, which changed a backyard local fruit into a highly profitable export item. Agroforestry, which can combat desertification and create sustainable systems, should adopt cacti for semiarid and arid zones. Australia experienced five consecutive years of drought from 1989, with devastating results for its beef and sheep industries (mainly in western New South Wales and southwestern Queensland). Adopting the Texan and the Mexican approaches to the use of various opuntias would have greatly improved the economic situation of such Australian ranchers. B. High-Input Systems Can new cactus crops compete with existing successful agricultural systems? In South Africa, cactus pear competes well with established orchard crops (Brutsch and Zimermann 1993). A major problem is the introduction of new products into markets that are saturated with the traditional crops. Who needs these strange-looking cacti? However, some markets continuously seek new products and often pay prime prices for them; adoptions in Europe of kiwifruit from New Zealand and avocado from Israel are examples. Cactus fruits such as the fruits of Selenicereus megalanthus and Hylocereus undatus are more attractive and better tasting than avocado and kiwifruit. In western societies, health foods are in demand, a niche that can also be filled by cacti. Irrigated orchards yield more than nonirrigated ones, and irrigation at the proper time can improve both fruit quantity and quality (Nerd et al. 1989), although much more research is required to optimize conditions for different species. As for other crops, NPK fertilization increases the yields as well as the number of new shoots for cacti (Nobel 1988). NPK fertilization and N alone can increase the number of floral buds for O. ficus-indica and even induce outof-season yields (Nerd et al. 1989, 1991b, 1993a). The myth that all cacti are slow growing is incorrect (Nobel 1991, 1994). Diseases and pests occur for cacti, which have not been researched as well as for other crops (Fucikovsky 1992, 1993). Also, like other crops, cacti need optimization of inputs to provide maximum income. In the case

6. CACTI AS CROPS

315

of the climbing cacti, two major expenses are expected: first, a trellis system to allow efficient management and harvesting, thereby maximizing yields and quality; and second, either nethouses and/or greenhouses to prevent damaging solar irradiation and/or temperatures. The Cactaceae have a unique and interesting biology, yet only a few scientists have done research to unravel their remarkable mechanisms of growth and survival in harsh and stressful environments. Outside of Mexico, even fewer scientists are involved with the agricultural aspects of cacti. Most research publications are unavailable to industrial and scientific policy makers worldwide. Resources are generally allocated to the well-known, well-established common crop plants, while the opportunities for new crops such as cactus pears, Hylocereus fruits, and nopalitos remain obscure. The Cactaceae not only have a special significance to countries where water is a major limiting factor and where desertification has taken its toll, but also offer economic opportunities to every entrepreneur willing to develop new consumer tastes capitalizing on the increasing worldwide acceptance of cactus products.

LITERATURE CITED Arad (Malis), S., and E. Cohen. 1991. Outdoor cultivation of micro algae in a closed system for production of valuable biochemicals. p. 301-316. In: D. Kamely, A. M. Chakrabarty, and S. E. Kornguth (eds.) Biotechnology: bridging research and applications. Kluwer Academic, Dordrecht, The Netherlands. Arcadio, L. B. 1986. Cultivo de la pitaya. Federacion de Cafeteros, Bogota, Colombia. Backeberg, C. 1984. Die Cactaceae. Gustav Fisher, Stuttgart, Germany. Barbeau, G. 1990. La pitaya rouge, un nouveau fruit exotique. Fruits 45:141-147. Barbera, G., and P. Inglese. 1993. La coltura del ficodindia. Edagricole, Bologna, Italy. Barbera, G., F. Carimi, and P. Inglese. 1991. The reflowering of prickly pear Opuntia ficus-indica (L.) Miller: influence of removal time and cladode load on yield and fruit ripening. Adv. Hort. Sci. 5:77-80. Barbera, G., P. Inglese, and E. Pimienta-Barrios (eds.). 1995. Agroecology, cultivation and uses of cactus pear. Food and Agriculture Organization of The United Nations, Rome, Italy. Berry, W. L., and P. S. Nobel. 1985. Influence of soil and mineral stresses on cacti. J. Plant Nutr. 8:697-696. Borrego-Escalante, F. 1992. Growing prickly pear for cochineal (grana) dye production. p. 45-48. In: Proc. Third Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville. Brutsch, M. 0., and M. B. Scott. 1991. Extending the fruiting season of spineless prickly pear Opuntia ficus-indica. J. South. Afr. Soc. Hort. Sci. 1:73-76.

316

Y. MIZRAHI, A. NERD, AND P. S. NOBEL

Brutsch, M. 0., and H. G. Zimmermann. 1993. The prickly pear (Opuntia ficusindica [Cactaceae]) in South Africa: Utilization of the naturalized weed, and of the cultivated plants. Econ. Bot. 47:154-162. Cacioppo, O. G. 1990. Pitaya: una de las mejores frutas productivas por Colombia. Informative Agro Economico de Colombia. February, p. 15-19. Cantwell, M. 1991. Quality and postharvest physiology of "nopalitos" and "tunas." p. 50-67. Proceedings of the Second Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville, Texas. Cantwell, M., A. Rodriguez-Felix, and F. Robles-Contreras. 1992. Postharvest physiology of prickly pear cactus stem. Sci. Hort. 50:1-9. Crosswhite, F. S. 1980. The annual saguaro harvest and crop cycle of the Papago with reference to ecology and symbolism. Desert Plants 2:2-61 Felger, R. S., and M. B. Moser. 1974. Columnar cacti in Seri Indian culture. Kiva 39:257-275. Felger, R. S., and M. B. Moser. 1976. Seri Indian food plants: desert subsistence without agriculture. J. Ecol. Food. Nutr. 5:13-27. Fernandez, M. L., A. Trejo, and D. J. McNamara. 1990. Pectin isolated from prickly pear (Opuntia sp.) modifies low density lipoprotein metabolism in cholesterolfed guinea pigs. J. Nutr. 120:1283-1290. Fernandez, M. 1., E. C. K. Lin, A. Trejo, and D. J. McNamara. 1992. Prickly pear (Opuntia spp.) pectin reverses low density lipoprotein receptor suppression induced by a hypercholesterolemic diet in guinea pigs. J. Nutr. 122:2330-2339. Flores, V. C. 1991. The present and potential market conditions of both cactus leaves and cactus pear in Mexico, and the exportation possibilities to the United States and other countries. p. 94-101. In: Proc. Second Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville. Flores, V. C. 1992. Growing, commercializing, and marketing cactus leaves in Mexico. p. 56-65. In: Proc. Third Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville. Frati, A. C. 1992. Medicinal implications of prickly pear cactus. p. 29-30. Proceedings of the Third Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville. Frati, A. c., J. A. Fernandez-Harp, H. De La Riva, R. Ariza-Andraca, and M. Del Carmen-Torres. 1983. Effect of nopal (Opuntia spp.) on serum lipids, glycemia and body weight. Arch. Invest. Med. Mexico 14:117-1125. Frati, A. C., B. E. Gordillo, P. A. Altamirano, and C. R. Ariza. 1988. Hypoglycemic effect of Opuntia streptacantha Lemaire in non-insulin-dependent diabetes. Diabetes Care 11:63-66. Frati, A. C., M. D. Valle-Martinez, C. R. Ariza, S. Islas, and A. Chavez-Negrete. 1989. Hypoglycemic effect of different doses of nopal (Opuntia streptacantha Lemaire) in patients with type II diabetes mellitus. Arch. Invest. Med. Mexico 20:197-201. Frati, A. c., B. E. Gordillo, P. A. Altamirano, C. R. Ariza, R. Cortes-Franco, and A. Chavez-Negret. 1990. Acute hypoglycemic effect of Opuntia streptacantha Lemaire in non-insulin-dependent diabetes. Diabetes Care 13:455-456. Fucikovsky, L. A. 1992. Review of the diseases of nopalitos and tunas and their control. p. 42-44. In: Proc. Third Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville. Fucikovsky, 1. A. 1993. Some bacterial, insect and bird problems of cactus in Mexico. p. 41-43. In: Proc. Fourth Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville.

6. CACTI AS CROPS

317

Fuentes-Rodriguez, J. 1991. A survey of the feeding practices, costs and production of dairy and beef cattle in Northern Mexico. p. 118-123. In: Proc. Second Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville. Fuentes-Rodriguez, J. 1992 Feeding prickly pear to dairy cattle in Northern Mexico. p. 31-34. In: Proc. Third Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville. Gibson, A. c., and P. S. Nobel. 1986. The cactus primer. Harvard Univ. Press, Cambridge, MA. Gil, G. F., and A. R. Espinosa. 1980. Fruit development in the prickly pear (Opuntia ficus-indica MilL) with preanthesis application of gibberellin and auxin. Ciencia Investigacion Agraria 7:141-147. Gil, G. F., M. Morales, and A. Momberg. 1977. Fruit set and development in the prickly pear (Opuntia ficus-indica Mill.) in relation to pollination and gibberellic and chlorethylphosphonic acids. Ciencia Investigacion Agraria 4:163-169. Haggerton, R. 1992. Cookin with cactus: a collection of favorite recipes from the Texas Prickly Pear Council, Kingsville. Hamilton, J. R. 1992. Planting and cultivating native cactus for cattle feed and wildlife utilization in South Texas. p. 35-41. In: Proc. Third Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville. Huang, B., and P. S. Nobel. 1994. Root hydraulic conductance and its components, with emphasis on desert succulents. Agron. J. 86:767-774. Ibanez-Camacho, R., and R. Roman-Ramos. 1979. Hypoglycemic effect of Opuntia cactus. Arch. Invest. Med. Mexico 10:223-230. Inglese, P., G. Barbera, and T. La Mantia. 1993. Research strategies and improvement of cactus pear (Opuntia ficus-indica) fruit quality and production. p. 2440. In: Proc. Fourth Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville. Loik, M. E., and P. S. Nobel. 1993. Freezing tolerance and water relations of Opuntia fragilis from Canada and the United States. Ecology 74:1722-1732. Maltsberger, W. A. 1991. Feeding and supplementing prickly pear cactus to beef cattle. p. 104-117. In: Proc. Second Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville. Mann, J. 1992. Murder, magic, and medicine. Oxford Univ. Press, Oxford, UK. Mass, E. V. 1986. Salt tolerance of plants. Appl. Agr. Res. 1:12-26. Master, R. W. P. 1959. Organic acid and carbohydrate metabolism in Nopalea cochenillifera. Experientia 15:30-31. Meckes-Lozoya, M., and R. Roman-Ramos. 1986. Opuntia streptacantha: a coadjutor in the treatment of diabetes mellitus. Am. J. Chinese Med. 14:116-118. Mick, R. J. 1991. Growing variety 1308 for year around production. p. 32-35. In: Proc. Second Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville. Mick, R. J. 1992. Growing and marketing the nopalito variety 1308 in Texas. p. 7- 9. In: Proc. Third Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville. Morton, J. F. 1987. Cactaceae, strawberry pear. p. 347-348. In: J. F. Morton (ed.), Fruits of warm climates. Morton, Miami, FL. Nerd, A., and Y. Mizrahi. 1992. Effect of fertilization on prickly pear production in Israel. p. 1-6. In: Proc. Third Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville.

318

Y. MIZRAHI, A. NERD, AND P. S. NOBEL

Nerd, A., and Y. Mizrahi. 1993. Cultural practices for cactus pear in Israel for yeararound production. p. 77-80. In: Proc. Fourth Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville. Nerd, A., and Y. Mizrahi. 1994. Effect offertilization and organ removal on rebudding in Opuntia ficus-indica (L.) Miller. Sci. Hart. 59:115-122. Nerd, A., and Y. Mizrahi. 1996. Reproductive biology of cactus fruit crops. Hart. Rev. 18:321-346. Nerd, A., A. Karadi, and Y. Mizrahi. 1989. Irrigation, fertilization and polyethylene covers influence bud development in prickly pear. HortScience 24:773-775. Nerd, A., J. A. Aronson, and Y. Mizrahi. 1990. Introduction and domestication of rare and wild fruit and nut trees for desert areas. p. 353-363. In: J. Janick, and J. E. Simon (eds.), Advances in new crops. Timber, Portland, OR. Nerd, A., A. Karadi, and Y. Mizrahi. 1991a. Salt tolerance of prickly pear cactus (Opuntia ficus-indica). Plant and Soil 137:201-207. Nerd, A., A. Karadi, and Y. Mizrahi. 1991b. Out-of-season prickly pear: fruit characteristics and effect of fertilization and short drought periods on productivity. HortScience 26:527-529. Nerd, A., R. Mesika, and Y. Mizrahi. 1993a. Effect of N fertilization on autumn flowering and N metabolism in prickly pear. J. Hart. Sci. 68:337-342. Nerd, A., E. Raveh, and Y. Mizrahi. 1993b. Adaptation of five columnar cactus species to various conditions in the Negev Desert of Israel. Econ. Bot. 47:304-311. Nobel, P. S. 1988. Environmental biology of agaves and cacti. Cambridge Univ. Press, New York. Nobel, P. S. 1991. Achievable productivities of certain CAM plants: basis for high values compared with Ca and C4 plants. New Phytol. 119:183-205, Nobel, P. S. 1994. Remarkable agaves and cacti. Oxford Univ. Press, New York. Nobel, P. S. 1996. Responses of some North American CAM plants to freezing temperatures and doubled CO 2 concentratiois: implications of global change for extending cultivation. J. Arid Environ., in press. Nobel, P. S., and M. Cui. 1992. Hydraulic conductances of the soil. the root-soil air gap, and the root: changes for desert succulents in drying soil. J. Expt. Bot. 43:319326. Nobel, P. S., and M. E. Loik. 1990. Thermal analysis, cell viability, and CO 2 uptake of a widely distributed North American cactus, Opuntia humifusa, at subzero temperatures. Plant Physiol. Biochem. 28:429-436. Nobel, P. S., U. Luttge, S. Heuer, and E. Ball. 1984. Influence of applied NaCI on Crassulacean acid metabolism and ionic levels in a cactus. Cereus validus. Plant Physiol. 75:799-803. Nobel, P. S., N. Wang, R. A. Balsamo, M. E. Loik. and M. A. Hawke. 1995. Lowtemperature tolerance and acclimation of Opuntia spp. after injecting glucose or methylglucose. Int. J. Plant Sci. 156:496-504. North, G. B., and P. S. Nobel. 1992. Drought-induced changes in hydraulic conductivity and structure in roots of Ferocactus acanthodes and Opuntia ficus-indica. New Phytol. 120:9-19. Perez-Reyes, C., and E. Pimienta-Barrios. 1995. Viabilidad de semillas y poliembrionia en morfoespecies cultivadas y silvestres de Opuntia. Agrociencia, in press. Pimienta-Barrios, E. 1990. El nopal tunero. Universidad de Guadalajara, Guadalajara, Jalisco.

6. CACTI AS CROPS

319

Pimienta-Barrios, K 1991. An overview of prickly pear production in the central part of Mexico. p. 1-15. In: Proc. Second Annual Texas Prickly Pear Council Meeting. Texas A & I Univ., Kingsville. Pimienta-Barrios, K 1993. Vegetable cactus (Opuntia). p. 177-192. In: J. T. Williams (ed.), Pulses and vegetables. Chapman & Hall, London. Pimienta-Barrios, E. 1994. Prickly pear (Opuntia spp.): a valuable fruit crop for semi-arid lands of Mexico. J. Arid Environ. 28:1-11. Pimienta-Barrios, K, and K M. Engelman. 1985. Desarrollo de la pulpa y proporcion en volumen, de los componentes del loculo rnaduro en tuna (Opuntia ficus-indica (1.) Miller). Agrociencia 62:51-56. Pimienta-Barrios, K, and P. S. Nobel. 1994. Pitaya (Stenocereus spp., Cactaceae): An ancient and modern fruit crop of Mexico. Econ. Bot. 48:76-83. Pimienta-Barrios, K, G. Barbera, and P. Inglese. 1993. Cactus pear (Opuntia spp., Cactaceae) International Network: an effort for productivity and environmental conservation for arid and semi-arid lands. Cactus Succulent J. 65:225-229. Raveh, K, J. Weiss, A. Nerd, and Y. Mizrahi. 1993. Pitayas (genus Hylocereus): A new fruit crop for the Negev Desert of Israel. p. 491-495. In: J. Janick and J. K Simon (eds.), New crops. Wiley, New York. Rengel, Z. 1992. The role of calcium in salt toxicity. Plant Cell Environ. 15:625-632. Rodriguez-Felix, A., and M. Cantwell. 1988. Developmental changes in composition and quality of prickly pear cactus cladodes (nopalitos). Plant Food Human Nutr. 38:83-93. Russell, C. K, and P. Felker. 1987a. The prickly pears (Opuntia spp., Cactaceae): a source of human and animal food in semiarid regions. Econ. Bot. 41:433-445. Russell, C. K, and P. Felker. 1987b. Comparative cold hardiness of Opuntia spp. and cvs. grown or fruit, vegetable and fodder production. J. Hart. Sci. 62:545550. Scheinvar, 1. 1985. Flora ilustrada Catarinese Cactaceae. Itajai. Santa Catarina, Brasil. Silverman, F. P., D . R. Young, and P. S. Nobel. 1988. Effect of applied NaCl on Opuntia humifusa. Physiol. Plant. 42:343-348. Vietmeyer, N. 1990. The new crops era. p. xviii-xxii. In: J. Janick and J. K Simon (eds.), Advances in new crops. Timber, Portland, OR. Wang, N., and P. S. Nobel. 1995. Phloem exudate collected via scale insect stylets for the CAM species Opuntia ficus-indica under current and doubled CO 2 concentrations. Ann. Bot. 75:525-532. Weiss, J., A. Nerd, and Y. Mizrahi. 1993a. Development of the cactus apple (Cereus peruvianus) as a new crop to the Negev Desert of Israel. p. 486-491. In: J. Janick and J. K Simon (eds.), New crops. Wiley, New York. Weiss, J., A. Nerd, and Y. Mizrahi. 1993b. Vegetative parthenocarpy in the cactus pear Opuntia ficus-indica (1.) Mill. Ann. Bot. 72:521-526. Weiss, J., A. Nerd, and Y. Mizrahi. 1994a. Flowering and pollination requirements in Cereus peruvianus cultivated in Israel. Israel J. Plant Sci. 42:149-158. Weiss, J., A. Nerd, and Y. Mizrahi. 1994b. Flowering behaviour and pollination requirements in climbing cacti with fruit crop potential. HortScience 29:1487-1492. Weiss, J., 1. Scheinvar, and Y. Mizrahi. 1995. Selenicereus megalanthus (the yellow pitaya): a climbing cactus from Colombia. Cactus Succulent J. 67:280-283. Wessels, A. B. 1988. Spineless prickly pear. First Perskor, Johannesburg, South Africa.

1. Young cladodes (nopalitos) of Opuntia ficus-indica in a traditional round pack in Milpa Alta, ready to be shipped to a Mexico City market. 2. Opuntia ficusindica growing in a soil bed with tunnel for year-round production of nopalitos in Milpa Alta, Mexico. 3. Flowering plant of Nopalea cochenillifera, whose spineless cladodes are used for nopalitos and whose glochid-containing flower bases (receptacles) are used as a delicate low-mucilage vegetable. 4. Harvesting fruit known as cactus pears from Opuntia ficus-indica in Sicily. 5. Colored fruits of Stenocereus queretaroensis (pitaya de Queretaro) in Oaxaca, Mexico. 6. Ripe fruits of Selenicereus megalanthus (pitaya amarillo).

7. Thirteen-month-old research plantation of Hylocereus undatus at Ben Gurian University greenhouse in Beer Sheva, Israel. 8. Commercial orchard of Hylocereus undatus near Nha Trang, Vietnam. 9. Fruits of Hylocereus undatus ("Dragon fruit") in Phan Rang market, Vietnam. 10. Ripening fruit of Hylocereus, probably a hybrid between H. undatus and H. polyrhizus. 11. Infestation of cochineal insects on clado des of Opuntia ficus-indica in Los Angeles, California.

12

13

14

Photographs of cactus fruits: 12. Opuntia ficus-indica ('Ofer'); 13. peruvian us; 14. Hylocereus costariscensis. Bar = 30 mm.

Cereus

15

16

Photographs of cactus fruits: 15. Bar = 30 mm.

Hylocereus undatus; 16. Hylocereus polyrhizus.

7

Reproductive Biology of Cactus Fruit Crops Avinoam Nerd The Institutes for Applied Research Ben-Gurian University of the Negev Beer-Sheva 84105, Israel Yosef Mizrahi The Institutes for Applied Research and the Department of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105, Israel 1.

II.

III.

IV. V.

Introduction Cultivated Species A. Growth Habit and Cultivation Areas B. Fruit Characteristics C. Nomenclature Flowers A. Flower-Bearing Parts B. Floral Bud Development C. Flower Structure D. Flowering Period E. Effects of Environmental Conditions on Flowering 1. Climate 2. Fertilization F. Effect of Organ Removal on Floral Bud Burst Pollination Requirements A. Day-Blooming Species B. Night-Blooming Species Fruit Development A. Fruit Structure

Horticultural Reviews, Volume 18, Edited by Jules Janick ISBN 0-471-57334-5 © 1997 John Wiley & Sons, Inc. 321

322

A. NERD AND Y. MIZRAHI

B. Fruit Growth and Maturation C. Fruit Weight D. Seed Set and Parthenocarpy E. Seed Dispersal Problem VI. Concluding Remarks Literature Cited

I. INTRODUCTION

The cactus family (Cactaceae) comprises more than 1500 species. In most of them the above-ground part of the plant consists of green succulent stems that store water for dry periods and function as the main photosynthetic organ. Leaves are often absent, and if they do exist they are small and are shed early (Gibson and Nobel 1986; Nobel 1988). A distinctive feature of the stems is the presence of areoles (lateral buds), which produce spines, trichomes, lateral shoots, and flowers. Cacti are native to the Americas and are known throughout the world as ornamental plants, appreciated for their beautiful flowers and bizarre vegetative features. Only a few cactus species are known as food crops, but ethnobotanical studies have revealed that many are utilized by local people in the Americas. Fruits are eaten fresh, dried, or fermented as alcoholic beverages, and flour is prepared from the seeds (Sanches-Mejorada 1984; Felger and Moser 1985; Bravo and Sanches-Mejorada 1991; Nobel 1994; Mizrahi et al. 1996). The recent upsurge of interest in cacti as fruit crops may be attributed to the search for new exotic fruit crops (Vietmeyer 1986; Nerd et al. 1990) or for crops with low water demands (Nobel 1994). In particular, the high productivity and the precocious yielding of some species, such as the cactus pear Opuntia ficus-indica (Nobel 1988, 1994), the columnar cactus Cereus peruvianus (Nerd et al. 1993b), and the climbing cactus Selenicereus megalanthus (Cacioppo 1990), are probably important factors in promoting their cultivation. The low water demand of stem cacti is related to their crassulacean acid metabolism (CAM) mode of photosynthesis; uptake of CO 2 occurs during the night when the stomata are open. Because night temperatures are lower than day temperatures, water loss by transpiration in CAM plants is lower than that in plants in which the stomata open during the day (C 3 and C4 plants). This results in a higher water use efficiency and lower water demand in CAM plants (Nobel 1988, 1994).

7.

REPRODUCTIVE BIOLOGY OF CACTUS FRUIT CROPS

323

II. CULTIVATED SPECIES

A. Growth Habit and Cultivation Areas Known cultivated species are described in Table 7.1. They include shrubs with flattened stem joints (cladodes) of the genus Opuntia (subfamily Opuntioidae); columnar shrubby or arborescent species of the genera Stenocereus and Cereus; and climbing (trailing) hemiepiphytes with slender ribbed stems of the genera Hylocereus and Selenicereus (subfamily Cactoidae). Opuntia spp., known as prickly pear or cactus pear, are the most widely cultivated cactus crops (Mizrahi et al. 1996). The main cultivated cactus pear is O. ficus-indica, which includes spineless (few spines) cultivars (Wessels 1988; Pimienta 1990, 1994; Barbera et al. 199Za). Another important species is O. amyc1aea, spiny plant with green fruits only (Pimienta 1990). Opuntias are usually cultivated in subtropical regions, O. ficus-indica on a wide scale in Mexico, the Mediterranean basin, and South Africa, and O. amyc1aea only in Mexico (Russel and Felker 1987). Columnar and climbing species are commercially cultivated to a far smaller extent than opuntias (Mizrahi et al. 1996). Most of the columnar crops belong to the genus Stenocereus and are cultivated in semiarid regions in central and southern Mexico (Castillo 1984; Cruz 1984; Liamas 1984; Bravo and Sanchez-Mejorada 1991; Nerd et al. 1993b; Nobel 1994; Pimienta and Nobel 1994). Among the columnar species, Cereus peruvianus has long been known as an ornamental in subtropical and tropical regions (Cullman et al. 1986), and recently it has been planted in orchards-both in Israel (Nerd et al. 1993b; Weiss et al. 1994a) and Southern California-for its fruits. The climbing cacti are native to warm, humid regions where they trail on trees or rocks (Haber 1983; Benzing 1990). They rely on roots anchored in the ground and also develop adventitious roots along the stems, which attach themselves to the natural supports and help the plant in absorbing water. These cacti are cultivated successfully in the open in tropical countries (Barbeau 1990; Cacioppo 1990; Mizrahi et al. 1996), but in subtropical countries they require both shading, because of their sensitivity to high irradiation, which damages the stems, and protection against low temperatures «3°C) in cold sites (Raveh et al. 1993). Large areas under cultivation have been reported for Selenicereus megalanthus in Colombia (Arcadio 1986; Cacioppo 1990) and for Hylocereus undatus in Nicaragua (Barbeau 1990) and Vietnam (Mizrahi et al. 1996).

A. NERD AND Y. MIZRAHI

324

Table 7.1. Fruit crops of the cactus family.

Common names

Species

Opuntia O. ficus-indica (1.) Miller

O. amyc1aea Tenore

Stenocereus S. griseus (Howarth) Buxbaum S. queretaroensis (Weber) Buxbaum S. stellatus (Pfeiffier) Riccobobo Cereus C. peruvianus (1.) Miller

Tuna, prickly, or cactus pear

Pitaya de Queretaro

Medium-size oblong fruit; peel and pulp colors are variations of green, yellow, and red; peel with glochids Fruits as above, but they are greenish Medium globose fruit; peel with spines; pulp yellow to red Medium globose fruit; peel with spines; pulp white, yellow, purple red or dark red Medium globose fruits; peel with spines; pulp red

Pitaya de Augusto

Columnar, shrub or small tree

Pitaya, apple cactus

Columnar, shrub or small tree

Medium-large oblong fruit; peel smooth, yellow, violet, or red; pulp white

Pitaya, pitahaya

Slender, triangular stems, climbing Slender, triangular stems, climbing Slender, triangular stems, climbing

Large globose fruit; peel dark red with large scales; pulp violet red Large oblong fruit; peel dark red with large scales; pulp red Large oblong fruit; peel dark red with large scales; pulp white

Slender, triangular stems, climbing

Medium oblong fruit; peel yellow with tubercles and spines; pulp white

Selenicereus S. megalanthus (Schun ex Vampel) Moran

Pitaya (pitahaya) amarilla, yellow pitaya

= 100-200 g;

Flattened stem joints, shrub

Columnar, shrub or small tree Columnar, shrub or small tree

Pitaya de Mayo

Pitaya (pitahaya) raja, red pitaya

Medium

Fruit characteristics z

As above, Tuna, prickly, or cactus pear spiny

Hylocereus H. costaricensis (Weber) Britton & Rose H. polyrhizus (Weber) Britton & Rose H. undatus (Haworth) Britton & Rose

z

Growth habit

Pitaya, pitahaya

large

= 300-600 g.

7.

REPRODUCTIVE BIOLOGY OF CACTUS FRUIT CROPS

325

B. Fruit Characteristics Fruits of the cactus pear are easily distinguishable from those of the columnar and climbing species (Color Plates 12-16). Cactus pear fruits have a relatively thick peel bearing areoles with glochids (small easily shed spines), and the flesh contains 50-300 rigid seeds with a rough coat (Pimienta 1990; Barbera et al. 1992a). Fruits of the other species of cactus do not have glochids and have a thin peel that may be smooth or may have scales and long spines. The flesh contains abundant small seeds that are soft and digestible, resembling those of the kiwifruit (Britton and Rose 1963; Morton 1987; Pimienta and Nobel 1994; Weiss et al. 1994a,b). In Latin America, the cactus pear type of fruit is known as tuna, and fruits of the columnar and climbing types as pitaya (Nobel 1994). The name pitahaya is also commonly used for fruits of some climbing cacti (Barbeau 1990; Bravo and Sanchez-Mejorada 1991). C. Nomenclature A scan of the literature pertaining to cacti reveals a certain amount of confusion about the classification and nomenclature of the cacti, a situation that reflects problems associated with the development of cactus systematics (Britton and Rose 1963; Bravo 1979; Cullman et al. 1986; Gibson and Nobel 1986). With respect to cactus fruit crops, we can cite two examples: (1) cultivated Opuntia spp. are in fact varieties, geographic forms, or hybrids (Pimienta 1994); and (2) the yellow-fruit climbing pitaya Selenicereus megalanthus, which is cultivated in Colombia, has been referred to as Hylocereus triangularis by Cacioppo (1991) and Nobel (1994). Recently, Weiss et al. (1995) have indicated that the species is, in fact, Mediocactus megalanthus later renamed Selenicereus megalanthus (K. Schum. ex Vaupel) Moran (Moran 1953). The botanical names in the present review are those used in recent publications. III. FLOWERS A. Flower-Bearing Parts In the cactus pear species, floral and vegetative buds emerge mainly on terminal cladodes formed during the preceding growth period and to a smaller extent on the underlying cladodes (Wessels 1988;

326

A. NERD AND Y. MIZRAHI

Nieddu and Spano 1992; Pimienta 1990; Barbera et al. 1992a; Brutsch and Scott 1991). Apical dominance appears to control bud emergence, since most of the buds arise from areoles located on the upper margin of the cladode. Shaded terminal cladodes are usually nonproductive, while in those exposed to light-the majority-the number of flower buds may reach 20 and more. We found that in a highly productive 8-year-old plantation approximately 80% of the terminal cladodes produced fruits in the spring, with an average of eight fruits per cladode (Nerd et al. 1993a). Fruiting of new cladodes is associated with dry weight accumulation (Nerd and Mizrahi 1994), and according to Garcia de Cotazar and Nobel (1992) the dry weight of bearing cladodes exceeds the minimum dry weight for a particular surface area by at least 33 g. In the pitayas (columnar and climbing cacti) the areoles are located on the edge of the stem ribs. Flower buds emerge from areoles located on segments of past years and are rarely found on the lower part of the plant (Pimienta and Nobel 1994; Weiss 1995). Sometimes, at the end of the growth season, the current year's growth also bears flowers in the climbing cacti.

B. Floral Bud Development For all the discussed species an areole may produce only one floral bud. In the cactus pear the first signs of floral structure can be detected under the microscope a few days after bud emergence (Rivera et al. 1981; Nieddu and Spano 1992). At this stage floral buds reach 4-5 mm in length and become spherical and easily distinguishable from the flat vegetative buds. In the spring the entire process of flower bud growth, from bud initiation to full development and anthesis, takes approximately 7 weeks (Pimienta 1990; Wessels and Swart 1990; Barbera et al. 1992b). Buds gain significant weight in this process, and bud final weight accounts for 25-30% of fruit fresh weight. Cumulative volume growth is sigmoidal, the phase of rapid growth beginning 2 weeks after emergence and ending 1 week before anthesis. Data on bud differentiation and growth in the pitaya crops are sparse, but it has been observed that the areolar buds are undifferentiated before bud emergence and that differentiation occurs after the buds emerge. The time between bud emergence and anthesis differs among the pitayas (Pimienta and Nobel 1994; Weiss 1995), being shorter in C. peruvianus and Hylocereus spp. (30-35 days) than in S. queretaroensis (40 days) and S. megalanthus (45-60 days).

7.

REPRODUCTIVE BIOLOGY OF CACTUS FRUIT CROPS

327

C. Flower Structure

Flowers of the above-mentioned species are sessile and hermaphroditic; the ovary is inferior and is engulfed by a thick receptacle; and abundant stamens, a single style, and perianth parts rise above the top of the ovary (Fig. 7.1). Investigations on the evolution of cactus flowers suggest that the ovary has been sunk into a modified stem, as is manifested by the existence of stem features, such as areoles, bracts, and spines on the surface of the receptacle, and later on the surface of the fruit (Gibson and Nobel 1986). Growth of the recep-

Fig. 7.1. Schematic longitudinal sections of cactus flower. From top to bottom: Opuntia ficus-indica (xO.5), Stenocereus queretaroensis (xO.5), Cereus peruvianus (xO.25), Selenicereus megalanthus (xO.25).

328

A. NERD AND Y. MIZRAHI

tacle tissue is very pronounced during the bud stage. At anthesis its length may reach 2-3 cm in the columnar cacti, 3-5 cm in the climbing cacti, and 4-5 cm in the cactus pears. The unilocular ovary derives from the 10Tver portions of the carpels, whereas the upper portion develops into the stamens and style. Numerous ovules are to be found in pariental placentation in vertical rows along the ovary wall (Boke 1980; Gibson and Nobel 1986). The number of ovules varies markedly among the flowers of the different types of cactus. An average of 270 ovules was counted in the Israeli cultivar of O. ficus-indica known as 'Ofer,' whereas ovaries of pitaya-type fruits from H. undatus contain an average of 7200 ovules. The ovules are connected by a very well developed funiculus to the ovary wall. For O. ficus-indica it has been shown that the funiculus grows around the entire ovule (Pimienta and Engelmann 1985) and for some pitaya fruits the funiculus was found to be branched (Kimnach 1967; Gibson and Nobel 1986). In the cactus pear the stamens and style are located in a pit surrounded on the top by a perianth composed of pale green and yellow leaf-like structures, which function as sepals and petals, respectively. In the columnar and the climbing cacti the stamens and the style are surrounded by a tube that ends in the perianth parts (Fig. 7.1). The flowers of the columnar cactus C. peruvianus and the climbing cacti Hylocereus spp. and S. megalanthus are among the largest in the cactus family, being distinguishable by their long tube and the wide whitish funnel produced by the perianth.

D. Flowering Period The appearance of flower buds marks the onset of the current reproductive period in both the cactus pear and the pitaya crops. In subtropical areas, the cactus pear O. ficus-indica produces a major flush of floral and vegetative buds in the spring, March-April in the northern hemisphere and September-October in the southern hemisphere (Wessels 1988; Pimienta 1990; Barbera et al. 1992a). However, under certain conditions (irrigation and fertilization), a second flush, generally smaller than the spring flush, appears in the autumn after the harvest of the summer crop produced by the spring flush (Nerd and Mizrahi 1993a,b). Bud burst occurs over a 4- to 5-week period, and thus fruit ripening is also spread over a period of several weeks. For the columnar cacti, there is very little information in the literature. Weiss et al. (1994a) found that for C. peruvianus cultivated in the Negev Desert of Israel there were one or two flushes of floral

7.

REPRODUCTIVE BIOLOGY OF CACTUS FRUIT CROPS

329

buds, each lasting several weeks. The first flush, which was the most important one, occurred for most of the investigated genotypes at the beginning of the summer (May-June) and the second flush occurred 2 months later. In a related species, C. jamacaru, native to northeastern Brazil (Scheinvar 1985), blooming stretches over the entire growth season. Species of Stenocereus growing in Israel differ in their flowering time, being spring for S. griseus (pitaya de Mayo) late summer for S. thurberi (organ pipe), and autumn for S. gummosus (pitaya agria). Climbing cacti grown in tropical countries produce several flushes of floral buds during the growth season, and the flowering cycle tends to be concentrated into a few days (Barbeau 1990; Haber 1983). In Israel, Hylocereus spp. produced two to three flushes during the summer-autumn, while S. megalanthus flowered mainly in the autumn (Weiss et al. 1994b). E. Effects of Environmental Conditions on Flowering Controlled experiments examining the effect of climatological factors on flowering in the species covered in this review are scarce, and for clues to inductive conditions we must look at their flowering response to various environmental situations. Data on the effect of fertilization on floral bud production are available only for O. ficus-indica and were obtained in experiments aimed at producing an out-of-season crop (Nerd et al. 1991. 1993a; Nerd and Mizrahi 1994). 1. Climate. The spring bud burst in O. ficus-indica follows the winter rest period; thus, chilling may be involved in bud break. The results of two studies carried out by us in Israel support this idea: (1) Covering plants in the field with a plastic cover for 4 weeks in the winter reduced floral bud burst (Nerd et al. 1989); and (2) a comparison of cladodes cut from the orchard at various times in the winter and placed in a heated greenhouse or in a shadehouse showed that floral bud burst occurred at the same time for late-cut cladodes in the greenhouse and for all the cladodes in the shadehouse as for bud burst in the orchard, but it was delayed for early-cut cladodes in the greenhouse (unpublished data). Average winter max/min temperatures were 30/17°C in the greenhouse and 20/10°C outdoors. The time of bud burst in the spring depends on warming up at the end of the winter; in Mexico (Pimienta 1990) and Israel budding is delayed at colder higher altitudes, occurring in Israel when the average monthly

330

A. NERD AND Y. MIZRAHI

temperature rises to 15°-16°C. Since main bud burst in O. ficus-indica occurs when day length is increasing it is possible that bud burst be photoperiod-dependent; however, there are no data available to support this premise. The climbing pitayas produce several floral flushes during the wet season in tropical areas (Arcadio 1986; Barbeau 1990; Cacioppo 1990). These cacti demonstrate autonomous flowering behavior, with flowers being produced under appropriate conditions. In Israel (Weiss et a1. 1994b), Hylocereus spp. tend to flower in the summer and S. megalanthus tends to flower in the autumn when temperatures decrease, indicating that high summer temperatures probably inhibit flowering in S. megalanthus. 2. Fertilization. Although floral bud burst in O. ficus-indica usually occurs in the spring, it may also take place in the summer and the autumn but to a lesser extent. Such a flowering pattern was observed in highly manured orchards located in an area with summer rainfall in northern California (Curtis 1977). In semiarid areas in Israel, late summer and autumn floral burst was stimulated by application of water together with high rates of N-P-K (Nerd et a1. 1991) or N fertilizers (Nerd et a1. 1993a; Nerd and Mizrahi 1994). Floral buds emerged a few weeks after fertigation, and the number of buds correlated with the concentration of different N fractions in the bearing cladodes. The main flush was observed on mature seasonal cladodes that had not experienced winter conditions. This indicates that chilling is not crucial for floral bud break in O. ficus-indica. However, chilling is more important to natural bud break than nutritional conditions (probably N), since the spring bud break is the common situation in O. ficus-indica and is not conditioned by nutrition (Nerd et a1. 1993a). The practice of inducing an autumn flush is widely used in Israel, because fruits that ripen in the winter and the spring command high prices-three to five times greater than those for the regular summer crop (Nerd and Mizrahi 1993b; Mizrahi et a1. 1996).

F. Effect of Organ Removal on Floral Bud Burst

Removing the natural spring flush of O. ficus-indica at bloom stimulates a second flush, which appears several weeks later (Barbera et a1. 1991; Brutsch and Scott 1991; Nerd and Mizrahi 1994). The reflowering ability of O. ficus-indica was known in Sicily as early as the eighteenth century, and the procedure is widely exploited there

7.

REPRODUCTIVE BIOLOGY OF CACTUS FRUIT CROPS

331

to produce a late crop (Barbera et a1. 1992a). The common treatment in Sicily, known as scozzolatura, involves the removal of new cladodes and flowers at full bloom. Studies on organ removal have revealed a number of factors that affect bud burst in O. ficus-indica. The new flush obtained by scozzolatura was closely associated with the natural flush (Barbera et a1. 1991); new floral buds were produced on the flower-bearing cladodes of the natural flush, and the number of new floral buds per cladode was correlated with the initial number of buds. In addition, flower production in the new flush had a low sensitivity to nitrogen fertilization, as is the case for the natural flush (Nerd et a1. 1993a; Nerd and Mizrahi 1994). The scozzalatura studies indicated that the first flush, flowers and new cladodes, further inhibited bud burst. Flowers probably imposed a stronger inhibitory effect than cladodes, since the removal of flowers was obligatory for a new floral bud burst, while removal of young cladodes simply increased the flush (Inglese et a1. 1994a; Nerd and Mizrahi 1994). There was also a seasonal effect on the flowering response to scozzolatura; if the treatment was applied before blooming, the number of new floral buds was close to that of the initial number, but if it was delayed to the end of blooming, budding decreased by more than 50% (Portolano 1962; Barbera et a1. 1991; Brutch and Scott 1991). When fruits were removed in the middle of the fruit development period, a significant number of floral buds was obtained only when the removal treatment was combined with the application of high rates of nitrogen (Nerd and Mizrahi 1994). Changes in climate, such as a rise in temperature, or accumulation of inhibitory metabolites in the bearing cladode may be associated with the tapering off of bud break with time. Gibberellins probably reduced the tendency for floral bud burst, since burst was arrested when GAs was injected into bearing cladodes a few days before or after the scozzolatura treatment (Barbera et a1. 1993). The latter study also showed that shading of bearing cladodes for few days shortly before or after the scozzolature treatment also arrested floral bud production; thus, exposure of bearing cladodes to high light probably promotes floral bud burst.

IV. POLLINATION REQUIREMENTS A. Day-Blooming Species Flowers of the Opuntia species open during the day. Studies performed on O. ficus-indica showed that most of the flowers open late

332

A. NERD AND Y. MIZRAHI

in the morning (type A), although some open in the afternoon (type B). All flowers close in the evening; this event marks the end of the

anthesis phase in type A flowers, but in type B anthesis resumes the following morning, with the flowers closing finally in the evening (Pimienta 1990; Weiss 1995). Flowers of O. ficus-indica display the attributes of insect-pollinated flowers (Portolano 1962; Pimienta 1990; Barbera et a1. 1992a). The perianth lobes are large and yellow, the central pistil possesses a rigid stigma that enables insects to land, the stigma is sticky, the pollen grains are large, and an abundant amount of nectar accumulates at the base of the corolla. Flowers are visited by bees of various species (Portolano 1962; Pimienta 1990; Barbera et a1. 1992a), which appear to be involved in pollination. For a number of Opuntia species native to the southwest United States, flowers were visited by various beetle and bee species, but only the bees were found to be efficient pollinators (Grant and Hurd 1979). The flowers may also be autogamous (self-pollinated), and bagged flowers set fruits. Dehiscence of anthers starts before anthesis, and anthers touch the pistil during the flower opening stage, laying pollen grains on the stigma (pseudocleistogamy) (Pimienta 1990). Stamen behavior of O. ficus-indica and other species of Opuntia probably facilitates insect pollination: When they are touched, they bend toward the style (Grant and Hurd 1979). Despite the autogamy, a visit of pollinators is necessary for efficient pollination; only a few seeds are produced in bagged flowers compared with uncovered ones (personal observation). Sicilian cultivars have been shown to be self-compatible (Damigella 1958). Since problems of fruit set are seldom encountered in vegetatively propagated plantations composed of a single cultivar or in single plants grown in backyards, it is safe to conclude that self-compatibility is a common phenomenon in O. ficus-indica. B. Night-Blooming Species Flowers of the pitaya crops Stenocereus spp., C. peruvian us, Hylocereus spp., and S. megalanthus are nocturnal and remain open for one night. Usually, the flowers begin to open near to sunset and close after sunrise (Gibson and Nobel 1986; Gibson 1989, 1990, 1991; Pimienta and Nobel 1994; Weiss et a1. 1994a,b). The flowers have traits of flowers pollinated by nocturnal animals (Grant and Grant 1979). They are strongly scented with a long floral tube-4-10 cm in Stenocereus spp. and 25-30 cm in C. peruvianus, Hylocereus spp., and S. megalanthus-and contain nectar chambers at the bottom of

7.

REPRODUCTIVE BIOLOGY OF CACTUS FRUIT CROPS

333

the tube. In Mexico, bats, wasps, and beetles visit the flowers of Stenocereus spp. (Gibson 1990; Pimienta and Nobel 1994), and in Central America bats and hawkmoths are known to visit the flowers of Hylocereus and Selenicereus (Barbeau 1990; Cacioppo 1990; Haber 1983). Anthesis phases as well as the breeding system and pollen vectors have been studied for C. peruvianus and the climbing cacti H. costaricensis, H. polyrhizus, H. undatus, and S. megalanthus under the conditions prevailing in Beer-Sheva, Israel (Weiss et al. 1994a,b, Weiss 1995). For all species, flowers started to open shortly before dark or at the beginning of the dark period and closed in the late morning (Fig. 7.2). Anther dehiscence occurred before flowers opened, and nectar secretion began just as the flowers opened. In vitro pollen germination studies showed that germinability tended to decrease during the anthesis period for most investigated species. In contrast to other species, S. megalanthus usually had low germination rate «4 %), which was associated with the low viability of its pollen. Hand pollination studies with self and foreign pollen showed that clones of C. peruvianus, H. polyrhizus, and H. costaricensis were self incompatible and those of H. undatus and S. megalanthus were self compatible (Weiss et al. 1994a,b; Weiss 1995). Genotypes of C. peruvianus raised from seeds were compatible with each other. However, for the self-incompatible climbing cacti, limited plant material, which consisted of one or two clones of each species, did not allow conclusions to be drawn concerning compatability within the species. The Hylocereus species investigated were compatible with one another, and the largest fruits in each of the Hylocereus species were obtained by specific cross-combination within the genus (Weiss et al. 1994b). Field experience also indicated that H. undatus and S. megalanthus are self-compatible, since solid clone orchards are productive (Barbeau 1990; Cacioppo 1990). It was also shown by Weiss et al. (1994b) that among the selfcompatible species S. megalanthus was autogamous and set fruits without a pollen vector, whereas H. undatus required a pollen vector to set fruits. Flower morphology was associated with autogamy; upper anthers touched the stigma lobes in S. megalanthus during flower closing, but anthers were separated spatially from the stigma in H. undatus. However, the involvement of a pollen vector in S. megalanthus was also important for fruit production, since heavier fruits were produced when flowers were hand self-pollinated. For species whose pollination depends on a pollen vectors, fruiting prob-

A. NERD AND Y. MIZRAHI

334

Plant No. 21 Visits by honeybees

I

I

:

:

I

I

I

Visits by honeybees

r--l

Flower open

I

--------~----------.,

: Flower opens

I I

Anther

I

f

I

18.00

!

!

20.00

I

I I I I I

I

I:

I I I I I

f!

!:

I I I

Flower closes:

I

Start of nectar secretion

de~is:ence

I I I

'I

22.00

24.00

2.00

4.00

I: I

6.00

I I I I

I

I I I I

I

I

8.00

Time of the day Plant NO.1 Visits by honeybees

n

I I

Visits by honeybees

Flower open

r--l

I I

I I

Flower opens i I I : Start of nectar

Flower closes: : ;I

I

Anther I dehiscence

I I

r

I I I

t

secre IOn

I ::

'f : 1/ Sunset ! !!L- !

18.00

20.00

Sunset \ I

I

22.00

I I

24.00

2.00

I

4.00

!

6.00

I I i

,

,

I I I I

I I

'I

I I

!

8.00

Time of the day

Fig. 7.2. Daily cycle events associated with anthesis in an early-opening plant (No. 21) and late-opening plant (No.1) of Cereus peruvianus.(Weiss et al. 1994b; reprinted with permission from Laser Pages Publishing, Jerusalem.) Observations were peformed in Beer-Sheva (south of Israel) in the summer. The diagram is also representative of climbing species in this area.

lems may rise in areas where the appropriate pollinators are absent. In Israel the visitors to C. peruvianus and the climbing species were confined to daily active visitors, the honeybee (Apis mellifera) and the carpenter bee (Xylocopa pubescens ) (Weiss et al. 1994a,b). The bees visited the flowers during the daylight hours, in the evening when the flowers opened, and in the morning when the flowers started to close. Fruit set and fruit weight were lower in bee-pollinated flowers (open pollination) than in hand cross-pollinated flow-

7.

REPRODUCTIVE BIOLOGY OF CACTUS FRUIT CROPS

335

ers, probably because the effectivity of bee pollination was limited. This may be related to the lack of specific adaptation of the bees to the flowers: During the process of collecting the pollen they usually touched the stigma, but they did not try to reach the nectar at the base of the long flower tube. In addition, the fact that the bees visited the flowers only for a short time, at the beginning and at the end of anthesis, may be a reason for the low pollination effectivity of the bees, especially in C. peruvianus and H. polyrhizus, for which pollen germinability decreased during the night. Honeybees, which are polytrophic pollinators that forage on a wide spectrum of flower types, have also been found to pollinate other nocturnal cacti, such as saguaro (Carnegiea gigantea) and the organ pipe cactus (S. thurberi) in their natural habitats, alongside doves and nocturnal bats (Alcorn et al. 1961, 1962; McGregor et al. 1962; Schmidt and Buchmann 1986). Evidence that genotypes of C. peruvianus that opened early in the evening had higher fruit set than those that opened at the onset of dark (Fig. 7 .2; Weiss et al. 1994a) suggests that selection of clones with flowers that open for a longer time in the afternoon and in the morning may improve the results of bee pollination. Since fruits of the climbing cacti demand high prices, hand pollination, which induces high fruit set and heavy fruits, is economically feasible. Studies in Israel showed that good results were obtained when pollination was performed during the entire period of anthesis, including in the morning when the flowers close (Weiss et al. 1994b), which is a time when hand pollination can be carried out conveniently. V. FRUIT DEVELOPMENT

A. Fruit Structure

Fruit of the species discussed here are of the berry type, developing from both the ovary and the receptacle that surrounds the ovary. The receptacle becomes the peel, and only a thin inner layer of peel is contributed by the pericarp (ovary wall). The pulp develops mainly from the funiculi that connect the ovuli to the ovary wall (Boke 1980; Gibson and Nobel 1986). Detailed anatomical studies have been made on the fruits of O. ficus-indica (Pimienta and Engelman 1985; Wessels 1992). It was shown that the ovule is completely surrounded by an envelope formed by the funiculus and that the main part of the pulp originates from the funicular envelopes and papillae that were an out-

336

A. NERD AND Y. MIZRAHI

growth of the dorsal epidermal cells of the funiculi and their envelopes. Similar observations were also reported for the prickly pear O. dillenii (Maheshwari and Chopra 1955). Since the pulp developed in O. ficus indica around empty seeds as well as around fullsized seeds, it was proposed that at least early seed development is necessary for fruit development (Pimienta and Engelman 1985). B. Fruit Growth and Maturation Changes in fruit size and fresh weight with time, measured in Sicilian and Israeli cultivars of O. ficus-indica, showed a double sigmoid growth curve consisting of three distinct phases: phase I, rapid growth, commencing shortly after anthesis; phase II, suspended growth; and phase III, a final growth spurt after the onset of color change, lasting several days (Barbera et a1. 1992b; Weiss et a1. 1993). In fruits that developed during the hot season, the three phases were approximately the· same length, and the entire fruit growth period lasted 80-90 days. A pronounced gain in fresh and dry weight occurred for the peel in phase I, for the seeds in phase II, and for the core at the end of phase II and in phase III. The duration of fruit development may depend on the cultivar; for example, in the Israeli parthenocarpic clone 'BS1' the period of fruit growth was shorter than that in the seeded cultivar 'Ofer.' The absence of the seed growth phase and early onset of the core development stage may be associated with the short fruit period in 'BS l' (Weiss et a1. 1993). The time of ripening has been documented for many cultivars grown in Mexico and South Africa (Wessels 1988; Pimienta 1990; Nerd and Mizrahi 1993b). Most of the cultivars had an intermediate ripening time, at the end of the summer, but some exhibited early or late ripening. For these cultivars there are no data pertaining to fruit development in terms of the time of flowering or the temperatures prevailing during fruit growth; it is, however, likely that these two factors determine the ripening time. In Israel it was found that the duration of fruit growth in lOfer' was 150 days in the winter, which was almost as twice as long as the summer duration. In addition, GA 3 has been found to affect fruit development: When it was applied to floral buds prior to anthesis, ripening was advanced (Nerd and Mizrahi 1993b) A number of chemical and physical changes associated with maturation have been shown to take place in O. ficus-indica during the stage of rapid core growth. The content of various core components, such as dry matter, crude fibers, pectins, total titratable acids eTTA), ash, fats, and proteins, was found to decrease until shortly before

7.

REPRODUCTIVE BIOLOGY OF CACTUS FRUIT CROPS

337

ripening (70 days after flowering), while the content of core total soluble solids (TSS), and total soluble sugars increased during this time. Sugars continued to accumulate during ripening, 85-100 days after flowering (Barbera et a1. 1992b). The major soluble sugars identified were glucose and fructose, and sucrose was present in small amounts (Sawaya et a1. 1983; Kuti and Galloway 1994). Fruit firmness decreased until ripening, then remained stable, decreasing again in fully ripened fruits. Peel color began to change 70 days after flowering, and both peel and core were fully ripened 85-100 days after flowering (Barbera et a1. 1992b). Cultivars of Opuntia spp. vary in peel and pulp colors (Wessels 1988; Pimienta 1990; Barbera et a1. 1992a). Common pulp colors are variations of yellow and red or pale green (whitish). In many cultivars the peel colors are similar to those of the core, but pigmentation starts earlier in the core. Vacuolar water-soluble pigments in cacti are nitrogenous compounds of the class betalins, which consists of betaxanthins having a yellow color and betacyanins having a red-violet color (Gibson and Nobel 1986). Pigments determined in O. ficus-indica included indicaxanthin in yellow fruits (Impellizeri and Piatelli 1972) and betanins and isobetanins in red fruits (Piatelli and Minale 1964). Changes in peel chlorophyll content were studied in developing fruits (Inglese et a1. 1994b). The concentration of chlorophyll a was 2.5 times higher than that of chlorophyll b in the younger fruits, and both chlorophylls decreased markedly toward the end of fruit growth, giving the peel the pale green color that indicates the beginning of ripening. Postharvest respiratory and ethylene studies carried out on fruits of other cactus pears, for example, O. robusta and O. amyclaea, revealed a nonclimacteric pattern, but a rise in respiration was measured at the ripening stage in O. amyc1aea (Lakshminarayana and Estrella 1978; Lakshminarayana et a1. 1979; Moreno-Rivera et a1. 1979; Cantwell 1991). A common index for fruit harvest is the beginning of peel color change. This may be supplemented by measurement of TSS. In many cultivars TSS values of 12-13 indicate maturity (Barbera et a1. 1992b; Kuti 1992; Weiss et a1. 1993). At later stages, when peel color becomes fully developed, fruits become soft and less suitable for processing and storage (Barbera et a1. 1992b), and some consumers consider them to be less appetizing. Even at the preripening stage, pulp acid content is very low (0.02-0.06% as citric acid) (Barbera et a1. 1992b; Kuti 1992), and it has therefore been suggested that acidity is a less useful indicator for harvest than other indices (Wessels 1988). Stages of fruit development were described by Weiss (1995) for the columnar cactus C. peruvian us. The duration of fruit growth was

338

A. NERD AND Y. MIZRAHI

7 to 8 weeks. The increase in fruit fresh weight could be divided into three phases: early (25-28 days) and late (15-18 days) rapid phases and an intermediate phase (10 days) during which growth was arrested. Peel growth occurred in the first phase, while pulp growth took place mainly in the third phase. Seeds developed during the first two phases and hardened in the second phase. During the third phase, TSS increased to 12-13%, acidity decreased to 0.3% (as citric acid), and peel color developed to full color by the end of the phase. Depending on the clone, peel color ranged from yellowish to violet-red, and pulp color was always white. Although fruits continued to grow at the end of the third phase and their taste was superior at this stage, fruits have to be harvested just before full color because at full color they tend to dehisce. Similarly in the col umnar species, S. queretaroensis, grown in Jalisco (Mexico), the duration of fruit growth is approximately 8 weeks (Lomeli and Pimienta 1993). Fruits in this area ripen in the spring and at the beginning of the summer. Depending on the cultivar, pulp color may be whitish, yellowish, red, or purple. Upon ripening, the long spines that cover the peel tend to abscise and the fruits dehisce. Dehisced fruit are often marketed, but they have a very short shelf life (Pimienta and Tomas 1993; Pimienta and Nobel 1994). Fruit composition is known for S.

queretaroensis (Pimienta and Tomas 1993), S. griseus, and S. stellatus

(Bravo and Sanchez-Mejorada 1991). The sugars are mainly reducing sugars and their level, 8-10%, is relatively lower than in the Opuntia fruit and many other fruits. Fruit acidity (as citric acid) ranged between 0.2% and 0.6%; the level depended on cultivar and probably also on the harvest time. Studies in climbing species made in Israel showed that fruits of H. undatus, H. costaricensis, and H. polyrhizus have a short growth period, about 7 weeks (summer and autumn), whereas fruits of S. megalanthus have a relatively long growth period of 13 to 14 weeks for the minor summer crop and 20 to 22 weeks for the major winter crop (Weiss et a1. 1994b; Weiss 1995). Measurement of fruit diameter and length gave of curvilinear curve for fruit growth of H. undatus and S. megalanthus. For both species ripening, as reflected in peel color change, occurred at the final phase of fruit growth when growth markedly decreased.

c.

Fruit Weight

Fruit weight in O. ficus-indica is influenced by cladode fruit load, seed content, and environmental factors. Thinning heavy fruiting cladodes at bloom or at first stage of fruit development to obtain

7.

REPRODUCTIVE BIOLOGY OF CACTUS FRUIT CROPS

339

large fruits (Wessels 1988; Pimienta 1990; Barbera et a1. 1992a) is common, the general practice being to leave approximately 8-10 fruits per cladode. The contribution of thinning to fruit weight was demonstrated by Inglese et a1. (1994b), who showed that fruit dry weight in cladodes bearing 10 and 15 fruits was lower by 16 and 25%, respectively, than in cladodes bearing 5 fruits. They also showed that the fruits, which exhibited CAM behavior, supplied only small part of the carbon demand, the main source of photosynthates being the bearing cladode (as well as other cladodes), especially for cladodes with more than 5 fruits. Recently Inglese et a1. (1955) reported that the time of thinning, prebloom or postbloom, did not affect fruit weight, percent flesh, TSS, and seed number. For Sicilian cultivars a high positive correlation was found between fruit or core fresh weight and the total number of seeds (full-sized seeds plus empty seeds) of the summer and autumn crops (Barbera et a1. 1994). However, the contribution of the full-sized seeds to core weight was much higher than that of the empty seeds, particularly in the autumn crop (obtained by scozzolatura), which produced heavier fruits. Since the ratio between core weight and seed number was higher in the late crop than in the early crop, it was suggested that favorable environmental conditions also promoted core growth in the late crop. Irrigation at the early and late stages of fruit development was very effective in increasing fruit size of the late crop in Sicily, without a negative effect on fruit quality (Barbera 1984). Farmers in Israel usually irrigate the plants in the summer at the last stage of fruit development in order to produce larger fruits. Fruits of the winter crop (autumn flush) tend to be heavier than those of the summer crop (Nerd et a1. 1991), the rise being due to an increase in peel weight. Clearly, some factor active in winter, such as low temperatures or high humidity, boosts peel growth. Data concerning factors affecting fruit weight in the pitaya crops are sparse. Weiss et a1. (1994a,b) showed that fruit weight in C. peruvianus, Hylocereus spp., and Selenicereus megalanthus depended on seed number; thus, appropriate pollination is required for large fruits. Fruits in C. peruvianus (Weiss 1995) contained underdeveloped brown seeds in addition to viable black seeds, but pulp originated only from the funiculi of the black seeds.

D. Seed Set and Parthenocarpy Seed number in O. ficus-indica varies from 50 to 300 (Wessels 1988; Barbera et a1. 1991,1994; Pimienta 1990; Weiss et a1. 1993). The high variability in seed number may be due to differences in the initial

340

A. NERD AND Y. MIZRAHI

number of ovules and in efficiency of pollination and fertilization. Counts of ovules in flowers of the Israeli cultivar 'Ofer' revealed considerable variation between flowers, with values ranging from 140 to 430 for an average of 268. Average seed number in various crops of 'Ofer' ranged between 80 and 180. A similar average number of ovules per flower with approximately 80% seed set was reported by Rosas and Pimienta (1986) for two Mexican cultivars. This indicates that only some of the ovules develop into seeds. Usually, some of the seeds (30-50%) are empty (Pimienta and Engelman 1985; Weiss et al. 1993; Barbera et al. 1994), with well-developed integuments. For 'Ofer' the rate of pollen germination on the stigma was 30%, and 100% of the pollen tubes reached the base of the style, but only 44% of the tubes reached the ovules; thus, some limitations to fertilization reduce seed set (Weiss et al. 1993). O. ficus-indica is an octoploid plant, 2n = 8x = 88 (Mazzola et al. 1988; Pimienta 1990), and cytogenetic studies have described it as an auto-allo-octoploid of two species of 44 somatic chromosomes each or as a segmental allo-octoploid (Jacobs and Kistner 1992). Probably, imbalanced gametes as result of polyploidy lead to the partial seed set in O. ficusindica. In general, cactus pears tend to produce nucellar embryos, probably also associated with their polyploidy (Maheshwari and Chopra 1955; Jacobs and Kistner 1992). Little is known about the effect of environmental conditions on pollen germination and fertilization. Recently, we found that in fruits of the early-winter-flowering crop of O. ficus indica (average maxi min temperatures 20/5°C) the posterior ovules had degenerated, the resulting space being filled with a brown substance. Since the pollen proved to be viable and the stigmas were covered with pollen, it seems likely that fertilization was adversely affected by the low winter temperatures, resulting in degeneration. The abundant hard, rough seeds of Opuntia reduce consumer acceptability of the cactus pear, particularly by those unfamiliar with it. Extensive studies aimed at producing parthenocarpic fruit were therefore carried out in Chile (Gil et al. 1977; Diaz and Gil 1978; Gil and Espinosa 1980). Emao;;culated flowers failed to set fruits, but treatment of emasculated flowers with GA 3 induced the development of normal-sized fruits containing empty seeds. Efficient GA 3 treatments consisted of spraying GA 3 at a concentration of 500 ppm at anthesis, or at a concentration of 100 ppm on three occasions-at anthesis, and 22 and 42 days after anthesis. In comparison with normal seeded fruits, GA 3 -induced fruits were longer, with a thicker peel, smaller core, lower TSS content, and delayed ripening time. Normal-sized

7.

REPRODUCTIVE BIOLOGY OF CACTUS FRUIT CROPS

341

fruits with a high content of empty seeds were obtained when intact floral buds were sprayed with GA 3 • These results indicate that GA 3 inhibits seed development and induces fruit growth. Natural parthenocarpic cultivars are not grown on a commercial scale. Vegetative-type parthenocarpy was demonstrated for the Israeli empty-seeded yellow clone 'BS1' (Weiss et al. 1993). It was found that normal-sized fruits were obtained when flowers were emasculated before anthesis, indicating that pollination is not required for set and development in this clone. The mechanism of 'BS1' parthenocarpy is still unclear, but there is evidence that it is associated with overgrowth of the ovules and the receptacle during the bud stage. Pollen was viable and stigma receptivity was high, but pollen tubes failed to reach the large ovules, so that seed set failed to occur. It was interesting to note that some fruit characteristics of 'BS1' were similar to those measured in GA 3 -induced fruits (Diaz and Gil 1978): Fruits were long and had a high peel/pulp ratio60% higher than in fruits of the seeded 'Ofer'. Very little is known about seed set in the pitaya crops. Under appropriate pollination most of the ovules set seeds in Hylocereus spp. but in S. megalanthus (self-pollenating), seed set reached only 25 %. In S. megalanthus this was associated with low pollen germination (Weiss et al. 1994b). E. Seed Dispersal Problem The juicy highly colored fruits attract birds, which eat the fruits. In adition to damage to the fruit, this may create a weed problem, particularly with spiny, fast growing species, by dispersal of seeds. Opuntia spp. have been declared weeds in Australia, and both the spiny Opuntia and C. peruvianus have infested wide areas in South Africa, where these species were naturalized (Brutsch and Zimerman 1993; Nobel 1994 ). Lack of natural biological control (diseases and pests) probably promoted their infestation there. Control was achieved by introduction of insect enemies of Opuntia and use of chemicals (Nobel 1994). VI. CONCLUDING REMARKS The data presented here reflect the evolution of the modern cultivation of cactus fruit crops. Horticultural research, including studies on reproductive biology have been largely dedicated to the oldest

342

A. NERD AND Y. MIZRAHI

cultivated cactus crops, the cactus pears, which were planted in modern plantations as long ago as the middle of the century. For the other cactus crops, the pitayas, which attracted the attention of modern growers only in the last decade the research is only in the initial stages and is likely to be linked to the economic importance of the crop. However, even for the cactus pears further studies are needed to bring knowledge of flower and fruit production to the level known for common commercial fruit crops. For example, various aspects of flowering control have been intensively investigated in common fruit crops, but only rarely in the cactus pears. We hope that this review, which covers the unknown rather than the known, will prove to be a useful reference to students, researchers, and growers to the challenging field of cactus fruit crops.

LITERATURE CITED Alcorn, S. M., S. E., McGregor, and G. Olin. 1961. Pollination of saguaro cactus by doves, nectar-feeding bats, and honey bees. Science 133:1594-1595. Alcorn, S. M., S. E. McGregor, and G. Olin. 1962. Pollination requirements of the organ pipe cactus. Cactus Succ. J. (Los Angeles), 34:134-139. Arcadio, L. B. 1986. Cultivo de la pitaya. Federacion Nacional de Cafeteros, Bogota, Colombia. Barbeau, G. 1990. La pitahaya rouge, un nouveau fruit exotique. Fruits 45:141-147. Barbera, G. 1984. Ricerche sull'irrigazione del ficodinia. Fruticoltura 46:49-55. Barbera, G., F. Carimi, and P. Inglese. 1991. The reflowering of prickly pear Opuntia ficus-indica (L.) Miller: influence of removal time and cladode load on yield and fruit ripening. Adv. Hort. Sci. 5:77-80. Barbera, G., F. Carimi, and P. Inglese. 1992a. Past and present role of the Indian-fig prickly-pear (Opuntia ficus-indica (L.) Miller, Cactaceae) in the agriculture of Sicily. Econ. Bot. 46:10-22. Barbera, G., F. Carimi, and M. Panno. 1992b. Physical, morphological and chemical changes during fruit development and ripening in three cultivars of prickly pear, Opuntia ficus-indica (L.) Miller. J. Hort. Sci. 67:307-312. Barbera, G., F. Carimi, and P. Inglese. 1993. Effects of GA 3 and shading on return bloom of prickly pear (Opuntia ficus-indica (L.) Miller). J. South African Soc. Hort. Sci. 3:9-10. Barbera, G., P. Inglese, and T. La Mantia. 1994. Seed content and fruit characteristics in cactus pear Opuntia ficus-indica Mill. Sci. Hort. 58:161-165. Benzing, D. H. 1990. Vascular epiphytes. Cambridge Univ. Press, Cambridge. Boke, N. H. 1980. Developmental morphology and anatomy in Cactaceae. BioScience 30:605-610. Bravo, H. H. 1979. Las Cactaceas de Mexico, 2d ed., vol. 1. Universidad Nacional Autonoma de Mexico, Mexico City. Bravo, H. H., and R.H. Sanchez-Mejorada. 1991. Utilidad de la Cactaceae. p. 501535. In: Las Cactaceas de Mexico, Vol. 3. Universidad Nacional Autonorna de Mexico, Mexico City.

7.

REPRODUCTIVE BIOLOGY OF CACTUS FRUIT CROPS

343

Britton, N. L., and J. N. Rose. 1963. The cactaceae: description and illustrations of plants of the cactus family, Vols. 1 and 2. Dover, New York. Brutsch, M. 0., and M. B. Scott. 1991. Extending the fruiting season of spineless prickly pear Opuntia ficus-indica. J. South African Soc. Hort. Sci. 1:73-76. Brutsch, M. 0., and G. Zimerman. 1993. The prickly pear (Opuntia ficus-indical Cactaceael) in South Africa: utilization of the naturalized weed and of the cultivated plants. Econ. Bot. 47:154-162. Cacioppo, O. G. 1990. Pitaya: una de las mejores frutas producida por Colombia. Informative Agroeconomico, Febrero: 15-19. Cantwell, M. 1991. Quality and postharvest physiology of "nopalitos" and "tunas." p. 50-66 In: P. Felker (ed.), Proc. 2d annual Texas prickly pear council, 16-17 August 1991, McAllen, TX. Castillo, H. A. 1984. Introduccion de pitaya (Stenocereus stellatus y S. marginatus) al valle del Mezquital en el edo. Hidalgo. p. 36-40. In: Memorias del Symposio sobre el Aprovechamiento del Pitayo. Universidad Autonoma MetropolitanaXochimilco, Oaxaca, Mexico. Cruz, H. P. 1984. Algunas caractaristicas del cultivo de la pitaya (Stenocereus spp.) p. 49-62. In: Memoria del Symposio sobre el Aprovechamiento del Pitayo. Universidad Autonoma Metropolitana-Xochimilco, Oaxaca, Mexico. Cullman, W., E. Goetz, and G. Groener. 1986. The encyclopedia of cacti. Timber, Portland, OR. Curtis, J. R. 1977. Prickly pear farming in the Santa Clara Valley, California. Econ. Bot. 31:175-179. Damigella, P. 1958. El fico d'india e Ie cultivar della Sicilia orientale. Tecnica Agricola 10:474-502. Diaz, F., and S. G. Gil. 1978. Effectiveness of dosage and methods of gibberellic acid application to induce parthenocarpy and stimulate fruit growth of prickly pear (Opuntia ficus-indica Mill.). Cienc. Inv. Agr. 5:109-117. Felger, R. S., and M. B. Moser. 1985. People of the desert and sea: ethnobotany of the Seri Indians. Univ. of Arizona Press, Tucson. Garcia de Cortazar, v., and P. S. Nobel. 1992. Biomass and fruit production of the prickly pear cactus Opuntia ficus-indica. J. Am. Soc. Hort. Sci. 117:558-562. Gibson, A. C. 1989. The systematics and evolution of the subtribe Stenocereinae, 6: Stenocereus stellatus and Stenocereus treleasei. Cactus Succ. J. (Los Angeles) 61:26-32. Gibson, A. C. 1990. The systematics and evolution of the subtribe Stenocereinae, 9: Stenocereus queretaroensis and its closest relatives. Cactus Succ. J. (Los Angeles) 62:170-176. Gibson, A. G. 1991. The systematics and evolution of the subtribe Stenocereinae, 10: the species group of Stenocereus griseus. Cactus Succ. J. (Los Angeles) 63:9299. Gibson, C. G., and P. S. Nobel. 1986. The cactus primer. Harvard Univ. Press, Cambridge, MA. Gil, G., and A. R. Espinosa. 1980. Fruit develoment in the prickly pear (Opuntia ficus-indica, Mill.) with preanthesis application of gibberellin and auxin. Cienc. Inv. Agr. 7:141-147. Gil, G., M. Morales, and A. Momberg. 1977. Fruit set and development in the prickly pear (Opuntia ficus-indica, Mill.) in relation to pollination and gibberellic and chlorethylphosphonic acids. Cienc. Inv. Agr. 4:163-169. Grant, V., and K. A. Grant. 1979. The pollination spectrum in the Southwestern American cactus flora. Plant Syst. Evol. 133:29-37.

344

A. NERD AND Y. MIZRAHI

Grant, V., and P. D. Hurd. 1979. Pollination of the Southwestern opuntias. Plant Syst. Evol. 133:15-28. Haber, W. A. 1983. Hylocereus costaricensis (pitahaya sylvestre, wild pitahaya). p. 252-253. In: D.H. Janjen (ed.), Costa Rican Natural History. Univ. of Chicago Press, Chicago. Impellizzeri, G., and M. Piattelli. 1972. Biosynthesis of indicaxanthin in Opuntia ficus-indica fruits. Biochemistry 11:2499-2502. Inglese, P., G. Barbera, and F. Carimi. 1994a. The effect of different amounts of cladode removal on reflowering of cactus pear (Opuntia ficus-indica (1.) Miller). J. Hort. Sci. 69:61-65. Inglese, P., A. Israel, and P. S. Nobel. 1994b. Growth and CO 2 uptake for cladode and fruit of the crassulacean acid metabolism species O. ficus-indica during fruit development. Physiol. Plant. 91:708-714. Inglese, P., G. Barbera, T. La Mantia and S. Portolano. 1995. Crop production, growth and ultimate size of cactus pear fruit following fruit thinning. HortScience 30:227230. Jacobs, D. H., and M. Kistner. 1992. Microsporogenesis in two Opuntia species with notes on its implication for breeding. II Congreso Int. de Tuna y Cochinilla, 2225 September, Santiago, Chile. Kimnach, M. 1967. Hylocereus calcaratus Cactus Succ. J. (Los Angeles) 39:102-105. Kuti, J. O. 1992. Growth and compositional changes during the development of prickly pear fruit. J. Hort. Sci. 67:861-868. Kuti, J. 0., and C. M. Galloway. 1994. Sugar composition and invertase activity in prickly pear fruit. J. Food Sci. 59:387-388, 393. Lakshminarayana, S., and 1. B. Estrella. 1978. Postharvest respiratory behavior of tuna (prickly pear) fruit (Opuntia robusta, Mill.). J. Hort. Sci. 53:327-330. Lakshminarayana, S., 1. Alvarado, Y. Sosa, and F. Barrientos Perez. 1979. The develpment and postharvest physiology of the fruit of prickly pear (Opuntia amyc1aea) p. 69-93. In: G. K Ingleft and G. Charalambous (eds.), Tropical foods: chemistry and nutrition, Vol 1. Academic, New York. Liamas, L. J. 1984. El cultivo del pitayo en Haujapan de Leon, Oax. p. 28-35. In: Symposio Memoria del Simposio sobre el Aprovechamiento del Pitayo. Universidad Autonoma Metropolitana-Xochimilco, Oaxaca, Mexico. Lomeli, M. E., and E. Pimienta. 1993. Demografia reproductiva del pitayo (Stenocereus quertaroensis (Web.) Buxbaum). Rev. Sociedad Mexicana Cactologia 29:9-10. Maheshwari, P., and R. N. Chopra. 1955. The structure and development of the ovule and seed of Opuntia dillenii Haw. Phytomorphology 5:112-122. Mazzola, P., S. Romano, and S. Fici. 1988. Contributo alIa conoscenza del genero Opuntia Miller, 1. dati cariologici e distributivi delle specie spontaneizzate e coltivate in Sicilia. Naturalista Sicil. IV, XII:159-168. McGregor, S. K, S. M. Alcorn, and G. Olin. 1962. Pollination and pollinating agents of the saguaro. Ecology 43:259-267. Mizrahi, Y., A. Nerd, and P. S. Nobel. 1996. Cacti as crops. Hort. Rev. 18:291-320. Moran, V. C. 1953. Selenicereus megalanthus (Schumann) Moran. Gent. Herb. 8:325. Moreno-Rivera, M.A., 1.S. Alvarado, and S. Laskshminaryana. 1979. Preharvest respiratory trend of the fruit of prickly pear (Opuntia amyc1aea). Proc. Flor. State Hort. Soc. 92:235-237. Morton, J. F. 1987. Fruits of warm climates. Morton, Miami, FL.

7.

REPRODUCTIVE BIOLOGY OF CACTUS FRUIT CROPS

345

Nerd, A., and Y. Mizrahi. 1993a. Modern cultivation of prickly pear in Israel: fertigation. Acta Hort. 349:235-237. Nerd, A., and Y. Mizrahi 1993b. Cultural practices for cactus pear in Israel for yearround production. p. 77-80 In: P. Felker and J. R. Moss (eds.), Proc. 4th Annual Texas Prickly Pear Council 13 & 14 August, Kingsville, Texas. Nerd, A., and Y. Mizrahi. 1994. Effect of nitrogen fertilization and organ removal on rebudding in Opuntia ficus-indica (L.) Miller. Sci. Hort. 59:115-122. Nerd, A., A. Karadi, and Y. Mizrahi. 1989. Irrigation, fertilization and polyethylene covers in prickly pear influence bud development. HortScience 24:773-775. Nerd, A., A. Aronson, and Y. Mizrahi. 1990. Introduction and domestication of rare and wild fruit and nut trees for desert areas. p. 353-365. In: J. Janick and J. E. Simon (eds.), Advances in new crops. Timber, Portland, OR. Nerd, A., A. Karadi, and Y. Mizrahi. 1991. Out-of-season prickly pear: fruit characteristics and effect of fertilization and short droughts on productivity. HortScience 26:527-529. Nerd, A., R. Mesika, and Y. Mizrahi. 1993a. Effect of N fertilizer on autumn floral flash and cladode N in prickly pear Opuntia ficus-indica (L.) Mill. J. Hort. Sci. 68:545-550. Nerd, A., E. Raveh, and Y. Mizrahi. 1993b. Adaptation of five columnar cactus species to various conditions in the Negev Desert of Israel. Econ. Bot. 47:304-311. Nieddu, J., and D. Spano. 1992. Flowering and fruit growth in Opuntia ficus-indica. Acta Hort. 296:153-159. Nobel, P. S. 1988. Environmental biology of agaves and cacti. Cambridge. Univ. Press, New York. Nobel, P. S. 1994. Remarkable agaves and cacti. Oxford Univ. Press, New York. Piatelli, M., and L. Minale. 1964. Pigments of centrospermae-I. betacyanins from Phyllocactus hybridus Hort. and Opuntia ficus-indica Mill. Phytochemistry 3:307311.

Pimienta, B. E. 1990. El nopal tunero. Univ. of Guadelajara, Guadaljara, Jalisco, Mexico. Pimienta, B. E. 1994. Prickly pear (Opuntia spp.), a valuable crop for the semi arid lands of Mexico. J. Arid Environ. 28:1-11. Pimienta, B. E., and E. M. Engelman. 1985. Desarrollo de la pulpa y proporcion en volumen, de los componentes del loculo maduro en tuna (Opuntia ficus-indica (L.) Miller). Agrociencia 62:51-56. Pimienta, B. E., and P. S. Nobel. 1994. Pitaya (Stenocereus spp., Cactaceae), an ancient and modern fruit crop of Mexico. Econ. Bot. 48:76-83. Pimienta, B. E., and M. L. Tomas V. 1993. Caracterizacion de la variacion en el peso y la composition quimica del fruto en variedades de pitayo (Stenocereus queretaroensis). Rev. Sociedad Mexicana Cactologia 38:82-88. Portolano, N. 1962. II fico d'India. Edagricole, Bologna, Italy. Raveh, E., J. Weiss, A. Nerd and Y. Mizrahi, 1993. Pitayas (genus Hylocereus): a new fruit crop for the Negev desert of Israel. p. 491-495. In: J. Janick and J. Simon (eds.), New crops. Wiley, New York. Rivera, O. S., G. Gil, G. Montenegro, and G. Avila. 1981. Stages of differentiation in floral buds of the prickly pear Opunia ficus-indica Mill. Cienc. Inv. Agr. 8:215219. Rosas, C. M. P., and B. E. Pimienta. 1986. Polinizacion y fase progamica en nopal (Opuntia ficus-indica (L) Miller) tunero. Fitotecnica 8:164-176. Russel, C. E., and P. Felker. 1987. The prickly pears (Opuntia spp. Cactaceae): a source of human and animal food in semiarid regions. Econ. Bot. 41:433-445.

346

A. NERD AND Y. MIZRAHI

Sawaya, W. N., H. A. Khatcadourian, W. M. Safi, and H. M. AI-Muhammad. 1983. Chemical characterization of prickly pear pulp, Opuntia ficus-indica and the manufacturing of prickly jam. J. Food Technol. 18:183-193. Sanchez-Mejorada R. H. 1982. Some prehispanicuses of cacti among the Indians of Mexico. Gobierno del Estado del Mexico.Toluca, Mexico. Sanchez-Mejorada R. H. 1984. Origen, taxonomia y distribucion de las pitayas en Mexico. p. 6-18. In: Memoria del Simposio sobre el Aprovechamiento del Pitayo. Universidad Autonoma Metropolitana-Xochimilco, Oaxaca, Mexico. Scheinvar, L. 1985. Flora Ilustrada Catarinese Cactaceas. Itajai, Santa Catarina, Brasil. Schmidt, J. 0., and S. L. Buchmann. 1986. Floral biology of the saguaro (Cereus giganteus) , I: Pollen harvest by Apis mellifera. Oecologia (Berlin) 69:491-498. Vietmeyer, N. D. 1986. Lesser known plants of potential use in agriculture and forestry. Science 232:1379-1381. Weiss, J. 1995. The biology of flowering, pollination and fruit development in nightblooming and day-blooming cacti. Thesis, Ben-Gurion University of the Negev, Beer-Sheva, Israel. Weiss J., A. Nerd, and Y. Mizrahi. 1993. Vegetative parthenocarpy in the cactus pear Opuntia ficus-indica (L.) Mill. Ann. Bot. 72:521-526. Weiss J., A. Nerd, and Y. Mizrahi. 1994a. Flowering and pollination requirements in Cereus peruvianus cultivated in Israel. Israel J. Plant Sci. 42:149-158. Weiss J., A. Nerd, and Y. Mizrahi. 1994b. Flowering behavior and pollination requirements in climbing cacti with fruit crop potential. HortScience 29:1487-1492. Weiss, J., L. Scheinvar, and Y. Mizrahi. 1995. Selenicereus megalanthus (the yellow pitaya), a climbing cactus from Colombia and Peru. Cactus Succ. J. (Los Angeles) 67:280-283. Wessels, A. B. 1988. Spineless prickly pear. Perskor, Johannesburg. Wessels, A. B. 1992. Development of the pulp of Opuntia ficus-indica (L.) Mill. fruit: a new look at fruit classification. In: II Congreso Int. de Tuna y Cochinilla, 22-25 September, Santiago, Chile. Wessels, A. B., and E. Swart. 1990. Morphogenesis of the reproductive bud and fruit of the prickly pear Opuntia ficus-indica (L.) Mill. cv. Morado. Acta Hort. 275:245253.

Subject Index B

G

Bulb crops, see Tulip genetics and breeding, 119-

Genetics and breeding, bulbs, flowering, 119-123

123

in vitro, 87-169 micropropagation, 89-113 virus elimination, 113-123

I

Industrial crops, cactus, 309312

In vitro, flowering bulbs, 87-

C

169

Cactus: crops, 291-320 reproductive biology, 321346

D

L

Light, tolerance, 215-246 M

Dedication, Looney, N.E., xiii Desiccation tolerance, 171-213 Disease, resistance, acquired, 247-289

F Feed crops, cactus, 298-300 Flower and flowering: cactus, 325-335 senescence, 1-85 water relations, 1-85 Fruit, cactus physiology, 335341

Fruit crops, cactus, 302-309

Micropropagation, see In vitro bulbs, flowering, 89-113

o Ornamental plants, flowering bulbs in vitro, 87-169 p

Physiology: cactus reproductive biology, 321-346

desiccation tolerance, 171213

347

SUBJECT INDEX

348

Physiology (cont'd) disease resistance, 247-289 light tolerance, 215-246 water relations of cut flowers, 1-85 Pollen, desiccation tolerance, 195

Pollination, cactus, 331-335

in vitro, 144-145

v

Vegetable crops, cactus, 300302

Vegetative tissue, desiccation tolerance, 176-195 Virus, elimination, 113-123

w

R

Root, cactus, 297-298

s

Water relations: cut flowers, 1-85 desiccation tolerance, 171213

Seed, desiccation tolerance, 196-203

Senescence, cut flowers, 1-85 T Tulip, see Bulb crops

X

Xanthophyll cycle, 226-239

Cumulative Subject Index (Volumes 1-18) A

Abscisic acid: chilling injury 15:78-79 cold hardiness, 11:65 dormancy,7:275-277 genetic regulation, 16:9-14, 20-21 mechanical stress, 17:20 rose senescence, 9:66 stress, 4:249-250 Abscission: anatomy and histochemistry, 1:172-203 citrus, 15:145-182 flower and petals, 3:104-107 regulation, 7:415-416 rose, 9:63-64 Acclimatization: foliage plants, 6:119-154 herbaceous plants, 6:379-395 micropropagation, 9:278-281, 316-317

Actinidia, 6:4-12 Adzuki bean, genetics, 2:373

Agaricus, 6:85-118 Agrobacterium tumefaciens, 3:34 Air pollution, 8:1-42 Almond: bloom delay, 15:100-101 in vitro culture, 9:313

A10casia, 8:46, 57. See also Aroids Alternate bearing: chemical thinning, 1:285-289 fruit crops, 4:128-173 pistachio, 3:387-388 Aluminum: deficiency and toxicity symptoms in fruits and nuts, 2:154 Ericaceae, 10:195-196 Amorphophallus, 8:46, 57. See also Aroids Anatomy and morphology: apple flower and fruit, 10:273-308 apple tree, 12:265-305 asparagus, 12:71 cassava, 13:106-112 citrus, abscission, 15:147156 embryogenesis, 1:4-21, 35-40 fig, 12:420-424 fruit abscission, 1 :172-203 fruit storage, 1:314 ginseng, 9:198-201 grape flower, 13:315-337 grape seedlessness, 11:16064 heliconia, 14:5-13 kiwifruit,6:13-50 orchid,5:281-283 349

CUMULATIVE SUBJECT INDEX

350

Anatomy and morphology (cont'd)

navel orange, 8:132-133 pecan flower, 8:217-255 petal senescence, 1:212-216 pollution injury, 8:15 Androgenesis, woody species, 10:171-173 Angiosperms, embryogenesis, 1:1-78 Anthurium, fertilization, 5:334335. See also Aroids, ornamental Antitranspirants, 7:334 cold hardiness, 11 :65 Apical meristem, cryopreservation, 6:357372 Apple: alternate bearing, 4:136-137 anatomy and morphology of flower and fruit, 10:273309 bitter pit, 11 :289-355 bioregulation, 10:309-401 bloom delay, 15:102-104 CA storage, 1:303-306 chemical thinning, 1:270-300 fertilization, 1:105 fire blight control, 1:423-474 flavor, 16:197-234 flower induction, 4:174-203 fruiting, 11:229-287 in vitro, 5:241-243; 9:319321 light, 2:240-248 maturity indices, 13:407-432 nitrogen metabolism, 4:204246 replant disease, 2:3 root distribution, 2:453-456 stock-scion relationships,

3:315-375 summer pruning, 9:351-375 tree morphology and anatomy, 12:265-305 vegetative growth, 11:229287 watercore, 6:189-251 yield,1:397-424 Apricot: bloom delay, 15:101-102 CA storage, 1 :309 Aroids: edible, 8:43-99; 12:166-170 ornamental,10:1-33 Arsenic, deficiency and toxicity symptoms in fruits and nuts, 2:154 Artichoke, CA storage, 1:349350 Asexual embryogenesis, 1:1-78; 2:268-310; 3:214-314; 7:163-168,171-173,176177,184,185-187,187188,189; 10:153-181; 14:258-259,337-339 Asparagus: CA storage, 1:350-351 fluid drilling of seed, 3:21 postharvest biology, 12:69155 Auxin: abscission, citrus, 15:161, 168-176 bloom delay, 15:114-115 citrus abscission, 15:161, 168-176 dormancy, 7:273-274 flowering, 15:290-291, 315 genetic regulation 16:5-6,14, 21-22 geotropism, 15:246-267 mechanical stress, 17:18-19

CUMULATIVE SUBJECT INDEX

petal senescence, 11:31 Avocado: flowering, 8:257-289 fruit development, 10:230238

fruit ripening, 10:238-259 rootstocks, 17:381-429 Azalea, fertilization, 5 :335-33 7

351

bearing Bioregulation, apple and pear, 10:309-401. See also Growth substances Bird damage, 6:277-278 Bitter pit in apple, 11 :289-355 Blackberry harvesting, 16:282298

Black currant, bloom delay, 15:104

B

Bloom delay, deciduous fruits,

Babaco, in vitro culture, 7:178 Bacteria: diseases of fig, 12:447-451 ice nucleating, 7:210-212, 11:69-71

pathogens of bean, 3:28-58 tree short life, 2:46-47 wilt of bean, 3:46-47 Bacteriocides, fire blight, 1:450-459

Bacteriophage, fire blight control, 1:449-450 Banana: CA storage, 1:311-312 fertilization, 1 :105 in vitro culture, 7:178-180 Bean: CA storage, 1:352-353 fluid drilling of seed, 3:21 resistance to bacterial pathogens, 3:28-58 Bedding plants, fertilization, 1:99-100; 5:337-341

Beet: CA storage, 1:353 fluid drilling of seed, 3:18-19 Begonia (Rieger), fertilization, 1:104

Biochemistry, petal senescence, 11:15-43

Biennial bearing, see Alternate

15:97

Blueberry: developmental physiology, 13:339-405

harvesting, 16:257-282 nutrition, 10:183-227 Botanic gardens, 15:1-62 Boron: deficiency and toxicity symptoms in fruits and nuts, 2:151-152 foliar application, 6:328 nutrition, 5:327-328 pine bark media, 9:119-122 Bramble, harvesting, 16:282298

Branching, lateral: apple, 10:328-330 pear, 10:328-330 Brassicaceae, in vitro, 5:232235

Breeding, see Genetics and breeding Broccoli, CA storage, 1:354-355 Brussels sprouts, CA storage, 1:355

Bulb crops, see Tulip genetics and breeding, 18:119-123

in vitro, 18:87-169 micropropagation, 18:89-113

352

Bulb crops (cont'd) root physiology, 14:57-88 virus elimination, 18:113123

c CA storage, see Controlledatmosphere storage Cabbage: CA storage, 1:355-359 fertilization, 1:117-118 Cactus: crops, 18:291-320 reproductive biology, 18:321346 Caladium, see Aroids, ornamental Calciole, nutrition, 10:183-227 Calcifuge, nutrition, 10:183-227 Calcium: bitter pit, 11 :289-355 cell wall, 5:203-205 container growing, 9:84-85 deficiency and toxicity symptoms in fruits and nuts, 2:148-149 Ericaceae nutrition, 10:196197 foliar application, 6:328-329 fruit softening, 10:107-152 nutrition, 5:322-323 pine bark media, 9:116-117 tipburn, disorder, 4:50-57 Calmodulin, 10:132-134, 137138 Carbohydrate: fig,12:436-437 kiwifruit partitioning, 12:318-324 metabolism, 7:69-108 partitioning, 7:69-108

CUMULATIVE SUBJECT INDEX

petal senescence, 11:19-20 reserves in deciduous fruit trees, 10:403-430 Carbon dioxide, enrichment, 7:345-398, 544-545 Carnation, fertilization, 1 :100; 5:341-345 Carrot: CA storage, 1:362-366 fluid drilling of seed, 3:13-14 Caryophyllaceae, in vitro, 5:237-239 Cassava, 12:158-166; 13:105129 Cauliflower, CA storage, 1:359362 Celeriac, CA storage, 1:366-367 Celery: CA storage, 1:366-367 fluid drilling of seed, 3:14 Cell culture, 3:214-314 woody legumes, 14:265-332 Cell membrane: calcium, 10:126-140 petal senescence, 11 :20-26 Cellular mechanisms, salt tolerance, 16:33-69 Cell wall: calcium, 10:109-122 hydrolases, 5:169-219 ice spread, 13:245-246 tomato, 13:70-71 Chelates, 9:169-171 Cherry: bloom delay, 15:105 CA storage, 1:308 Chestnut: blight, 8:281-336 in vitro culture, 9:311-312 Chicory, CA storage, 1:379 Chilling: injury, 4:260-261,15:63-95

CUMULATIVE SUBJECT INDEX

pistachio, 3:388-389 Chlorine: deficiency and toxicity symptoms in fruits and nuts, 2:153 nutrition, 5:239 Chlorosis, iron deficiency induced, 9:133-186 Chrysanthemum fertilization, 1:100-101; 5:345-352 Citrus: abscission, 15:145-182 alternate bearing, 4:141-144 asexual embryogenesis, 7:163-168 CA storage, 1:312-313 chlorosis, 9:166-168 cold hardiness, 7:201-238 fertilization, 1:105 flowering, 12:349-408 honey bee pollination, 9:247248 in vitro culture, 7:161-170 navel orange, 8:129-179 nitrogen metabolism, 8:181 rootstock, 1 :23 7-269 Cloche (tunnel), 7:356-357 Coconut palm: asexual embryogenesis, 7:184 in vitro culture, 7:183-185 Cold hardiness, 2:33-34 apple and pear bioregulation, 10:374-375 citrus, 7:201-238 factors affecting, 11:55-56 herbaceous plants, 6:373-417 injury,2:26-27 nutrition, 3:144-171 pruning, 8:356-357 Co10casia, 8:45, 55-56. See also Aroids Common blight of bean, 3:45-46

353

Compositae, in vitro, 5:235-237 Container production, nursery crops, 9:75-101 Controlled-atmosphere (CA) storage: asparagus, 12:76-77, 127-130 chilling injury, 15:74-77 flowers, 3:98; 10:52-55 fruit quality, 8:101-127 fruits, 1:301-336; 4:259-260 pathogens, 3:412-461 seeds, 2:134-135 tulip, 5:105 vegetable quality, 8:101-127 vegetables, 1:337-394; 4:259260 Controlled environment agriculture, 7:534-545. See also Greenhouse and greenhouse crops; Hydroponic culture; Protected crops Copper: deficiency and toxicity symptoms in fruits and nuts, 2:153 foliar application, 6:329-330 nutrition, 5:326-327 pine bark media, 9:122-123

Corynebacterium flaccumfaciens, 3:33,46 Cowpea: genetics, 2:317-348 U.S. production, 12:197-222 Cranberry: fertilization, 1:106 harvesting, 16:298-311 Cryphonectria parasitica, see

Endothia parasitica

Cryopreservation: apical meristems, 6:357-372 cold hardiness, 11 :65-66 Crytosp erm a, 8:47, 58. See also

354

Crytosperma (cont'd) Aroids Cucumber, CA storage, 1:367368 Currant, harvesting, 16:311-327 Cytokinin: cold hardiness, 11:65 dormancy,7:272-273 floral promoter, 4:112-113 flowering, 15:294-295,318 genetic regulation, 16:4-5, 14,22-23 grape root, 5:150, 153-156 lettuce tipburn, 4:57-58 petal senescence, 11 :30-31 rose senescence, 9:66 D

Date palm: asexual embryogenesis, 7:185-187 in vitro culture, 7:185-187 Daylength, see Photoperiod Dedication: Bailey, L.H., l:v-viii Beach, S.A., l:v-viii Bukovac, M.J., 6:x-xii Cummins, J.N., 15:xii-xv Faust, Miklos, 5:vi-x Hackett, W.P., 12:x-xiii Halevy, A.H., 8:x-xii Hess, C.E., 13:x-xii Kader, A.A., 16:xii-xv Looney, N.E., 18:xiii Magness, J.R., 2:vi-viii Moore, J.N., 14:xii-xv Proebsting, Jr., E.L., 9:x-xiv Rick, Jr., C.M., 4:vi-ix Sansavini, S., 17:xii-xiv Smock, R.M., 7:x-xiii Weiser, C.J., 11:x-xiii

CUMULATIVE SUBJECT INDEX

Whitaker, T.W., 3:vi-x Wittwer, S.H., 10:x-xiii Deficiency symptoms, in fruit and nut crops, 2:145-154 Defoliation, apple and pear bioregulation, 10:326-328 'Delicious' apple, 1:397-424 Desiccation tolerance, 18:171213 Dieffenbachia, see Aroids, ornamental Dioscorea, see Yam Disease: and air pollution, 8:25 aroids, 8:67-69; 10:18; 12:168-169 bacterial, of bean, 3:28-58 cassava, 12:163-164 control by virus, 3:399-403 controlled-atmosphere storage, 3:412-461 cowpea, 12:210-213 fig, 12:447-479 flooding, 13:288-299 hydroponic crops, 7:530-534 lettuce, 2:187-197 mycorrhizal fungi, 3:182-185 ornamental aroids, 10:18 resistance, acquired, 18:247289 root, 5:29-31 stress, 4:261-262 sweet potato, 12:173-175 tulip, 5:63, 92 turnip moasic virus, 14:199238 yam (DioscoreaJ, 12:181-183 Disorder, see Postharvest physiology bitterpit, 11:289-355 fig, 12:477-479 pear fruit, 11:357-411

CUMULATIVE SUBJECT INDEX

watercore, 6:189-251; 11:385-387 Dormancy,2:27-30 blueberry, 13:362-370 release in fruit trees, 7:239300 tulip, 5:93 Drip irrigation, 4:1-48 Drought resistance, 4:250-251 cassava, 13:114-115 Dwarfing: apple, 3:315-375 apple mutants, 12:297-298 by virus, 3:404-405 E

Easter lily, fertilization, 5:352355 Embryogenesis, see Asexual embryogenesis Endothia parasitica, 8:291-336 Energy efficiency, in greenhouses, 1:141-171; 9:1-52 Environment: air pollution, 8:20-22 controlled for agriculture, 7:534-545 controlled for energy efficiency' 1:141-171, 9:1-52 embryogenesis, 1:22, 43-44 fruit set, 1:411-412 ginseng, 9:211-226 greenhouse management, 9:32-38 navel orange, 8:138-140 nutrient film technique, 5:13-26 Epipremnum, see Aroids, ornamental Erwinia: amylovora, 1:423-474

355

lathyri, 3:34 Essential elements: foliar nutrition, 6:287-355 pine bark media, 9:103-131 plant nutrition 5:318-330 soil testing, 7:1-68 Ethylene: abscission, citrus, 15:158161,168-176 apple bioregulation, 10:366369 avocado, 10:239-241 bloom delay, 15:107-111 CA storage, 1:317-319, 348 chilling injury, 15:80 citrus abscission, 15:158161,168-176 cut flower storage, 10:44-46 dormancy, 7:277-279 flowering, 15:295-296, 319 flower longevity, 3:66-75 genetic regulation, 16:6-7, 14-15,19-20 kiwifruit respiration, 6:47-48 mechanical stress, 17:16-17 petal senescence, 11:16-19, 27-30 rose senescence, 9:65-66 F

Feed crops, cactus, 18:298-300 Fertilization and fertilizer: anthurium, 5:334-335 azalea, 5:335-337 bedding plants, 5:337-341 blueberry, 10:183-227 carnation, 5:341-345 chrysanthemum, 5:345-352 controlled release, 1:79-139; 5:347-348 Easter lily, 5:352-355

CUMULATIVE SUBJECT INDEX

356

Fertilization and fertilizer

(cont'd)

Ericaceae, 10:183-227 foliage plants, 5:367-380 foliar, 6:287-355 geranium, 5:355-357 greenhouse crops, 5:317-403 lettuce, 2:175 nitrogen, 2:401-404 orchid,5:357-358 poinsettia, 5:358-360 rose, 5:361-363 snapdragon, 5:363-364 soil testing, 7:1-68 trickle irrigation, 4:28-31 tulip,5:364-366 Vaccinium, 10:183-227 Fig: industry, 12:409-490 ripening, 4:258-259 Filbert, in vitro culture, 9:313314 Fire blight, 1:423-474 Flooding, fruit crops, 13:257313 Floricultural crops, see indi-

vidual crops

fertilization, 1:98-104 growth regulation, 7:399-481 heliconia, 14:1-55 postharvest physiology and senescence, 1:204-236; 3:59-143; 10:35-62; 11:1543 Florigen, 4:94-98 Flower and flowering: alternate bearing, 4:149 apple anatomy and morphology, 10:277-283 apple bioregulation, 10:344348 aroids, ornamental, 10:19-24

avocado, 8:257-289 blueberry development, 13:354-378 cactus, 18:325-335 citrus, 12:349-408 control, 4:159-160; 15:279334 fig, 12:424-429 grape anatomy and morphology, 13:354-378 honey bee pollination, 9:239243 induction, 4:174-203,254256 initiation, 4:152-153 in vitro, 4:106-127 kiwifruit, 6:21-35; 12:316318 orchid,5:297-300 pear bioregulation, 10:344348 pecan, 8:217-255 perennial fruit crops, 12:223264 phase change, 7:109-155 photoperiod,4:66-105 pistachio, 3:378-387 postharvest physiology, 1:204-236; 3:59-143; 10:35-62; 11:15-43 protea leaf blackening, 17:173-201 pruning,8:359-362 raspberry, 11 :187-188 regulation in floriculture, 7:416-424 rhododendron, 12:1-42 rose, 9:60-66 senescence, 1:204-236; 3:59143; 10:35-62; 11 :15-43; 18:1-85; sugars, 4:114

CUMULATIVE SUBJECT INDEX

thin cell layer morphogenesis, 14:239-256 tulip,5:57-59 water relations, 18:1-85 Fluid drilling, 3:1-58 Foliage plants: acclimatization, 6:119-154 fertilization, 1:102-103; 5:367-380 Foliar nutrition, 6:287-355 Freeze protection, see Frost, protection Frost: apple fruit set, 1:407-408 citrus, 7:201-238 protection, 11:45-109 Fruit: abscission, 1:172-203 citrus, 15:145-182 apple anatomy and morphology, 10:283-297 apple bioregulation, 10:348374 apple bitter pit, 11 :289-355 apple flavor, 16:197-234 apple maturity indices, 13:407-432 apple ripening and quality, 10:361-374 avocado development and ripening, 10:229-271 bloom delay, 15:97-144 blueberry development, 13:378-390 cactus physiology, 18:335341 CA storage and quality, 8:101-127 chilling injury, 15:63-95 diseases in CA storage, 3:412-461 drop, apple and pear, 10:359-

357

361 fig, 12:424-429 kiwifruit, 6:35-48; 12:316318 maturity indices, 13:407-432 navel orange, 8:129-179 nectarine, postharvest, 11:413-452 peach, postharvest, 11 :413452 pear: bioregulation, 10:348-374 fruit disorders, 11:357-411 pear maturity indices, 13:407-432 pear ripening and quality, 10:361-374 pistachio, 3:382-391 quality and pruning, 8:365367, ripening, 5:190-205 set, 1:397-424; 4:153-154 set in navel oranges, 8:140142 size and thinning, 1:293-294; 4:161 softening, 5:109-219; 10:107152 strawberry growth and ripening, 17:267-297 thinning, apple and pear, 10:353-359 tomato parthenocarpy, 6:6584 tomato ripening, 13:67-103 Fruit crops: alternate bearing, 4:128-173 apple bitter pit, 11 :289-355 apple flavor, 16:197-234 apple growth, 11:229-287 apple maturity indices, 13:407-432

358

Fruit crops (cont'd) avocado flowering, 8:257-289 avocado rootstocks, 17: 381429 berry crop harvesting, 16:255-382 bloom delay, 15:97-144 blueberry developmental physiology, 13:339-405 blueberry harvesting, 16:257282 blueberry nutrition, 10:183227 bramble harvesting, 16:282298 cactus, 18:302-309 carbohydrate reserves, 10:403-430 CA storage, 1:301-336 CA storage diseases, 3:412461 chilling injury, 15:145-182 chlorosis, 9:161-165 citrus abscission, 15:145-182 citrus cold hardiness, 7:201238 citrus flowering, 12:349-408 cranberry harvesting, 16:298311 currant harvesting, 16:311327 dormancy release, 7:239-300 Ericaceae nutrition, 10:183227 fertilization, 1:104-106 fig, industry, 12:409-490 fireblight, 11:423-474 flowering, 12:223-264 foliar nutrition, 6:287-355 frost control, 11 :45-109 grape flower anatomy and morphology, 13 :315-33 7

CUMULATIVE SUBJECT INDEX

grape harvesting, 16:327-348 grape nitrogen metabolism, 14:407-452 grape pruning, 16:235-254, 336-340 grape root, 5:127-168 grape seedlessness, 11:164176 grapevine pruning, 16:235254,336-340 honey bee pollination, 9 :244250,254-256 jojoba, 17:233-266 in vitro culture, 7:157-200; 9:273-349 kiwifruit, 6:1-64; 12:307-347 longan, 16:143-196 lychee, 16:143-196 muscadine grape breeding, 14:357-405 navel orange, 8:129-179 nectarine postharvest, 11:413-452 nutritional ranges, 2:143-164 orange, navel, 8:129-179 orchard floor management, 9:377-430 peach origin, 17:331-379 peach postharvest, 11:413452 pear fruit disorders, 11:357411 pear maturity indices, 13:407-432 pecan flowering, 8:217-255 photosynthesis, 11:111-157 Phytophthora control, 17:299-330 pruning,8:339-380 rambutan, 16:143-196 raspberry, 11:185-228 roots, 2:453-457

CUMULATIVE SUBJECT INDEX

sapindaceous fruits, 16:143196 short life and replant problem, 2:1-116 strawberry fruit growth, 17:267-297 strawberry harvesting, 16:348-365 summer pruning, 9:351-375 Vaccinium nutrition, 10:183227 water status, 7:301-344 Fungi: fig,12:451-474 mushroom, 6:85-118 mycorrhiza, 3:172-213; 10:211-212 pathogens in postharvest storage, 3:412-461 truffle cultivation, 16:71-107 Fungicide, and apple fruit set, 1:416

G Garlic, CA storage, 1:375 Genetics and breeding: aroids (edible), 8:72-75; 12:169 aroids (ornamental), 10:1825 bean, bacterial resistance, 3:28-58 bloom delay in fruits, 15:98107 bulbs, flowering, 18:119-123 cassava, 12:164 chestnut blight resistance, 8:313-321 citrus cold hardiness, 7:221223 embryogenesis, 1:23

359

fig, 12:432-433 fire blight resistance, 1:435436 flowering, 15:287-290, 303305,306-309,314-315 flower longevity, 1:208-209 ginseng, 9:197-198 in vitro techniques, 9:318324; 18:119-123 lettuce, 2:185-187 muscadine grapes, 14:357405 mushroom, 6:100-111 navel orange, 8:150-156 nitrogen nutrition. 2:410-411 plant regeneration. 3:278-283 pollution insensitivity, 8:1819 potato tuberization, 14:121124 rhododendron, 12:54-59 sweet potato, 12:175 tomato parthenocarpy, 6:6970 tomato ripening, 13:77-98 tree short life, 2:66-70 Vigna, 2:311-394 woody legume tissue and cell culture, 14:311-314 yam (Dioscorea), 12:183 Genetic variation: alternate bearing, 4:146-150 photoperiodic response, 4:82 pollution injury, 8:16-19 temperature-photoperiod interaction, 17:73-123 Geophyte, see Bulb, tuber Geranium, fertilization, 5:355357 Germination, seed, 2:117-141, 173-174 Germplasm preservation:

CUMULATIVE SUBJECT INDEX

360

Germplasm preservation (cont'd) cryopreservation, 6:357-372 in vitro, 5:261-264; 9:324325

Gibberellin: abscission, citrus, 15:166167

bloom delay, 15:111-114 citrus, abscission, 15:166167

cold hardiness, 11 :63 dormancy, 7:270-271 floral promoter, 4:114 flowering, 15:219-293, 315318

genetic regulation, 16:15 grape root, 5:150-151 mechanical stress, 17:19-20 Ginseng, 9:187-236 Girdling, 4:251-252 Graft and grafting: incompatibility, 15:183-232 phase change, 7:136-137, 141-142

rose, 9:56-57 Grape: CA storage, 1 :308 chlorosis, 9:165-166 flower anatomy and morphology, 13:315-337 harvesting, 16:327-348 muscadine breeding, 14:357405

nitrogen metabolism, 14:407452

pollen morphology, 13:331332

pruning, 16:235-254,336340

root, 5:127-168 seedlessness, 11:159-187 sex determination, 13:329-

331

Gravitropism, 15:233-278 Greenhouse and greenhouse crops: carbon dioxide, 7:357-360, 544-545

energy efficiency, 1:141-171; 9:1-52

growth substances, 7:399-481 nutrition and fertilization, 5:317-403

pest management, 13:1-66 Growth regulators, see Growth substances Growth substances, 2:60-66. See also Abscisic acid; Auxin; Cytokinin; Ethylene; Gibberellin abscission, citrus, 15:157176

apple bioregulation, 10:309401

apple dwarfing, 3:315-375 apple fruit set, 1 :417 apple thinning, 1:270-300 aroids, ornamental, 10:14-18 avocado fruit development, 10:229-243

bloom delay, 15:107-119 CA storage in vegetables, 1:346-348

cell cultures, 3:214-314 chilling injury, 15:77-83 citrus abscission, 15:157-176 cold hardiness 7:223-225; 11:58-66

dormancy, 7:270-279 embryogenesis, 1:41-43; 2:277-281

floriculture, 7:399-481 flower induction, 4:190-195 flowering, 15:290-296

CUMULATIVE SUBJECT INDEX

flower storage, 10:46-51 genetic regulation, 16:1-32 ginseng, 9:226 grape seedlessness, 11:177180

in vitro flowering, 4:112-115 mechanical stress, 17:16-21 meristem and shoot-tip culture, 5:221-227 navel oranges, 8:146-147 pear bioregulation, 10:309401

petal senescence, 3:76-78 phase change, 7:137-138, 142-143 raspberry, 11:196-197 regulation, 11:1-14 rose, 9:53-73

seedlessness in grape, 11:177-180 triazole, 10:63-105

361

4:179-184. See also Anatomy and morphology Honey bee, 9:237-272 Horseradish, CA storage, 1:368 Hydrolases, 5:169-219 Hydroponic culture, 5:1-44; 7:483-558

Hypovirulence, in Endothia parasitica,8:299-310 I

Ice, formation and spread in tissues, 13:215-255 Ice-nucleating bacteria, 7:210212; 13:230-235

Industrial crops, cactus, 18:309--:312

Insects and mites: aroids, 8:65-66 avocado pollination, 8:275277

H

Halo blight of beans, 3:44-45 Hardiness, 4:250-251 Harvest: flower stage, 1:211-212 index, 7:72-74 lettuce, 2:176-181 mechanical of berry crops, 16:255-382

Hazelnut, see Filbert Heliconia, 14:1-55 Herbaceous plants, subzero stress, 6:373-417 Herbicide-resistant crops, 15:371-412

Histochemistry: flower induction, 4:177-179 fruit abscission, 1:172-203 Histology, flower induction,

fig, 12:442-447 hydroponic crops, 7:530-534 integrated pest management, 13:1-66

lettuce, 2:197-198 ornamental aroids, 10:18 tree short life, 2:52 tulip, 5:63, 92 Integrated pest management, greenhouse crops, 13:1-66 In vitro: abscission, 15:156-157 apple propagation, 10:325326

aroids, ornamental, 10:13-14 bulbs, flowering, 18:87-169 cassava propagation, 13:121123

cellular salinity tolerance,

362

In vitro (con t'd) 16:33-69 cold acclimation, 6:382 cryopreservation, 6:357-372 embryogenesis, 1:1-78; 2:268-310; 7:157-200; 10:153-181 environmental control, 17:123-170 flowering,4:106-127 flowering bulbs, 18:87-169 pear propagation, 10:325326 phase change, 7:144-145 propagation, 3:214-314; 5:221-277; 7:157-200; 9:57-58,273-349; 17:125172 thin cell layer morphogenesis, 14:239-264 woody legume culture, 14:265-332 Iron: deficiency chlorosis, 9:133186 deficiency and toxicity symptoms in fruits and nuts, 2:150 Ericaceae nutrition, 10:193195 foliar application, 6:330 nutrition, 5:324-325 pine bark media, 9:123 Irrigation: drip or trickle, 4:1-48 frost control, 11:76-82 fruit trees, 7:331-332 grape root growth, 5:140141 lettuce industry, 2:175 navel orange, 8:161-162 root growth, 2:464-465

CUMULATIVE SUBJECT INDEX

J Jojoba, 17:233-266 Juvenility, 4:111-112 pecan, 8:245-247 tulip, 5:62-63 woody plants, 7:109-155 K

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

Lamps, for plant growth, 2:514531 Leaves: apple morphology, 12:283288 flower induction, 4:188-189 Leek: CA storage, 1:375 fertilization, 1:118 Leguminosae, in vitro, 5:227229; 14:265-332 Lemon, rootstock, 1:244-246. See also Citrus Lettuce: CA storage, 1:369-371 fertilization, 1:118 fluid drilling of seed, 3:14-17 industry, 2:164-207 tipburn, 4:49-65 Light: fertilization, greenhouse crops, 5:330-331 flowering, 15:282-287, 310312 fruit set, 1:412-413

CUMULATIVE SUBJECT INDEX

lamps, 2:514-531 nitrogen nutrition, 2:406-407 orchards, 2:208-267 ornamental aroids, 10:4-6 photoperiod,4:66-105 photosynthesis, 11:117-121 plant growth, 2:491-537 tolerance, 18:215-246 Longan, see Sapindaceous fruits Lychee, see Sapindaceous fruits M

Magnesium: container growing, 9:84-85 deficiency and toxicity symptoms in fruits and nuts, 2:148 Ericaceae nutrition, 10:196198 foliar application, 6:331 nutrition, 5:323 pine bark media, 9:117-119 Male sterility, temperaturephotoperiod induction, 17:103-106 Mandarin, rootstock, 1:250-252 Manganese: deficiency and toxicity symptoms in fruits and nuts, 2:150-151 Ericaceae nutrition, 10:189193 foliar application, 6:331 nutrition, 5:235-326 pine bark media, 9:123-124 Mango: alternate bearing, 4:145-146 asexual embryogenesis, 7:171-173 CA storage, 1:313

363

in vitro culture, 7:171-173 Mechanical harvest, berry crops, 16:255-382 Mechanical stress regulation, 17:1-42 Media: fertilization, greenhouse crops, 5:333 pine bark, 9:103-131 Meristem culture, 5:221-277 Metabolism: flower, 1:219-223 nitrogen in citrus, 8:181-215 seed,2:117-141 Micronutrients: container growing, 9:85-87 pine bark media, 9:119-124 Micropropagation, see In vitro, propagation bulbs, flowering, 18:89-113 environmental control, 17:125-172 nuts, 9:273-349 rose, 9:57-58 temperate fruits, 9:273-349 tropical fruits and palms, 7:157-200 Microtus, see Vole Moisture, and seed storage, 2:125-132 Molybdenum nutrition, 5:328329 Monocot, in vitro, 5:253-257 Monstera, see Aroids, ornamental Morphology: navel orange, 8:132-133 orchid, 5:283-286 pecan flowering, 8:217-243 Moth bean, genetics, 2:373-374 Mung bean, genetics, 2:348-364

364

Mushroom: CA storage, 1:371-372 spawn, 6:85-118 Muskmelon, fertilization, 1:118-119 Mycoplasma-like organisms, tree short life, 2:50-51 Mycorrhizae: container growing, 9:93 Ericaceae, 10:211-212 fungi,3:172-213 grape root, 5:145-146 N

Navel orange, 8:129-179 Nectarine: bloom delay, 15:105-106 CA storage, 1:309-310 postharvest physiology, 11:413-452 Nematodes: aroids, 8:66 fig, 12:475-477 .lettuce, 2:197-198 tree short life, 2:49-50 NFT (nutrient film technique), 5:1-44 Nitrogen: CA storage, 8:116-117 container growing, 9:80-82 in embryogenesis, 2:273-275 deficiency and toxicity symptoms in fruits and nuts, 2:146 Ericaceae nutrition, 10:198202 fixation in woody legumes, 14:322-323 foliar application, 6:332 metabolism in apple, 4:204246

CUMULATIVE SUBJECT INDEX

metabolism in citrus, 8:181215 metabolism in grapevine, 14:407-452 nutrition, 2:395,423; 5:319320 pine bark media, 9:108-112 trickle irrigation, 4:29-30 Nursery crops: fertilization, 1:106-112 nutrition, 9:75-101 Nut crops: chestnut blight, 8:291-336 fertilization, 1:106 honey bee pollination, 9:250251 in vitro culture, 9:273-349 nutritional ranges, 2:143-164 pistachio culture, 3:376-396 Nutrient: concentration in fruit and nut crops, 2:154-162 film technique, 5:1-44 foliar-applied, 6:287-355 media: for asexual embryogenesis, 2:273-281 for organogenesis, 3:214314 plant and tissue analysis, 7:30-56 solutions, 7:524-530 uptake, in trickle irrigation, 4:30-31 Nutrition (human): aroids, 8:79-84 CA storage, 8:101-127 Nutrition (plant): air pollution, 8:22-23, 26 blueberry, 10:183-227 calcifuge, 10:183-227 cold hardiness, 3:144-171

CUMULATIVE SUBJECT INDEX

container nursery crops, 9:75-101 embryogenesis, 1:40-41 Ericaceae, 10:183-227 fire blight, 1:438-441 foliar, 6:287-355 fruit and nut crops, 2:143164 ginseng, 9:209-211 greenhouse crops, 5:317-403 kiwifruit, 12:325-332 mycorrhizal fungi, 3:185-191 navel orange, 8:162-166 nitrogen in apple, 4:204-246 nutrient film techniques, 5:18-21,31-53 ornamental aroids, 10:7-14 pine bark media, 9:103-131 raspberry,11:194-195 slow-release fertilizers, 1:79139

o Oil palm: asexual embryogenesis, 7:187-188 in vitro culture, 7:187-188 Okra, CA storage, 1:372-373 Olive, alternate bearing, 4:140141 Onion: CA storage, 1:373-375 fluid drilling of seed, 3:17-18 Orange, see Citrus alternate bearing, 4:143-144 sour, rootstock, 1:242-244 sweet, rootstock, 1:252-253 trifoliate, rootstock, 1:247250 Orchard and orchard systems: floor management, 9:377-430

365

light, 2:208-267 root growth, 2:469-470 water, 7:301-344 Orchid: fertilization, 5:357-358 physiology,5:279-315 Organogenesis, 3:214-314. See also In vitro; Tissue, culture Ornamental plants: chlorosis, 9:168-169 fertilization, 1:98-104, 106116 flowering bulb roots, 14:5788 flowering bulbs in vitro, 18:87-169 foliage acclimatization, 6:119-154 heliconia, 14:1-55 protea leaf blackening, 17:173-201 rhododendron, 12:1-42 p

Paclobutrazol, see Triazole Papaya: asexual embryogenesis, 7:176-177 CA storage, 1:314 in vitro culture, 7:175-178 Parsley: CA storage, 1:375 drilling of seed, 3:13-14 Parsnip, fluid drilling of seed, 3:13-14 Parthenocarpy, tomato, 6:65-84 Passion fruit, in vitro culture, 7:180-181 Pathogen elimination, in vitro, 5:257-261

366

Peach: bloom delay, 15:105-106 CA storage, 1:309-310 origin, 17:333-379 postharvest physiology, 11:413-452 short life, 2:4 summer pruning, 9:351-375 Peach palm (Pejibaye), in vitro culture, 7:187-188 Pear: bioregulation, 10:309-401 bloom delay, 15:106-107 CA storage, 1:306-308 decline, 2:11 fire blight control, 1:423-474 fruit disorders, 11:357-411 in vitro, 9:321 maturity indices, 13:407-432 root distribution, 2:456 short life, 2:6 Pecan: alternate bearing, 4:139-140 fertilization, 1:106 flowering, 8:217-255 in vitro culture, 9: 314-315 Pejibaye (peach palm), in vitro culture, 7:189 Pepper (Capsicum): CA storage, 1:375-376 fertilization, 1:119 fluid drilling in seed, 3:20 Persimmon: CA storage, 1:314 quality, 4:259 Pest control: aroids (edible), 12:168-169 aroids (ornamental), 10:18 cassava, 12:163-164 cowpea, 12:210-213 fig,12:442-477 fire blight, 1:423-474

CUMULATIVE SUBJECT INDEX

ginseng,9:227-229 greenhouse management, 13:1-66 hydroponics, 7:530-534 sweet potato, 12:173-175 vertebrate, 6:253-285 yam (Dioscorea), 12:181-183 Petal senescence, 11:15-43 pH: container growing, 9:87-88 fertilization greenhouse crops, 5:332-333 pine bark media, 9:114-117 soil testing, 7:8-12, 19-23 Phase change, 7:109-155 Phenology: apple, 11:231-237 raspberry, 11:186-190 Philodendron, see Aroids, ornamental Phosphonates, Phytophthora control, 17:299-330 Phosphorus: container growing, 9:82-84 deficiency and toxicity symptoms in fruits and nuts, 2:146-147 nutrition, 5:320-321 pine bark media, 9:112-113 trickle irrigation, 4:30 Photoautotrophic micropropagation, 17:125172 Photoperiod, 4:66-105,116117; 17:73-123 flowering, 15:282-284, 310312 Photosynthesis: cassava, 13:112-114 efficiency, 7:71-72; 10:378 fruit crops, 11:111-157 ginseng, 9:223-226

CUMULATIVE SUBJECT INDEX

light, 2:237-238 Physiology, see Postharvest physiology bitter pit, 11 :289-355 blueberry development, 13:339-405 cactus reproductive biology, 18:321-346 calcium, 10:107-152 carbohydrate metabolism, 7:69-108 cassava, 13:105-129 citrus cold hardiness, 7:201238 conditioning 13:131-181 cut flower, 1:204-236; 3:59143; 10:35-62 desiccation tolerance, 18:171-213 disease resistance, 18:247289 dormancy, 7:239-300 embryogenesis, 1:21-23; 2:268-310 flowering,4:106-127 fruit ripening, 13:67-103 fruit softening, 10:107-152 ginseng, 9:211-213 heliconia, 14:5-13 juvenility, 7:109-155 light tolerance, 18:215-246 male sterility, 17:103-106 mechanical stress, 17:1-42 ni trogen metabolism in grapevine, 14:407-452 nutritional quality and CA storage, 8:118-120 orchid,5:279-315 petal senescence, 11 :15-43 photoperiodism, 17:73-123 pollution injury, 8:12-16 polyamines, 14:333-356

367

potato tuberization, 14:89188 pruning, 8:339-380 raspberry, 11:190-199 regulation, 11:1-14 root pruning, 6:158-171 roots of flowering bulbs, 14:57-88 rose, 9:3-53 salinity hormone action, 16:1-32 salinity tolerance, 16:33-69 seed,2:117-141 seed priming, 16:109-141 subzero stress, 6:373-417 summer pruning, 9:351-375 thin cell layer morphogenesis, 14:239-264 tomato fruit ripening, 13:67103 tomato parthenocarpy, 6:7174 triazole, 10:63-105 tulip, 5:45-125 vernalization, 17:73-123 volatiles, 17:43-72 watercore, 6:189-251 water relations of cut flowers, 18:1-85 Phytohormones, see Growth substances Phytophthora control, 17:299330 Phytotoxins, 2:53-56 Pigmentation: flower, 1:216-219 rose, 9:64-65 Pinching, by chemicals, 7:453461 Pineapple: CA storage, 1:314 in vitro culture, 7:181-182

368

Pine bark, potting media, 9:103-131 Pistachio: alternate bearing, 4:137-139 culture, 3:376-393 in vitro culture, 9: 315 Plantain, in vitro culture, 7:178-180 Plant protection, short life, 2:79-84 Plum, CA storage, 1:309 Poinsettia, fertilization, 1:103104; 5:358-360 Pollen, desiccation tolerance, 18:195 Pollination: apple, 1:402-404 avocado, 8:272-283 cactus, 18:331-335 embryogenesis, 1:21-22 fig, 12:426-429 fruit crops, 12:223-264 fruit set, 4:153-154 ginseng, 9:201-202 grape, 13:331-332 heliconia, 14:13-15 honey bee, 9:237-272 kiwifruit, 6:32-35 navel orange, 8:145-146 orchid,5:300-302 petal senescence, 11 :33-35 protection, 7:463-464 rhododendron, 12:1-67 Pollution, 8:1-42 Polyamines, 14:333-356 chilling injury, 15 :80 Polygalacturonase, 13:67-103 Postharvest physiology: apple bitter pit, 11 :289-355 apple maturity indices, 13:407-432 aroids, 8:84-86

CUMULATIVE SUBJECT INDEX

asparagus, 12:69-155 CA storage and quality, 8:101-127 cut flower, 1:204-236; 3:59143; 10:35-62 foliage plants, 6:119-154 fruit, 1:301-336 fruit softening, 10:107-152 lettuce, 2:181-185 low-temperature sweetening, 17:203-231 navel orange, 8:166-172 nectarine, 11:413-452 pathogens, 3:412-461 peach,11:413-452 pear disorders, 11:357-411 pear maturity indices, 13:407-432 petal senescence, 11 :15-43 protea leaf blackening, 17:173-201 seed,2:117-141 tomato fruit ripening, 13:67103 vegetables, 1:337-394 watercore, 6:189-251; 11:385-387 Potassium: container growing, 9:84 deficiency and toxicity symptoms in fruits and nuts, 2:147-148 foliar application, 6:331-332 nutrition, 5:321-322 pine bark media, 9:113-114 trickle irrigation, 4:29 Potato: CA storage, 1:376-378 fertilization, 1:120-121 low temperature sweetening, 17:203-231 tuberization, 14:89-198

CUMULATIVE SUBJECT INDEX

369

Propagation, see In vitro apple, 10:324-326; 12:288295 aroids, ornamental, 10:12-13 cassava, 13:120-123 floricultural crops, 7:461-462 ginseng, 9:206-209 orchid,5:291-297 pear, 10:324-326 rose, 9:54-58 tropical fruit, palms 7:157200 woody legumes in vitro, 14:265-332 Protea, leaf blackening, 17:173201 Protected crops, carbon dioxide, 7:345-398 Protoplast culture, woody species, 10:173-201 Pruning, 4:161, 8:339-380 apple, 9:351-375 apple training, 1:414 chemical, 7:453-461 cold hardiness, 11:56 fire blight, 1:441-442 grapevines, 16:235-254 light interception, 2:250251 peach, 9:351-375 phase change, 7:143-144 root, 6:155-188 Prunus, see Almond; Cherry; Nectarine; Peach; Plum in vitro, 5:243-244; 9:322 root distribution, 2:456

Pseudomonas: pbaseolicola, 3:32-33 39 44-45

'

solanacearum, 3:33 syringae, 3:33, 40; 7:210212

,

R

Rabbit,6:275-276 Radish, fertilization, 1:121 Rambutan, see Sapindaceous fruits Raspberry: harvesting, 16:282-298 productivity, 11:185-228 Rejuvenation: rose, 9:59-60 woody plants, 7:109-155 Replant problem, deciduous fruit trees, 2:1-116 Respiration: asparagus postharvest, 12:7277 fruit in CA storage, 1:315316 kiwifruit,6:47-48 vegetables in CA storage, 1:341-346 Rhizobium, 3:34,41 Rhododendron, 12:1-67 Rice bean, genetics, 2:375-376 Root: apple, 12:269-272 cactus, 18:297-298 diseases, 5:29-31 environment, nutrient film technique, 5:13-26 Ericaceae, 10:202-209 grape, 5:127-168 kiwifruit,12:310-313 physiology of bulbs, 14:5788 pruning,6:155-188 raspberry, 11:190 rose, 9:57 tree crops, 2:424-490 Root and tuber crops: aroids, 8:43-99; 12:166-170

370

Root and tuber crops (coni'd) cassava, 12:158-166 low-temperature sweetening, 17:203-231 minor crops, 12:184-188 potato tuberization, 14:89188 sweet potato, 12:170-176 yam (Dioscorea), 12:177-184 Rootstocks: alternate bearing, 4:148 apple, 1:405-407; 12:295-297 avocado, 17:381-429 citrus, 1:237-269 cold hardiness, 11:57-58 fire blight, 1:432-435 light interception, 2:249-250 navel orange, 8:156-161 root systems, 2:471-474 stress, 4:253-254 tree short life, 2:70-75 Rosaceae, in vitro, 5:239-248 Rose: fertilization, 1:104; 5:361363 growth substances, 9:3-53 in vitro, 5:244-248

s Salinity: air pollution, 8:25-26 soils, 4:22-27 tolerance, 16:33-69 Sapindaceous fruits, 16:143196 Scoring, and fruit set, 1:416417 Seed: abortion, 1 :293-294 apple anatomy and morphology, 10:285-286

CUMULATIVE SUBJECT INDEX

conditioning,13:131-181 desiccation tolerance, 18:196-203 environmental influences on size and composition, 13:183-213 flower induction, 4:190-195 fluid drilling, 3:1-58 grape seedlessness, 11:159184 kiwifruit, 6:48-50 lettuce, 2:166-174 priming,16:109-141 rose propagation, 9:54-55 vegetable, 3:1-58 viability and storage, 2:117141 Secondary metabolites, woody legumes, 14:314-322 Senescence: cut flower, 1:204-236; 3:59143; 10:35-62; 18:1-85 petal, 11:15-43 rose, 9:65-66 whole plant, 15:335-370 Sensory quality, CA storage, 8:101-127 Shoot-tip culture, 5:221-277. See also Micropropagation Short life problem, fruit crops, 2:1-116 Small fruit, CA storage, 1:308 Snapdragon fertilization, 5:363364 Sodium, deficiency and toxicity symptoms in fruits and nuts, 2:153-154 Soil: grape root growth, 5:141-144 management and root growth, 2:465-469 orchard floor management,

CUMULATIVE SUBJECT INDEX

9:377-430 plant relations, trickle irrigation, 4:18-21 stress, 4:151-152 testing, 7:1-68; 9:88-90 Soilless culture, 5:1-44 Solanaceae, in vitro, 5:229-232 Somatic embryogenesis, see Asexual embryogenesis Spathiphyllum, see Aroids, ornamental Stem, apple morphology, 12:272-283 Storage, see Controlled-atmosphere (CA) storage; Postharvest physiology cut flower, 3:96-100; 10:3562 rose plants, 9:58-59 seed,2:117-141 Strawberry: fertilization, 1:106 fruit growth and ripening, 17:267-297 harvesting, 16:348-365 in vitro, 5:239-241 Stress: benefits of, 4:247-271 climatic, 4:150-151 flooding, 13:257-313 mechanical, 17:1-42 petal, 11:32-33 plant, 2:34-37 protection, 7:463-466 subzero temperature, 6:373417 Sugar beet, fluid drilling of seed,3:18-19 Sugar, see Carbohydrate allocation, 7: 74-94 flowering,4:114 Sulfur:

371

deficiency and toxicity symptoms in fruits and nuts, 2:154 nutrition, 5:323-324 Sweet potato: culture, 12 :170-176 fertilization, 1:121 Symptoms, deficiency and toxicity symptoms in fruits and nuts, 2:145-154 Syngonium, see Aroids, ornamental T Taro, see Aroids, edible Temperature: apple fruit set, 1:408-411 bloom delay, 15:119-128 CA storage of vegetables, 1:340-341 chilling injury, 15:67-74 cut flower storage, 10:40-43 cryopreservation, 6:357-372 fertilization, greenhouse crops, 5:331-332 fire blight forecasting, 1:456459 flowering, 15 :284-28 7, 312313 interaction with photoperiod, 4:80-81 low ternperature sweetening, 17:203-231 navel orange, 8:142 nutrient film technique, 5:21-24 photoperiod interaction, 17:73-123 photosynthesis, 11:121-124 plant growth, 2:36-37 seed storage, 2:132-133

CUMULATIVE SUBJECT INDEX

372

subzero stress, 6:373-417 Thinning, apple, 1:270-300 Tipburn, in lettuce, 4:49-65 Tissue, see In vitro culture, 1:1-78; 2:268-310; 3:214-314; 4:106-127; 5:221-277; 6:357-372; 7:157-200; 8:75-78; 9:273349; 10:153-181 dwarfing, 3:347-348 nutrient analysis, 7:52-56; 9:90

Tomato: CA storage, 1:380-386 fertilization, 1:121-123 fluid drilling of seed, 3:19-20 fruit ripening, 13:67-103 galacturonase, 13:67-103 parthenocarpy, 6:65-84 Toxicity symptoms in fruit and nut crops, 2:145-154 Transport, cut flowers, 3:100104

Tree decline, 2:1-116 Triazole, 10:63-105 chilling injury, 15:79-80 Trickle irrigation, 4:1-48 Truffle cultivation, 16:71-107 Tuber, potato, 14:89-188 Tuber and root crops, see Root and tuber crops Tulip, see Bulb crops fertilization, 5 :364-366 in vitro, 18:144-145 physiology, 5:45-125 Tunnel (cloche), 7:356-357 Turfgrass, fertilization, 1:112117

Turnip, fertilization, 1:123124

Turnip Mosaic Virus, 14:199238

U

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

v Vaccinium, 10:185-187. See also Blueberry; Cranberry Vase solutions, 3:82-95; 10:4651

Vegetable crops: aroids, 8:43-99; 12:166-170 asparagus postharvest, 12:69 155

cactus, 18:300-302 cassava, 12:158-166; 13:105129

CA storage, 1:337-394 CA storage diseases, 3:412461

CA storage and quality, 8:101-127

chilling injury, 15:63-95 fertilization, 1:117-124 fluid drilling of seeds, 3:1-58 greenhouse pest management, 13:1-66 honey bee pollination, 9:251254

hydroponics, 7:483-558 low-temperature sweetening, 17:203-231

minor root and tubers, 12:184-188

mushroom spawn, 6:85-118 potato tuberization, 14:89188

seed conditioning, 13:131-181 seed priming, 16:109-141 sweet potato, 12:170-176 tomato fruit ripening, 13: 67-103

CUMULATIVE SUBJECT INDEX

tomato parthenocarpy, 6:6584

truffle cultivation, 16:71-107 yam (Dioscorea), 12:177-184 Vegetative tissue, desiccation tolerance, 18:176-195 Vernalization, 4:117, 15 :284287; 17:73-123

Vertebrate pests, 6:253-285 Vigna, see Cowpea genetics, 2:311-394 U.S. production, 12:197-222 Virus: benefits in horticulture,

373

pear, 11:385-387 Watermelon, fertilization, 1:124 Weed control, ginseng, 9:228229

Weeds: lettuce research, 2:198 virus, 3:403 Woodchuck, 6:276-277 Woody species, somatic embryogenesis, 10:153-181

x Xantbomonas pbaseo1i, 3 :29-

3:394-411

elimination, 7:157-200; 9:318; 18:113-123

fig, 12:474-475 tree short life, 2:50-51 turnip mosaic, 14:199-238 Volatiles, 17:43-72 Vole, 6:254-274

w Walnut, in vitro culture, 9:312 Water relations: cut flower, 3:61-66; 18:1-85 desiccation tolerance, 18:171-213

fertilization, greenhouse crops, 5:332 fruit trees, 7:301-344 kiwifruit, 12:332-339 light in orchards, 2:248-249 photosynthesis, 11:124-131 trickle irrigation, 4:1-48 Watercore, 6:189-251

32,41,45-46

Xanthophyll cycle, 18:226-239 Xantbosoma, 8:45-46, 56-57. See also Aroids

y Yam (Dioscorea), 12:177-184 Yield: determinants, 7:70-74,97-99 limiting factors, 15:413-452

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

Cumulative Contributor Index (Volumes 1-18) Adams, W.W., III, 18:215 Aldwinckle, H.S., 1:423; 15:xiii Anderson, J.1., 15:97 Anderson, P.C., 13:257 Andrews, P.K., 15:183 Ashworth, E.N., 13:215 Asokan, M.P., 8:43 Atkinson, D., 2:424 Aung, 1.H., 5:45 Bailey, W.G., 9:187 Baird, 1.A.M., 1:172 Barden, J.A., 9:351 Barker, A.V., 2:411 Bass, 1.N., 2:117 Becker, J.S., 18:247 Beer, S.V., 1:423 Bennett, A.B., 13:67 Benschop, M., 5:45 Ben-Ya'acov, A., 17:381 Benzioni, A., 17:233 Bewley, J.D., 18:171 Binzel, M.L., 16:33 Blanpied, G.D., 7:xi Bliss, F.A., 16:xiii Borochov, A., 11:15 Bower, J.P., 10:229 Bradley, G.A., 14:xiii Brennan, R., 16:255 Broschat, T.K., 14:1 Brown, S. 15:xiii Buban, T., 4:174

Bukovac, M.J., 11:1 Burke, M.J., 11:xiii Buwalda, J.G., 12:307 Byers, R.E., 6:253 Caldas, 1.S., 2:568 Campbell, L.E., 2:524 Cantliffe, D.J., 16:109, 17:43 Carter, J.V., 3:144 Cathey, H.M., 2:524 Chambers, R.J., 13:1 Charron, C.S., 17:43 Chin, C.K., 5:221 Cohen, M., 3:394 Collier, G.F., 4:49 Collins, W.L., 7:483 Compton, M.E., 14:239 Conover, C.A., 5:317; 6:119 Coyne, D.P., 3:28 Crane, J.C., 3:376 Criley, R.A., 14:1 Crowly, W., 15:1 Cutting, J.G., 10:229 Daie, J., 7:69 Dale, A., 11:185; 16:255 Darnell, R.1., 13:339 Davenport, T.1., 8:257; 12:349 Davies, F.S., 8:129 Davies, P.J., 15:335 Davis, T.D., 10:63 DeGrandi-Hoffman, G., 9:237 375

376

De Hertogh, A.A., 5:45; 14:57; 18:87 Deikman, J., 16:1 DellaPenna, D., 13:67 Demmig-Adams, B., 18:215 Dennis, F.G., Jr., 1:395 Doud, S.L., 2:1 Duke, S.D., 15:371 Dunavent, M.G., 9:103 Dyer, W.E., 15:371 Early, J.D., 13:339 Elfving, D.C., 4:1; 11:229 EI-Goorani, M.A., 3:412 Esan, E.B., 1:1 Evans, D.A., 3:214 Ewing, E.E., 14:89 Faust, M., 2:vii, 142; 4:174; 6:287; 14:333, 17:331 Fenner, M., 13:183 Ferguson, A.R., 6:1 Ferguson, LB., 11:289 Ferguson, L., 12:409 Ferree, D.C., 6:155 Fery, R.L., 2:311; 12:157 Fischer, R.L., 13:67 Flick, C.E., 3:214 Flore, J.A., 11:111 Forshey, e.G., 11:229 Fujiwara, K., 17:125 Geisler, D., 6:155 Geneve, R.L., 14:265 George, W.L., Jr., 6:25 Gerrath, J.M., 13:315 Giovannetti, G., 16:71 Giovannoni, J.J., 13:67 Glenn, G.M., 10:107 Goldschmidt, E.E., 4:128 Goldy, R.G., 14:357 Goren, R., 15:145

CUMULATIVE CONTRIBUTOR INDEX

Goszczynska, D.M., 10:35 Grace, S.C., 18:215 Graves, C.J., 5:1 Gray, D., 3:1 Grierson, W., 4:247 Griffen, G.J., 8:291 Grodzinski, B., 7:345 Guest, D.l., 17:299 Guiltinan, M.J., 16:1 Hackett, W.P., 7:109 Halevy, A.H., 1:204; 3:59 Hammerschmidt, R., 18:247 Hanson, E.J., 16:255 Heath, R.R., 17:43 Helzer, N.L., 13:1 Hendrix, J.W., 3:172 Henny, R.J., 10:1 Hergert, G.B., 16:255 Hess, F.D., 15:371 Heywood, V., 15:1 Hogue, E.J., 9:377 Holt, J.S., 15:371 Huber, D.J., 5:169 Hutchinson, J.F., 9:273 Isenberg, F.M.R., 1;337 Iwakiri, B.T., 3:376 Jackson, J.E., 2:208 Janick, J., l:ix; 8:xi; 17:xiii Jensen, M.H., 7:483 Jeong, B.R., 17:125 Joiner, J.N., 5:317 Jones, H.G., 7:301 Jones, J.B., Jr., 7:1 Jones, R.B., 17:173 Kagan-Zur, V., 16:71 Kang, S.-M., 4:204 Kato, T., 8:181 Kawa, L., 14:57

CUMULATIVE CONTRIBUTOR INDEX

377

Kawada, K., 4:247 Kelly, J.F., 10:ix Khan, A.A., 13:131 Kierman, J., 3:172 Kim, K.-W., 18:87 Kinet, J.-M., 15:279 King, G.A., 11:413 Kingston, C.M., 13:407-432 Kliewer, W.M., 14:407 Knox, R.B., 12:1 Kofranek, A.M., 8:xi Korcak, R.F., 9:133; 10:183 Kozai, T., 17:125 Krezdorn, A.H., l:vii

Mika, A., 8:339 Miller, S.S., 10:309 Mills, H.A., 9:103 Mitchell, C.A., 17:1 Mizrahi, Y., 18:291, 321 Molnar, J.M., 9:1 Monk, G.J., 9:1 Moore, G.A., 7:157 Mor, Y., 9:53 Morris, J.R., 16:255 Mills, H.A., 2:411 Monselise, S.P., 4:128 Murashige, T., 1:1 Myers, P.N., 17:1

Lakso, A.N., 7:301; 11:111 Lamb, R.C., 15:xiii Lang, G.A., 13:339 Larsen, R.P., 9:xi Larson, R.A., 7:399 Ledbetter, C.A., 11:159 Li, P.H., 6:373 Lill, R.E., 11:413 Lipton, W.J., 12:69 Litz, R.E., 7:157 Lockard, R.G., 3:315 Loescher, W.H., 6:198 Lorenz, O.A., 1:79

Neilsen, G.H., 9:377 Nerd, A., 18:291, 321 Niemiera, A.X., 9:75 Nobel, P.S., 18:291

Maraffa, S.B., 2:268 Marangoni, A.G., 17:203 Marini, R.P., 9:351 Marlow, G.C., 6:189 Maronek, D.M., 3:172 Martin, G.G., 13:339 Mayak, S., 1:204; 3:59 Maynard, D.N., 1:79 McConchie, R., 17:173 McNicol, R.J., 16:255 Merkle, S.A., 14:265 Michailides, T.J., 12:409 Michelson, E., 17:381

Ogden, R.J., 9:103 O'Donoghue, E.M., 11:413 O'Hair, S.K., 8:43; 12:157 Oliveira, C.M., 10:403 Oliver, M.J., 18:171 Ormrod, D.P., 8:1 Palser, B.F., 12:1 Parera, C.A., 16:109 Pegg, K.G., 17:299 Pellett, H.M., 3:144 Perkins-Veazil, P., 17:267 Ploetz, R.C., 13:257 Pokorny, F.A., 9:103 Poole, R.T., 5:317;6:119 Poovaiah, B.W., 10:107 Porter, M.A., 7:345 Possingham, J.V., 16:235 Pratt, C., 10:273; 12:265 Preece, J.E., 14:265 Priestley, C.A., 10:403 Proctor, J.T.A., 9:187

378

Quamme, H., 18:xiii Raese, J.T., 11:357 Ramming, D.W., 11:159 Reddy, A.S.N., 10:107 Reid, M., 12:xiii, 17:123 Reuveni, M., 16:33 Richards, D., 5:127 Rieger, M., 11 :45 Roth-Bejerano, N., 16:71 Roubelakis-Angelakis, K.A., 14:407 Rouse, J.L., 12:1 Rudnicki, R.M., 10:35 Ryder, E.J., 2:164; 3:vii Sachs, R., 12:xiii Sakai, A., 6:357 Salisbury, F.B., 4:66; 15:233 San Antonio, J.P., 6:85 Sankhla, N., 10:63 Saure, M.C., 7:239 Schaffer, B., 13:257 Schneider, G.W., 3;315 Schuster, M.L., 3:28 Scorza, R., 4:106 Scott, J.W., 6:25 Sedgley, M., 12:223 Seeley, S.S., 15:97 Serrano Marquez, C., 15:183 Sharp, W.R., 2:268; 3:214 Shattuck, V.I., 14:199 Shear, C.B., 2:142 Sheehan, T.J., 5:279 Shorey, H.H., 12:409 Sklensky, D.E., 15:335 Smith, G.S., 12:307 Smock, R.M., 1:301 Sommer, N.F., 3:412 Sondahl, M.R., 2:268 Sopp, PJ., 13:1 Soule, J., 4:247

CUMULATIVE CONTRIBUTOR INDEX

Sparks, D., 8:217 Splittstoesser, W.E., 6:25; 13:105 Srinivasan, C., 7:157 Stang, E.J., 16:255 Steffens, G.L., 10:63 Stevens, M.A., 4:vii Struik, P.C., 14:89 Stutte, G.W., 13:339 Styer, D.J., 5;221 Sunderland, K.D., 13:1 Swanson, B., 12:xiii Swietlik, D., 6:287 Syvertsen, J.P., 7:301 Tibbitts, T.W., 4:49 Timon, B., 17:331 Tindall, H.D., 16:143 Tisserat, B., 1:1 Titus, J.S., 4:204 Trigiano, R.N., 14:265 Tunya, G.O., 13:105 van Doorn, W.G., 17:173; 18:1 Veilleux, R.E., 14:239 Wallace, A., 15:413 Wallace, D.H., 17:73 Wallace, G.A., 15:413 Wang, C.Y., 15:63 Wang, S.Y., 14:333 Wann, S.R., 10:153 Watkins, C.B., 11:289 Watson, G.W., 15:1 Webster, B.D., 1:172; 13:xi Weichmann, J., 8:101 Wetzstein, H.Y., 8:217 Whiley, A.W., 17:299 Whitaker, T.W., 2:164 White, J.W., 1:141 Williams, E.G., 12:1 Williams, M.W., 1:270

CUMULATIVE CONTRIBUTOR INDEX

Wismer, W.V., 17:203 Wittwer, S.H., 6:xi Woodson, W.R., 11:15 Wright, R.D., 9:75 Wutscher, H.K., 1:237 Yada, R.Y., 17:203 Yadava, D.L., 2:1 Yahia, E.M., 16:197

379

Yan, W., 17:73 Yarborough, D.E., 16:255 Yelenosky, G., 7:201 Zanini, E., 16:71 Zieslin, N., 9:53 Zimmerman, R.H., 5:vii; 9:273 Zucconi, F., 11:1