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

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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.

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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.

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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

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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:

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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

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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

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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

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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).

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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.

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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

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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

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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

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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

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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.

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(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