Horticultural Reviews (Volume 19)

HORTICULTURAL REVIEWS Volume 19

Horticultural Reviews is sponsored by: American Society for Horticultural Science

Editorial Board, Volullle 19 Wanda W. Collins Wouter G. van Doorn Yosef Mizrahi

HORTICULTURAL REVIEWS Volume 19

edited by

Jules Janick Purdue University

John Wiley 8' 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 0-471-16529-8 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.

IV. V. VI. VII.

2.

Introduction Pollination-Induced Perianth Senescence Pollination Regulation of Floral Pigmentation Changes Pollination Regulation of Ovary Development Pollination Regulation of Male and Female Reproductive Development Coordinate Regulation of Postpollination Gametophyte Development Conclusion Literature Cited

Speciality Mushrooms and Their Cultivation Daniel J. Royse I. II. III. IV. V.

3.

xiii

Postpollination Flower Development Sharman D. O'Neill and Jeanette A. Nadeau I. II. III.

ix

1

2 4 25 26 28 42 43 44

59

Introduction Spawn Mushroom Production Technology Marketing Future Prospects Literature Cited

60 61 62 90 92 93

Glucosinolates in Crop Plants E. A. S. Rosa, R. K. Heaney, G. R. Fenwick, and C. A. M. Portas

99

v

vi

CONTENTS

Introduction Biodistribution, Chemistry, and Biochemistry Analysis Levels of Glucosinolates in Plants Factors Associated With Glucosinolate Variation Biological Effects Flavor Coneluding Remarks/Prospects Literature Cited

100 101 116 124 132 148 180 182 183

4. Fruit Skin Splitting and Cracking Linus U. Opara, Clifford J. Studman, and Nigel H. Banks

217

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

I. II. III. IV. V. 5.

Origin and Dissemination of Cherry Miklos Faust and Dezso Suranyi

I. II. III. IV. V. VI. VII. VIII. IX. X. 6.

Introduction Types of Cracking Causes of Cracking and Splitting Reducing Fruit Cracking Summary and Conelusions Literature Cited

Introduction Classification of Cultivated Cherries The Native Home of Sweet and Sour Cherries Early Records of Cherry Cultivars The Modern Era, Cherries in the Twentieth Century Cultivar Improvement Rootstock Improvement Japanese Cherries and Their Movement Into America Worldwide Dissemination and Production of Cherries Conelusion Literature Cited

Artemisia annua: Botany, Horticulture, Pharmacology Jorge F. S. Ferreira, James E. Simon, and Jules Janick I.

Introduction

218 225 227 242 248 252 263

264 264 271 278 298 303 305 307 309 311 313 319

320

CONTENTS

II. III. IV. V. 7.

vii

Botany Horticulture Pharmacology of Artemisinin and Derivatives Conclusions Literature Cited

Opium Poppy (Papaver somniferum): Botany and Horticulture

322 337 346 359 362

373

Peter Tetenyi L II. III. IV.

Introduction Botany Horticulture Summary and Prospects Literature Cited

Subject Index Cumulative Subject Index Cumulative Contributor Index

373 375 391 404 405 409

411 437

Contributors

Nigel H. Banks, Department of Plant Science, Massey University, Private Bag 11-222, Palmerston North, New Zealand Miklos Faust, Fruit Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, Beltsville, Maryland 20705 G. R. Fenwick, Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich Research Park, NR4 TUA, United Kingdom Jorge F. S. Ferreira, Center for New Crops and Plant Products, Department of Horticulture, Purdue University, West Lafayette, Indiana 47907-1165 R. K. Heaney, Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich Research Park, NR4 TUA, United Kingdom Jules Janick, Center for New Crops and Plant Products, Department of Horticulture, Purdue University, West Lafayette, Indiana 47907 Robert J. Knight, University of Florida, Tropical Research and Education Center, 18905 SW 280 St., Homestead, Florida 33031 Jeanette A. Nadeau, Department of Plant Biology, Ohio State University, 1735 Neil Avenue, Columbus, Ohio 43210-1293 Sharman D. O'Neill, Section of Plant Biology, Division of Biology Sciences, University of California, Davis, California 95616 Linus U. Opara, Department of Agricultural Engineering, Massey University, Private Bag 11-222, Palmerston North, New Zealand C. A. M. Portas, Horticulture Section, Instituto Superior de Agronomia, 1100 Lisbon, Portugal E. A. S. Rosa, Horticulture Section, Universidade de Tras-os-Montes e Alto Douro, Apt. 202, 5001 Vila Real, Portugal Daniel J. Royse, Mushroom Research Center, Department of Plant Pathology, 316 Buckhout Laboratory, Pennsylvania State University, University Park, Pennsylvania 16802-4508 James E. Simon, Center for New Crops and Plant Products, Department of Horticulture, Purdue University, West Lafayette, Indiana 47907-1165

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CONTRIB UTORS

Clifford J. Studman, Department of Agricultural Engineering, Massey University, Private Bag 11-222, Palmerston North, New Zealand Dezso Suranyi, Fruit Research Station, Cegled, Hungary Peter Tetenyi, Research Institute for Medicinal Plants, Budakalasz H-1143 Budapest, Stefania Str. 9, Hungary

HORTICULTURAL REVIEWS Volume 19

Carl w. Campbell

Dedication: Carl W. Campbell Probably no horticulturist of this generation in North America has made a greater contribution to the collection and dissemination of current information on tropical and subtropical fruit crops than Carl Campbell, a contribution that happily continues. An authority on tropical fruit cultivars and mango culture, Carl Campbell has assumed a leadership role in promoting horticultural education and understanding throughout tropical America. Carl Walter Campbell was born in Decatur, Illinois, in 1929 and was raised on a grain and cattle farm. He received a B.Sc.Ed. degree from Illinois State University in 1951 and a masters degree from Kansas State University in 1952. Carl fulfilled his military service obligation as a soldier-scientist at the Army's biology laboratory in Fort Detrick, Maryland, for two years and then entered Purdue University, where he was awarded the Ph.D. degree in 1957 under Carl Leopold. In 1957, Dr. Campbell joined USDA's Agricultural Marketing Service at Chapman Field, Miami (then called the U.S. Plant Introduction Station), where he received his first exposure to the vast array of tropical fruits grown in subtropical Florida. This was a case of the right person coming to the right place at the right time. Acreages in limes, avocados, mangos, and other tropical crops were increasing rapidly and there was a corresponding need for more intensive horticultural research. As a part of the expansion of the University of Florida's work at Homestead, Dr. Campbell joined the Subtropical Experiment Station as a horticultural researcher in 1960, and in 1981 assumed extension duties as well. Carl retired in 1988, but continues as an active consultant in over 20 countries. His activity as a crops consultant in Latin America has had a major influence on production of the mango, now available in international markets throughout most of the year. While at Homestead, Carl introduced improved cultivars of avocado, carambola, guava, sapodilla and mamey sapote, and authored publications covering most aspects of tropical fruit culture. He continues to provide valuable information on cultivars and on the production of tropical fruits hitherto little known in the xiii

xiv

DEDICATION

United States. The unadorned style of his prose has made his publicatons particularly valuable for educating students new to tropical horticulture as well as for growers in need of straight facts. In 1981 and 1988 he was visiting professor at the Escuela Agricola Panamericana at Zamorano, Honduras, episodes mutually gratifying to the Campbell family and the students there. While at Homestead he taught a popular biennial summer course in tropical fruit production, attended by a mixed clientele of students of local, national, and international origin. Many of his students have since assumed important positions in education, public service, and commerce throughout the Americas. Carl Campbell is a renaissance man. He came to Florida competent in French and German and is now sufficiently fluent in Spanish to deliver technical lectures and conduct meetings. He is an authority on native Americans and their artifacts and has become an authority on the local flora and fauna of southern Florida. For some years he was an active member of an informal group called the Native Plant Workshop, and was involved with the Nature Conservancy. For his positive effect in the community, he received the Dade County AGRlcouncil HAg Pioneer" Award in 1996. Carl has been active in the Florida State Horticultural Society, was its president in 1984, and received its Presidential Gold Medal in 1988. He was elected a Fellow of the American Society for Horticultural Science in 1986. In addition, he plays the acoustic guitar well and, with encouragement, can sing a wide variety of ballads and traditional songs with the ring of authenticity. Carl has been an influential member of the Tropical Region (formerly Caribbean Region) of the American Society for Horticultural Science, known since 1986 as the Interamerican Society for Tropical Horticulture. The organization would not have survived in its present healthy condition without the active support and nurture of Carl and his wife Becky who have toiled in that vineyard for 20 years. Carl and Becky are blessed with five children, and their three sons, Rob L. Campbell, Craig A. Campbell, and Richard J. Campbell, are professional horticulturists. Carl Campbell's long and productive career continues still. We are pleased and honored to dedicate this volume of Horticultural Reviews in recognition of his outstanding contributions to tropical horticulture. Robert J. Knight University of Florida, Homestead

1 Postpollination Flower Development Sharman D. O'Neill and Jeanette A. Nadeau * Division of Biological Sciences Section of Plant Biology University of California Davis, California, USA I. II.

III. IV. V.

VI. VII.

Introduction Pollination-Induced Perianth Senescence A. Ethylene Coordination of Pollination-Induced Flower Senescence B. Signaling Processes Involved in Pollination-Induced Senescence 1. Primary Pollination Signals 2. Secondary Pollination Signals C. Regulation of Ethylene Biosynthesis in Pollinated Flowers 1. Regulation of ACC Synthase Gene Expression in Pollinated and Senescing Flowers 2. Regulation of ACC Oxidase Gene Expression in Pollinated and Senescing Flowers D. Regulation of Gene Expression in Senescing Flowers E. Model for Pollination-Regulated Flower Senescence Pollination Regulation of Floral Pigmentation Changes Pollination Regulation of Ovary Development Pollination Regulation of Male and Female Reproductive Development A. Female Reproductive Development in Orchids 1. Range of Developmental States Prior to Pollination 2. Significance of Delayed Female Reproductive Development 3. Ultrastructural, Biochemical, and Genetic Studies of Ovule Development 4. Orchids as a Model System to Study Ovule Development B. Other Examples of Postpollination Female Reproductive Development C. Male Reproductive Development Coordinate Regulation of Postpollination Gametophyte Development Conclusion Literature Cited

* The authors express their appreciation to A. H. Halevy, W. R. Woodson, and J. A. Arditti for their helpful comments and review.

Horticultural Reviews, Volume 19, Edited by Jules Janick ISBN 0-471-16529-8 © 1997 John Wiley & Sons, Inc.

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I. INTRODUCTION

Flower development can be readily divided into prepollination and postpollination stages. These two major stages are defined both temporally and functionally in that the first stage of prepollination prepares the floral organs for pollen dispersal and reception while the second postpollination stage sheds certain floral organs and prepares others for fertilization, embryogenesis, and fruit development. The postpollination stage includes a suite of developmental changes, including perianth senescence, pigmentation changes, ovary maturation, and, in some species, ovule differentiation and female gametophyte development. Each of these processes is central to successful sexual plant reproduction and because they occur in distinct floral organs implicate the involvement of interorgan signals that serve to coordinate the overall process of postpollination development. Figure 1.1 summarizes the physiological processes that are pollination regulated in the three major sets of floral organs and illustrates the interorgan signaling that occurs to coordinate these developmental events. In some species of flowering plants, postpollination development is entirely dependent on pollination, whereas in many others, pollination serves to accelerate rather than induce developmental changes that are already occurring in the ovary and perianth and that will proceed anyway in the unpollinated flower. Certain orchid species represent examples of flowers in which postpollination development is precisely and completely triggered by pollination. This postpollination developmental syndrome of orchids includes the induction of perianth senescence, the induction of ovary and ovule development in preparation for fertilization, as well as changes in the pigmentation of floral organs, presumably to signal pollinators that a flower has already been visited (Van der Pijl and Dodson 1966; Arditti 1979; Dressler 1982). In the absence of pollination, the orchid flower will not exhibit these developmental changes as it ages and in the absence of pollination, many orchid flowers exhibit a lifespan of many days or months, depending on the species, prior to wilting (Goh et al. 1985). This unusual and extreme dependence on pollination to induce perianth senescence led Charles Darwin to propose in his treatise on orchid flower pollination by insects that the extraordinary longevity of orchid flowers was an adaptation to pollination by highly specific insect vectors, which increased the likelihood that a specific pollinator would eventually find the particular flower with which it had coevolved (Darwin 1862). The highly regulated postpollination developmental responses of orchid flowers has provided

1.

POSTPOLLINATION FLOWER DEVELOPMENT

I.-S_t_i9_m_al.-l---1

Perianth

1

Ethylene biosynthesis Changes in protein profile and gene expression Hyponastic behavior Changes in pigmentation Protein degradation Ubiquitin Other systems Loss of membrane integrity Abscission

3

Changes in secretions Swelling of cells Ethylene biosynthesis Changes in protein profile and gene expression Abscission of stigma

Ovary

1

Morphological changes associated with fruit development Hair cell growth Swelling of ovary wall cells Changes in pigmentation Changes in protein profile and gene expression Ovule development

Fig. 1.1. The effect of pollination on physiological and developmental processes in different organs of the flower.

an excellent biological model to elucidate the mechanisms of interorgan floral regulation. Although orchids are extreme in their complete dependence on pollination to trigger postpollination developmental events, many flowers also exhibit an acceleration of developmental events, such as perianth senescence, following pollination. The information obtained from orchid flowers has thus provided valuable insights that

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appear to be applicable to a wide variety of flowers. In this review, we have surveyed information from a variety of flowers, but, for the reasons discussed above, this review relies to a great extent on data and reports that utilize orchid flowers as the experimental system. Because one of the important postpollination developmental events is perianth senescence, this review will overlap with previous reviews on the senescence and postharvest physiology of flowers that have appeared in earlier volumes of this series (Halevy and Mayak; 1979; 1981; Borochov and Woodson 1989). II. POLLINATION-INDUCED PERIANTH SENESCENCE

Senescence is a developmentally regulated process that terminates the life of floral organs by initiating a coordinated change in cellular metabolism that ultimately leads to loss of cellular compartmentation and death (Nooden 1988). Senescence of certain floral organs, most notably the perianth, is a prominent part of the postpollination syndrome of developmental events. Perianth senescence is not pollination-triggered in all flowers; instead, mostflowers appear to senesce as part of a temporal program. For example, daylily flowers senesce within 12 to 18 h after flower opening regardless of their pollination status (Lukaszewski and Reid 1989; Lay-Yee et al. 1992). Other flowers, such as petunia, gradually senesce over a period of days after flower opening, but this process is accelerated by pollination (Halevy and Mayak 1979; Halevy 1986; Stead 1992). The type of response to pollination varies; typical responses include flowers in which perianth parts wilt but do not abscise (Cymbidium, Dianthus, Lupinus, Phalaenopsis and related hybrids, and Vandal, flowers in which the perianth wilts and later abscises (Ipomoea, Petunia, Pharbitis, and Rosa), and flowers in which the perianth abscises before wilting (Alstroemeria, Antirrhinum, Cyclamen, Delphinium, Digitalis, Geranium, Lathyrus, Linum, Mimulus, Papaver, Paphiopedilum, and Pelargonium). As with other aspects of postpollination development, the most extreme example of pollination-regulated flower senescence is found in certain orchid species where the perianth' of the flowers remains fresh for many days, weeks, or even months in the absence of pollination but begins to senesce shortly after pollination (noticeably within 24 h). In flowers that exhibit either pollination-dependent or pollination-accelerated senescence, pollination leads to a rapid increase in ethylene production resembling the climacteric peak observed in ripening fruits (Halevy et al. 1984; Brady 1987;

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POSTPOLLINATION FLOWER DEVELOPMENT

5

O'Neill et al. 1993). Thus, it is possible that the mechanisms underlying pollination-regulated senescence in various species are fundamentally similar. The senescence and postharvest physiology of flowers have been comprehensively reviewed in several earlier volumes of this series (Halevy and Mayak 1979,1981; Borochov and Woodson 1989). This part of the review focuses specifically on the relationship between pollination and the initiation of floral organ senescence with specific reference to orchid flowers. A. Ethylene Coordination of Pollination-Induced Flower Senescence Although some flowers senesce in an ethylene-independent manner (Halevy 1986), in many plants, including petunia, carnation, cyclamen, and orchids, flower senescence is accompanied by a sudden and rapid increase in endogenous ethylene production (Nichols 1966; Hall and Forsyth 1967; Nichols 1968; Bufler et al. 1980; Whitehead et al. 1983b; Nichols et al. 1983; Halevy et al. 1984; Porat et al. 1994a). This is certainly the case in many orchid flowers, and indeed orchid species have figured prominently in the study of the role of ethylene in flower senescence. One of the earliest reports linking ethylene production and flower senescence involved the production of ethylene by fading flowers of the orchid Yanda cv. Miss Agnes Joaquim (Akamine 1963). In this Yanda cultivar, the color of the perianth changes from pinkish purple to white during normal senescence, constituting a floral color fading. This natural floral color fading was accelerated by exogenous ethylene; thus it was proposed that endogenous ethylene production triggered the color fading. Akamine (1963) measured the ethylene production by the flower using the perchlorate manometric method and correlated changes in ethylene production with the degree of color fading. Following emasculation to induce premature fading, ethylene production increased for 32 h, reaching a peak rate of 3400 nL/g h- 1 at a time that correlated with the maximum degree of color fading. The high rate of ethylene production reported by Akamine (1963) in the fading Yanda flower is often cited as the highest reported rate of ethylene evolution of any plant organ or tissue (Arditti and Harrison 1979; Goh et al. 1985), although attempts to corroborate this high level of ethylene production in Yanda cv. Miss Agnes Joaquim using gas chromatographic assays of ethylene indicated that actual rates of ethylene production by this flower are actually about 100-fold lower than were previously reported (Goh et al. 1985). The effects of exogenous ethylene in this Yanda cultivar could be mimicked by emasculation or by

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pollination alone, treatments that stimulate ethylene production in this experimental system (5. D. O'Neill, unpublished). Ethylene plays an important role in coordinating senescence-related processes in many other orchid species as well. This is supported by the findings that emasculation, pollination, and treatment with auxin, ACC, or by wounding of orchid flowers stimulate ethylene production and induce floral color change in several orchid genera, including Arachnis, Aranda, Cattleya, Cymbidium, Dendrobium, Paphiopedilum, Phalaenopsis and Vanda (Davidson 1949; Oertli and Kohl 1960; Akamine 1963; Burg and Dijkman 1967; Dijkman and Burg 1970; Arditti et al. 1973; Goh et al. 1985; Nair and Fong 1987; Yip and Hew 1988; Woltering 1989; Hew et al. 1989; Nair 1990; Nair et al. 1991; O'Neill et al. 1993; Woltering 1994). Orchids have generally been considered to be among the most sensitive flowers to ethylene. This high sensitivity was first noted by Davidson (1949), who reported that treatment of two species of Cattleya flowers with as little as 2 ppb of ethylene for 24 h induced senescence symptoms that appeared identical to "dried-sepal injury," which developed on the perianth parts of orchid flowers and presumably was caused by contaminating ethylene in the greenhouse atmosphere. More comprehensive studies have demonstrated large variability in sensitivity to ethylene among different orchid species (Goh et al. 1985). Among those species examined, Vanda cv. Miss Joaquim was reported to be the most sensitive to ethylene treatment and, interestingly, it also produced the highest levels of endogenous ethylene as discussed previously. Cymbidium, Cattleya, and Paphiopedilum were moderately sensitive to ethylene, while Dendrobium and Oncidium were relatively insensitive. These results confirmed the role of ethylene in regulating orchid flower senescence but did not support the general notion that orchids are particularly sensitive to ethylene, relative to other flowers. In several studies propylene was used to determine the effects of ethylene on the senescence of excised flowers. Propylene, an ethylene analog, is believed to interact with the ethylene receptor with an approximately 100-fold reduced affinity and has been used to induce ethylene responses in plant tissues while still monitoring endogenous ethylene production (Burg and Burg 1966; Burg and Dijkman 1967; McMurchie et al. 1972). In carnation and Phalaenopsis flowers, treatment with exogenous propylene promoted a pattern of sustained ethylene production and symptoms ofperianth senescence that were similar to the endogenous ethylene production induced by pollination (Nichols 1977; O'Neill et al. 1993). These results sup-

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POSTPOLLINATION FLOWER DEVELOPMENT

7

port the conclusion that ethylene alone, or its analog, is sufficient to promote the pattern of ethylene production and perianth senescence observed in pollinated Phalaenopsis flowers as well as in aging carnation flowers. Pollination also provides the signal for rapid petal or corolla abscission, Le., "shattering," in a number of horticulturally important species. Flowers exhibiting pollination-related abscission include Cyclamen, Digitalis, and Geranium and its close relative, Pelargonium (Stead and Moore 1979, 1983; Halevy et al. 1984; Deneke et al. 1990; Evensen 1991). This subject has been reviewed earlier (Reid 1985; Leshem et al. 1986) and is presented only briefly here. As is the case for pollination-regulated perianth senescence, there appears to be an equally strong correlation between ethylene production and petal or corolla abscission. One of the best examples of this phenomenon is that of Digitalis purpurea (Stead and Moore 1979, 1983). In Digitalis, unpollinated flowers undergo an endogenous aging process, losing corolla composition and turgor for a period of approximately 6 days before corolla abscission occurs. Pollination, on the other hand, results in rapid corolla abscission. Pollination of the receptive stigma of the flower leads to a rapid and progressive weakening of the abscission zone between the corolla and receptacle and the final abscission of the fully turgid corolla by 24 h after pollination (Stead and Moore 1979). Pollination-dependent changes in the strength of the abscission zone were first observed within 8 h of pollination, at the time when the pollen tubes were still growing through the stigma but had not yet reached the ovules for fertilization. It was proposed that an abscission zone "weakening substance" or "stimulus" is transmitted from the stigma, via the style and ovary, to the abscission zone at a rate of 4 mm per hour (Stead and Moore 1979). Surgical manipulation experiments suggested that the stimulus is produced in the stigma and style tissue, a situation analogous to that proposed for the pollination-induced production of a "wilting factor" in Petunia (Gilissen 1976). Interestingly, weakening of the abscission zone required a continuous supply of weakening substance up to 14 h after pollination, suggesting that critical synthesis and translocation of the stimulus have already been completed by that point. As with pollination-induced perianth senescence in general, ethylene can mimic the effect of pollination by inducing petal or corolla abscission. This is most clearly observed in species that exhibit high sensitivity to ethylene, for example, in Pelargonium and the related genus, Geranium. In both these cases, petal abscission

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begins within one to a few hours after treatment with exogenous ethylene (Sexton et al. 1983; Deneke et al. 1990). Ethylene production from the gynoecium was markedly stimulated by pollination and preceded petal abscisssion (Deneke et al. 1990). In the most extreme example of Pelargonium xdomesticum, petal abscission began approximately 1 h after ethylene exposure (Evensen 1991). From these studies it can be inferred that ethylene is the coordinating signal involved in the pollination-induced abscission response. B. Signaling Processes Involved in Pollination-Induced Senescence

The stigma is the initial site of perception of the pollination event, suggesting that a pollen-born molecule or associated event is responsible for triggering pollination responses, including ethylene production and the initiation of perianth senescence. The initial perception of the pollination event is accompanied by a rapid increase in ethylene production by the stigma and style within hours after pollination (Lipe and Morgan 1973; Gilissen 1976, 1977; Nichols 1977; Mor et al. 1985; Lovell et al. 1987a, b; Gard 1994). In self-incompatible species, compatible pollination results in a second phase of ethylene production in the perianth, which triggers wilting and senescence of perianth organs distal to the stigma (Linskens 1974; Singh et al. 1992). Thus, the primary pollen signal results in rapid ethylene production in the stigma and style, but this early signal must be transduced and translocated to effect senescence processes in the more distal organs of the flower, such as the perianth. It is possible, and indeed likely, that the primary pollination signal perceived in the stigma is distinct from the subsequent signal, a secondary signal, that transmits and amplifies this primary pollen signal, thus serving to coordinate interorgan postpollination responses (O'Neill 1994). Figure 1.2 illustrates the site of primary pollen signal perception on the stigmatic surface of an orchid flower and the transmission of secondary pollination signals that bring about developmental changes in the perianth and ovary. The identity and role of the primary and secondary pollination signals are reviewed separately below. 1. Primary Pollination Signals. The initial perception of pollination by the stigma has been proposed to result from physical messengers such as pollen contact and pollen tube penetration of the stigma (Gilissen 1976, 1977) or from chemical messengers present on the pollen surface (Whitehead et al. 1983b; Hoekstra and van

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Fig. 1.2. Coordinate regulation of postpollination flower behavior is a two-step process: (A) perception of the primary signal; (B) transmission of the secondary signal to the petals, sepals, and ovary.

Roekel 1988; Singh et al. 1992). In petunia, carnation, orchid, and tobacco flowers, the initial response to pollination is a burst of ethylene production by the stigma. In each case, the production of pollination-associated ethylene precedes germination of the pollen tube, suggesting that physical growth of the pollen tube cannot be the primary signal responsible for the induction of ethylene biosynthesis (Burg and Dijkman 1967; Hall and Forsyth 1967; Nichols 1977; Gilissen and Hoekstra 1984; Hill et al. 1987; O'Neill et al. 1993). Mock pollination using latex beads as a substitute for pollen grains has been reported in a study of the role of the stylar matrix in pollen tube extension in several different higher plants (Sanders and Lord 1989). Mock pollination of orchid flowers with latex beads to the stigma and transmitting tract failed to trigger ethylene production or any other pollination responses, which indicates that physical contact alone is not a sufficient signal to induce any component of the postpollination syndrome (Zhang and O'Neill 1993). While physical contact may contribute to primary pollen perception in some species, the preponderance of results suggest that physical contact between the pollen and stigma or wounding reactions associated with pollen tube growth are not the primary pollination signals that initiate perianth senescence. Considerable experimental attention has thus focused on the identification of a pollen-borne chemical that

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functions as a primary pollination signal. Potential primary pollen signals include 1-aminocyclopropane-1-carboxylic acid (ACC), auxin, proteins, lipids, pectic oligosaccharides, flavonoids, and methyl jasmonate, all of which have been detected in pollen and some of which are also known .inducers of ethylene biosynthesis, making them attractive candidates for the primary pollen signal molecule (Knox and Heslop-Harrison 1970; van der Donk 1975; Ueda and Kato 1981; Yamane et al. 1982; Anderson et al. 1982; Wiermann and Vieth 1983; Weidhase et al. 1987; Dobson 1989; Kandasamy et al. 1990; Ryan and Farmer 1991; Felix et al. 1991; Mo et al. 1992; Vogt et al. 1994). Shortly after the identification of ACC as the immediate biochemical precursor of ethylene (Adams and Yang 1979), several reports identified its presence in pollen and suggested that ACC may be the primary pollen signal (Whitehead et al. 1983a; Reid et al. 1984). The role of pollen-borne ACC in triggering the initial burst of ethylene production and its potential translocation to other floral organs has now been extensively studied, particularly in petunia and carnation (Whitehead et al. 1983a, b; Whitehead et al. 1984a, b). In spite of its presence in relatively large quantities in some pollen (Whitehead et al. 1983a; Reid et al. 1984; Singh et al. 1992), the role of ACC in supporting the initial ethylene production in the stigma has been questioned on several grounds. ACC content of pollen from various sources is not well correlated with the level of ethylene production following pollination (Hoekstra and Weges 1986). In addition, treatment of the stigma with an inhibitor of ACC synthase, aminoethoxyvinylglycine (AVG), prior to pollination prevented pollination-induced ethylene production (Hoekstra and Weges 1986; Zhang and O'Neill 1993; O'Neill et al. 1993; Woltering et al. 1993), suggesting that pollination-induced ethylene production is derived from endogenous production of ACC by the ACC synthase enzyme rather than from exogenous pollen-borne ACC. It has also been suggested that the quantity of pollen-borne ACC would be vastly insufficient to support the amount of ethylene produced in the stigma following pollination (Stead 1992). Two reports also indicate that diffusion of ACC from the pollen is likely to be restricted under conditions that prevail in vivo, which would further restrict its availability to support ethylene production in the stigma (Pech et al. 1987; Hoekstra and van Roekel 1988). These results contrast with those reported by Singh et al. (1992), who demonstrated that an initial peak of pollination-induced ethylene production was not inhibited by a different inhibitor of ACC synthase, aminooxyacetic acid (AOA).

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In addition, wide variations in pollen-borne ACC content in different petunia genotypes were shown to be correlated with the magnitude of the peak of initial ethylene biosynthesis, suggesting that pollen-borne ACC was the substrate for this initial ethylene production (Singh et al., 1992). Although the preponderance of data suggests that pollen-borne ACC is not directly responsible for all of the pollination-associated ethylene production in the stigma, there are conflicting data. It appears likely that exogenous pollen-borne ACC may be responsible for initating ethylene production in the stigma, which is then enhanced by autocatalytic endogenous production of ACC. The role of pollen-borne ACC in promoting the full syndrome of perianth senescence has also been tested by direct application of ACC to the stigma (Reid et al. 1984; Hoekstra and Weges 1986; O'Neill et al. 1993) and in each case, it promoted an initial burst of ethylene production but did not accelerate wilting and perianth senescence unless extremely high concentrations were used. A rigorous analysis of the ACC content of orchid pollen has not been reported. Thus far it has not been detected in Phalaenopsis pollen in spite of the fact that pollination induces rapid and high levels of ethylene production in the stigma (O'Neill et al. 1993). In petunia, Singh et al. (1992) reported that compatible pollination elicited two phases of ethylene production and caused rapid perianth senescence (36 h), whereas incompatible pollination triggered only the first phase of ethylene production and perianth senescence was delayed. Because the pollen used for both compatible and incompatible pollinations contained ACC, other factors appear to be important for the induction of perianth senescence. Collectively, the data suggest that pollen-borne ACC is not a universal primary pollen signal, because it appears to be absent in pollen of some flowers, such as orchid, that exhibit strong postpollination responses. In spite of its presence at relatively high levels in the pollen of certain species, it appears unlikely to be freely diffusible in vivo or to be present in quantities sufficient enough to serve directly as the sole substrate for the early phase of ethylene production following pollination. Nevertheless, even small amounts of ACC may trigger autocatalytic ethylene production in the stigma, and thus pollen-borne ACC may play an important role in some species in triggering the initial burst of ethylene production. Using the Phalaenopsis orchid flower as a model system to bioassay for the primary pollen signal, a number of potential primary pollen signals have been tested for their capacity to induce ethylene production and stigma closure, two early postpollination responses

12

S. D. O'NEILL AND ]. A. NADEAU

(J. A. Nadeau and S. D. O'Neill, unpublished). Using this system, pollen-derived proteins, systemin (Pearce et a1. 1991), certain flavonoids, methyl jasmonate, and jasmonic acid have been eliminated as likely candidates because they failed to elicit ethylene production and/or stigma closure within a time frame consistent with a role in pollination signaling. Only auxin, provided as either IAA or NAA, was active in triggering ethylene production and stigma closure in this orchid bioassay (Zhang and O'Neill 1993). This result is consistent with a long history of the association of auxin with orchid pollen, beginning with the report of Fitting (1909, 1910) who recognized that Phalaenopsis pollen contained a substance that caused wilting of the flower which he termed the "pollenhormon." The orchid pollen extract was later shown to contain auxin, and it is likely that this is the active component of Fitting's "pollenhormon" (Muller 1953). Even before auxin was identified in orchid pollen, it was demonstrated that exogenous application of auxin could mimic pollination and initiate most postpollination developmental events (Hubert and Maton 1939; Curtis 1943). The capacity of auxins to initiate postpollination developmental events in orchids has also been demonstrated in numerous subsequent studies (Curtis 1943; Burg and Dijkman 1967; Arditti and Knauft 1969; Arditti 1971; Arditti et a1. 1971, 1973; Chadwick et a1. 1980; Strauss and Arditti 1984). Indeed, Burg and Dijkman (1967) provided experimental evidence that auxin provided the necessary physiological link between pollination and ethylene production that led to floral color fading in Vanda orchids. A model of auxin as the primary pollination signal was suggested by Burg and Dijkman (1967), who proposed that, following pollination, auxin was transferred from the pollen to the stigma and eventually diffused to the column and labellum where it promoted ethylene production that became autocatalytic throughout the flower, leading to perianth senescence. Thus, auxin was viewed as the primary pollen signal with auxin-stimulated ethylene production being the causal agent in perianth senescence. Similar evidence that auxin stimulated ethylene production in orchids (Arditti et a1. 1973; Chadwick et a1. 1980) further supported the Burg and Dijkman (1967) hypothesis of auxin as the primary pollen signal in orchid perianth senescence. While the role of auxin as the primary pollen signal has been consistently supported, recent evidence argues against the idea that auxin diffuses to the perianth where it directly stimulates ethylene production. In spite of the evidence that auxin is present in orchid pollen and that it may act as the primary pollen signal, the levels of free auxin

1.

POSTPOLLINATION FLOWER DEVELOPMENT

13

(Le., unconjugated) in orchid pollen may be insufficient to be the sole primary pollen signal. For example, we have found that exogenous application of 10 nmol of IAA per stigma is required to induce complete stigma closure and rates of ethylene production comparable to those induced by pollination, yet approximately 100 times less free IAA was eluted from Phalaenopsis pollinia pairs (S. D. O'Neill, unpublished). It is possible that pollen contains other forms of auxin, such as auxin conjugates, or other pollen-borne factors, that may participate synergistically with auxin to elicit postpollinaHon responses in orchid. As suggested by Arditti (1971), the "pollenhormon" described by Fitting (1909) may indeed be a mixture of biologically active molecules. In this regard, a number of candidate signal molecules should be examined, including jasmonic acid, which has recently been demonstrated to hasten flower senescence by enhancing ethylene production (Porat et al. 1993) and pectic cell wall fragments, potentially produced by pollen-derived polygalacturonase and pectate lyase or deposited by the tapetum (Wing et al. 1989; Brown and Crouch 1990), and which has been shown to elicit ethylene production (Tong et al. 1986). 2. Secondary Pollination Signals. Following the initial perception of pollination at the stigma, the signal is transduced and translocated to bring about metabolic changes in the perianth and ovary. This serves to coordinate senescence of the perianth with growth of the pollinated ovary. Such interorgan regulation of the postpollination response suggests the production of a transmissible signal that moves from the stigma, through the style, to other floral organs. Early evidence for this secondary pollination signal came from surgical experiments demonstrating that a mobile wilting factor is transmitted through the style to the corolla of petunia flowers within 6 h after pollination (Gilissen and Hoekstra 1984). Stylar exudates have also been reported to promote perianth senescence in petunia and carnation, implicating the role of a chemical messenger either produced in the style or translocated through it (Sacalis et al. 1983; Gilissen and Hoekstra 1984). Because auxin, ethylene, and ACC have all been implicated in primary pollen signaling, these same molecules have been extensively evaluated as potential transmissible signals in pollinated flowers. The model of Burg and Dijkman (1967), which emphasized the role of auxin as the primary pollen signal in Yanda orchid flowers, also proposed that auxin diffused to the labellum where it triggered ethylene biosynthesis. It was further proposed that ethylene produced

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in the labellum triggered autocatalytic ethylene production in the petals and sepals. This model was supported by the demonstration that [14C] IAA applied to the stigma was mobilized to the column and labellum but very little reached the sepals or petals. Subsequent studies in other orchid species indicated that [14C] IAA applied to Angraecum and Cattleya stigmas was largely immobilized at the point of application with translocation being primarily to the ovary. Because the pollination signal spreads to distal floral organs (petals and sepals) much faster than exogenously applied [14C]IAA it was concluded that auxin is unlikely to be the secondary pollination signal that regulates perianth senescence (Strauss and Arditti 1982). However, the data that auxin is translocated primarily to the ovary (Strauss and Arditti 1982) are consistent with other evidence that auxin translocated from the stigma may specifically contribute to the regulation of postpollination developmental events in that organ. Zhang and O'Neill (1993) examined factors responsible for pollination induction of ovary development by monitoring the differentiation of hair cells from the inner epidermis of the ovary wall, the earliest morphological change associated with pollination. Treatment of the stigma with ACC alone stimulated ethylene production, and eventually senescence of the ovary, but did not promote hair cell growth, whereas treatment with NAA promoted hair cell elaboration in the ovary (Zhang and O'Neill 1993). The effect of pollination or NAA application on ovary hair cell elaboration was reversed by simultaneous treatment with N-1-naphlylphthalamic acid (NPA), an inhibitor of polar auxin transport, indicating that auxin exerts its effect on ovary development by translocation from the stigma to the ovary (X. S. Zhang and S. D. O'Neill, unpublished). Interestingly, the effect of NAA on ovary hair cell elaboration was also partially reversed by simultaneous application of AVG, an inhibitor of ACC synthase activity (Zhang and O'Neill 1993). This latter result suggested that auxin and ethylene are both required to initiate ovary development following pollination. Thus, in this limited context of ovary development, auxin together with ethylene appears to be an important transmissible signal that propagates the primary pollination signal. Ethylene itself is a potential transmissible signal to floral parts distal from the stigma that could act to promote a number of postpollination developmental events, including perianth senescence. It has been demonstrated that aspiration of ethylene produced in the gynoecium did not delay petal senescence in carnation, indicating that volatile ethylene produced as a result of the primary polli-

1.

POSTPOLLINATION FLOWER DEVELOPMENT

15

nation signal was not the transmissible signal responsible for regulating perianth senescence (Reid et al. 1984). It has been recently suggested, however, that diffusion of ethylene within the intercellular spaces (interstitial ethylene) may function in interorgan communication (Woltering 1990a, 1995; Larsen et al. 1993; Woltering et al. 1994). Internal ethylene in the Cymbidium flower central column was measured at levels up to 15 ppm and treatment of the column with exogenous ethylene increased ethylene concentration in the perianth, consistent with the proposal that interstitial ethylene from the column is translocated directly to the perianth (Larsen et al. 1993). In addition, exogneously applied ACC or its analog, aminoisobutyric acid (AlB) was shown to be essentially immobile in Cymbidium flowers, and excisions in the Cymbidium lip that were designed to aspirate interstitial ethylene delayed pollination-induced changes in flower coloration. These results questioned the role of ACC as a translocatable wilting factor and suggested that ethylene could be translocated between floral organs within tissue interstitial spaces (reviewed for Cymbidium in Woltering [1994]). In spite of the suggestions that ethylene, rather than ACC, is translocated between floral organs, there is substantial experimental support of the proposal that ACC is an important secondary pollination signal that coordinates interorgan postpollination responses. Translocation of ACC was first demonstrated in waterlogged tomato plants and illustrated the potential for transport of this hormone precursor to effect interorgan regulation of growth responses (Bradford and Yang 1980). This experimental result provided a mechanism for interorgan regulation of ethylene-dependent responses that relied on a soluble and translocatable hormone precursor rather than transport of the ethylene itself, which is less amenable to targeted translocation processes because of its gaseous state. Similarly, the translocation of ACC in pollinated flowers provides a potential mechanism for the targeted translocation of a secondary pollination signal. Nichols et al. (1983) measured increased levels of ACC in all flower parts following pollination and suggested that this may result from ACC translocation. Reid et al. (1984) showed that [14C] ACC exogenously applied to the stigma of carnation flowers resulted in the production of 14C-Iabeled ethylene in the gynoecium and perianth. This provided compelling evidence that ACC was translocated from the stigma to the gynoecium and perianth and that it served as a substrate for ethylene biosynthesis in those organs. A detailed study of emasculation-induced senescence of Cymbidium flowers demonstrated that changes in lip coloration, an early morphological marker

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of ethylene-regulated senescence, were triggered by ACC translocated from the central column (Woltering 1990b). In the same series of experiments, measurements of ethylene production at various times following emasculation in excised floral organs indicated that ethylene production increased only in the central column. In contrast, measurements of ethylene production in each floral organ in situ demonstrated that ethylene production increased in all floral organs. The discrepancy between ethylene production observed in situ or in excised floral organs suggests that excision of floral organs removed them from a source of ACC for ethylene production, or of ethylene. A similar observation was made in Phalaenopsis orchid flowers, where ethylene production by the intact flower significantly exceeded the sum of ethylene production by excised floral organs, again suggesting that ethylene production by some organs is dependent on ACC import from other floral parts (O'Neill et al. 1993). Collectively, the results from several systems provide strong evidence that ACC is translocated between floral organs and that it functions as a secondary pollination signal in coordinating postpollination responses. Attempts to identify other potential secondary pollination signals have focused on the identification of pollination-induced ethylene "sensitivity factors." A sensitivity factor can be defined as a physical substance that renders the floral tissue more susceptible to the senescence-inducing effects of exogenous ethylene. The presence of such factors were inferred from experiments that indicated that pollination-induced abscission of Cyclamen petals was prevented by the ethylene response inhibitor silver thiosulfate, but could not be induced by ethylene or ACC treatment of unpollinated flowers (Halevy et al. 1984). These results suggest that transmissible pollination factors are responsible for stimulation of ethylene sensitivity as well as the promotion of ethylene production. Research to characterize transmissible ethylene "sensitivity factors" led to the identification of two short-chain fatty acids, decanoic and octanoic acid, in petunia style eluates which possessed senescence-inducing properties (Whitehead and Halevy 1989). Subsequent reports have not confirmed the effects of octanoic and decanoic acid on senescence of petunia, carnation, or orchid flowers, and the role of these putative ethylene sensitivity factors remains unresolved (Woltering et al. 1993). Pollination-induced ethylene sensitivity was reported to occur in Phalaenopsis flowers within 4 h after pollination, whereas ethylene production could not be detected until 8 to 10 h later (Porat et al. 1994a, 1995). Related studies demonstrated that pollination-

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POSTPOLLINATION FLOWER DEVELOPMENT

17

enhanced ethylene sensitivity in Phalaenopsis petals could be increased by treatment with compounds that modulate the activity of GTP-binding proteins or by treatment with calcium ions and the calcium ionophore A23187 (Porat et al. 1994b). Furthermore, pollination was shown to lead to elevated levels of protein phosphorylation, particularly of a 30-kD polypeptide. These results were interpreted to indicate an involvement of GTP-binding proteins and protein phosphorylation in the regulation of pollination-induced ethylene sensitivity in Phalaenopsis flowers (Porat et al. 1994b). Most recently, research to identify the ethylene sensitivity factor associated with pollination-induced sensitivity has led to the proposal that short-chain saturated fatty acids (SCSFA) are involved (Halevy et aI. 1996). Following pollination in Phalaenopsis flowers, there was a significant increase in the endogenous content of fatty acids in the column and perianth, with octanoic acid being the main SCSFA involved. Also, application of SCSFA to the stigma increases their sensitivity to ethylene in the same way as did pollination itself, leading to the suggestion that SCSFA may be the "sensitivity factors" produced following pollination. Its mode of action would involve affects on membrane fluidity, consequently altering membrane action (Halevy et aI., 1996). The role of increased sensitivity to ethylene following pollination is likely to play an important role in postpollination responses. Although the precise mechanisms underlying the regulation of ethylene sensitivity in pollinated flowers are not yet known, it is likely that the elucidation of the molecular components involved in ethylene perception and ethylene-mediated signal transduction (Chang et aI. 1993; Kieber et aI. 1993; Porat et aI. 1994b) will contribute to a better understanding of this phenomenon.

c.

Regulation of Ethylene Biosynthesis in Pollinated Flowers

The regulation of ethylene biosynthesis is central to the control of perianth senescence in pollinated flowers, and the induction of ethylene production by pollination is well documented. The recent cloning of genes encoding the major biosynthetic steps in ethylene biosynthesis have provided the basis for examining the temporal and spatial regulation of ethylene biosynthesis in flowers and specifically in response to both primary and secondary pollination signals. In addition to providing tools to better understand the regulation of this pathway, the cloning of ethylene biosynthetic genes have provided the means to genetically engineer flowers for enhanced lon-

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gevity as has been done for ripening fruit (Hamilton et al. 1990; Oeller et al. 1991; Theologis 1992). The ethylene biosynthetic pathway as elucidated by Adams and Yang (1979) proceeds by the methylation of methionine to Sadenosylmethionine (AdoMet), transamination of AdoMet to produce ACC, followed by oxidation of ACC to ethylene (Met~AdoMet~ ACC~ethylene). S-Adenosylmethionine serves as a methyl donor in a wide variety of cellular reactions, with a relatively small proportion of the cellular pool of AdoMet being used for ethylene production, even during periods of high demand. Thus, the activity of AdoMet synthetase is generally not considered to be limiting for ethylene production (Lieberman 1979; Yang and Hoffman 1984). This was confirmed in carnation flowers where it was shown that AdoMet synthetase mRNA declined dramatically in aging flowers (Woodson et al. 1992). This decline in AdoMet synthetase gene expression may reflect an overall decline in methyl transferase reactions that rely on AdoMet as the methyl donor. The key enzyme in the ethylene biosynthetic pathway, ACC synthase, is widely regarded as the ratecontrolling step (Kende 1989, 1993). This step is highly regulated at the level of enzyme activity and at the level of gene expression (Kende 1989, 1993). While considered to be constitutive in many tissues, the final enzyme, ACC oxidase, is also regulated at the level of enzyme activity and at the level of gene expression (Kende 1989,1993). The regulation of ACC synthase and ACC oxidase gene expression in pollinated and senescing flowers is summarized below. 1. Regulation of ACC Synthase Gene Expression in Pollinated and Senescing Flowers. Because of its central role in the regulation of ethylene production, ACC synthase has been the subject of intense interest with regard to the senescence of flowers. A number of eDNA clones encoding ACC synthase have been isolated from apple (Dong et al. 1992; Kim et al. 1992), arabidopsis (Liang et al. 1992), carnation (Park et al. 1992), orchid (O'Neill et al. 1993), petunia (Michael et al., 1993), rice (Zarembinski and Theologis 1993), tomato (Lincoln et al. 1987; Sato and Theologis 1989; Van der Straeten et al. 1990; Olson et al. 1991), winter squash (Nakajima et al. 1990), and zucchini (Huang et al. 1991). In nearly all cases, ACC synthase has been found to be encoded by multiple genes which exhibit differential tissue specificity or differential regulation by environmental or hormonal stimuli (Kende 1993). The expression of ACC synthase(s) at the level of mRNA abundance has been studied in senescing carnation flowers and in orchids

1.

POSTPOLLINATION FLOWER DEVELOPMENT

19

in relation to pollination and perianth senescence (Park et al. 1992; Woodson et al. 1992; O'Neill et al. 1993; Henskens et al. 1994). In carnation flowers, ACC synthase mRNA was undetectable in all floral organs immediately after harvest but increased dramatically after 5 to 6 days in petals coincident with the increase in ethylene production. ACC synthase activity in senescing styles was approximately 6-fold greater than in petals, whereas ACC synthase mRNA accumulated to similar levels in both tissues (Woodson et al. 1992). Following pollination, ACC synthase mRNA increased within 12 to 24 h in the gynoecium and petals (Woodson et al. 1992). A subsequent study reported the presence of a second ACC synthase mRNA in carnation flowers that was expressed predominantly in the styles and is likely to account for the apparent discrepancy between stylar ACC synthase mRNA and activity levels relative to that observed in petals (Henskens et al. 1994). Both carnation ACC synthase cDNAs (termed CARACC3 and CARAS1) corresponded to genes whose expression was stimulated by ethylene, suggesting that these genes participate in autocatalytic ethylene production. Relatively low levels of both ACC synthase mRNAs (corresponding to CARACC3 and CARAS1) were detected in ovaries, in spite of high levels of ethylene production in this organ. This result suggests the existence of a third ACC synthase gene that is predominantly expressed in this floral organ and illustrates the complexity in understanding the detailed regulation of ethylene production in floral tissues (Henskens et al. 1994). Expression of ACC synthase genes in pollinated orchid flowers was qualitatively different than that observed in aging carnation flowers (O'Neill et al. 1993). Two highly homologous ACC synthase cDNAs (OASl and OAS2) were initially isolated from orchid flowers and used to probe ACC synthase mRNA abundance following pollination. The results indicated that ACC synthase mRNA corresponding to both OASl and OAS2 accumulated to high levels in the gynoecium by 18 h after pollination, accumulated weakly in labellum tissue over the same time course, but was undetectable in the perianth (excluding the labellum) even at 72 h after pollination (O'Neill et al. 1993). These results were surprising because the perianth is the site of substantial ethylene production during perianth senescence, as determined by an examination of organ ethylene production (O'Neill et al. 1993). ACC synthase activity was also assayed in perianth tissues and found to also be absent, indicating that the failure to detect ACC synthase mRNA in this tissue was not due to failure of the ACC synthase probe to hybridize to a divergent gene

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family member in perianth tissue (A. Q. Bui and S. D. O'Neill, unpublished). Because of the importance of auxin in regulating postpollination responses in orchid flowers and because auxin has been reported to induce expression of certain ACC synthase gene family members in tomato and winter squash (Van der Straeten et a1. 1990; Nakajima et a1. 1990), regulation of the orchid OAS1/0AS2 ACC synthase genes by NAA was evaluated. Although exogenous application of NAA to the stigma strongly induced the accumulation of ACC synthase mRNA, this induction was completely reversed by pretreatment with AVG, indicating that the OAS1 and OAS2 mRNAs were regulated by auxin-induced ethylene and not directly by auxin. This was confirmed by experiments that demonstrated that pollination induction of ACC synthase mRNAs was inhibited by the ethylene antagonist 2,5-norbornadiene (O'Neill et a1. 1993). Because a substantial body of evidence implicates auxin as an important primary pollination signal in orchids, the presence of additional ACC synthase mRNAs expressed in the stigma was exhaustively tested. A divergent ACC synthase cDNA was cloned that corresponds to an auxin-regulated ACC synthase gene in orchid that is expressed in the stigma upon pollination, in response to auxin, and in the presence of pollen or auxin and 2,5-norbornadiene (A. Q. Bui and S. D. O'Neill, unpublished). Taken together, there appear to be at least two different types of ACC synthase genes expressed in pollinated orchid flowers that differ markedly in their spatial and hormonal regulation: one that responds to the primary pollination signal (tentatively identified as auxin), and another that serves to amplify that signal by triggering, and in some instances sustaining, autocatalytic ethylene production. 2. Regulation of ACC Oxidase Gene Expression in Pollinated and

Senescing Flowers. ACC oxidase in most instances is considered to be constitutive (Kende 1989, 1993). However, increases in ACC oxidase have been reported in senescing carnation flower petals (Manning 1985), and ACC oxidase mRNA levels are clearly induced by pollination in Phalaenopsis flowers (Nadeau et a1. 1993), indicating that ACC oxidase levels are highly regulated in floral organs. A carnation cDNA clone (SR120) isolated on the basis of its elevated mRNA abundance during petal senescence was determined to encode ACC oxidase (Wang and Woodson 1991). Accumulation of carnation ACC oxidase mRNA was strongly induced by senescence and ethylene in all floral organs, indicating that it is not constitutive in carnation.

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The ACC oxidase mRNA was undetectable in presenescent petals, ovaries, and receptacles, but was present at significant levels in presenescent styles; transcript levels increased dramatically in abundance following pollination (Woodson et al. 1992). These results are consistent with previous reports that ACC oxidase is present in stigmas prior to pollination and therefore capable of oxidizing pollenborne ACC to ethylene. Pech et al. (1987) reported that ACC oxidase levels in petunia flowers increased markedly during flower development and were maximal shortly after flower opening and prior to the onset of senescence. ACC oxidase activity was most abundant in the petunia flower stigma, and pollination had no effect on its activity. These results were interpreted in light of the potential role of pollen-borne ACC as a primary pollen signal and indicated that the stigma was capable of converting exogenous ACC to ethylene. In contrast to this report of the lack of regulation of ACC oxidase levels in the petunia gynoecium during flower senescence, a recent report indicated that ACC oxidase levels in the petunia flower corolla increased dramatically during senescence (Tang et al. 1993). Recently, a family of petunia ACC oxidase genes have been cloned (Tang et al. 1993) and their expression in petunia flowers has been studied in detail (Tang et al. 1994). Four ACC oxidase genes were characterized but only three were found to be actively transcribed. These expressed ACC oxidase genes were termed AC01, AC03, and AC04. In agreement with Pech et al. (1987), each of the three ACC oxidase mRNAs accumulated in the gynoecium during early flower development, reaching their maximal levels at the time of flower opening (Tang et al. 1994). In addition, ethylene treatment enhanced the accumulation of all three ACC oxidase mRNAs in the pistil tissues. Interestingly, the AC01 mRNA transcript also accumulated in all other floral organs (sepals, corolla, anthers, and ovary) in response to ethylene, but accumulation of the AC03 and AC04 mRNA transcripts was restricted to the pistil (Tang et al. 1994). Spatial localization of the ACC oxidase mRNAs was determined by in situ hybridization and found to be expressed in the stigmatic region of the pistil and in secretory cells associated with the nectary and receptacle. In the presence of ethylene, ACC oxidase mRNA levels increased dramatically, and in situ hybridization indicated that the localization of ethylene-induced ACC oxidase mRNAs was more uniformly distributed throughout the pistil tissue (Tang et al. 1994). The effect of pollination on the expression of the petunia ACC oxidase genes was not investigated.

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S. D. O'NEILL AND ]. A. NADEAU

The regulation of ACC oxidase activity and of ACC oxidase mRNA accumulation has also been characterized in orchid flowers (Nadeau et al. 1993; O'Neill et al. 1993). Unlike petunia, ACC oxidase activity was not detected in mature flowers prior to pollination but was induced rapidly following pollination (Nadeau et al. 1993). Following pollination, orchid ACC oxidase (OA01) mRNA was found to accumulate to high levels in all floral organs within 48 h (Nadeau et al. 1993). This result contrasted with the expression of ACC synthase mRNA in orchid flowers, which failed to accumulate in the perianth (O'Neill et al. 1993) and suggested that the sepals and petals developed the capacity to oxidize ACC to ethylene but could not synthesize ACC endogenously (Nadeau et al. 1993). Further experiments indicated that ACC oxidase mRNA levels, as with ACC synthase mRNA accumulation, were regulated by ethylene produced in the flower following pollination (O'Neill et al. 1993). Spatial localization of ACC oxidase mRNA accumulation by in situ hybridization indicated that the transcript accumulated in all living cells of the orchid gynoecium, including the stigmatic surface, transmitting tract cells, parenchyma, phloem tissues, and epidermal papillae (Nadeau et al. 1993). Careful analysis of in situ hybridization sections also indicated that low levels of ACC oxidase mRNA were present in the stigma of unpollinated flowers that were not detectable by RNA gel blot hybridization analysis. Thus, by inference, it is likely that a low level of ACC oxidase activity was present in the stigma prior to pollination. D. Regulation of Gene Expression in Senescing Flowers

Perianth senescence is a coordinated developmental process that serves, in part, to remobilize nutrients from the petals to the developing ovary. This process is active and is brought about by changes in gene expression (Woodson and Lawton 1988; Borochov and Woodson 1989; Lawton et al. 1989, 1990; O'Neill et al. 1993). Three cDNAs (SR5, SR8, and SR12) were initially isolated from senescing carnation petals. Two of these were shown to be regulated by ethylene and the third was shown to be regulated by both ethylene and by temporal cues (Lawton et al. 1989, 1990). Based on sequence similarities, two of the senescence-related cDNAs are likely to encode a p-galactosidase (SR12) and a glutathione s-transferase (SR5) (Meyer et al. 1991). The potential role ofp-galactosidase in senescing flower petals is likely to be in cell wall disassembly that accompanies most senescence processes. The role of glutathione s-transferase was sug-

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POSTPOLLINATION FLOWER DEVELOPMENT

23

gested to be detoxification of lipid and DNA hydroperoxides associated with senescence-induced oxidative processes (Sylvestre et al. 1989). Overall, these results indicate that specific biochemical events are induced at the level of gene expression in senescing petals and are likely to mediate the senescence process in floral organs. It is likely that the current information related to senescence-related gene expression in flowers reflects only the most abundant mRNAs and that, underlying this process, is a plethora of genes whose expression brings about the full metabolic program of organ senescence. E. Model for Pollination-Regulated Flower Senescence

Although there are significant differences between flowers in relation to the apparent mechanisms of pollination perception and its regulation of senescence, there are also a number of similarities that suggest a general model of the interorgan regulation of pollinationinduced floral organ senescence. Because this process is primarily regulated by ethylene, the model focuses on the interorgan regulation of ethylene biosynthesis in pollinated flowers which has been elucidated by examining the expression of genes encoding ethylene biosynthetic enzymes in flowers. This model (Figure 1.3) empha-

A

? Wound? ACC .-} ...... Emascuiation Auxin

--+--------------......".._

Ovary

8 ACC

SAM

ACC

t Ethylene s

Ethylene o

Fig. 1.3. Model of interorgan regulation of ethylene biosynthesis in the flower.

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sizes the role of pollen and a pollen-borne factor as the primary stimulus in triggering floral organ senescence. In orchid flowers, an important component of the primary pollen signal is auxin, whereas in other flowers, such as petunia, it is likely that ACC participates in primary pollen signaling. Other factors, such as physical signals of emasculation-induced wounding or pollen tube penetration or as yet unidentified chemical messengers, are likely to also participate in primary pollen signaling. These factors may influence the sensitivity to ethylene that is the earliest response to pollination. The first observable change in gene expression following pollination is the induction of ethylene production in the stigma and style. This ethylene production may utilize pollen-borne ACC directly as substrate or, more likely, relies on the rapid induction of ACC synthase in the stigma. In orchid flowers, an auxin-regulated ACC synthase is rapidly induced in the stigma, which can account for the rapid transduction of the primary pollen signal, auxin, to the initial burst of ethylene production. In other flowers, such as petunia, a trace amount of pollen-borne ACC may be converted directly to ethylene, which leads to localized autocatalytic ethylene production in the stigma. The presence of ACC oxidase mRNA in presenescent petunia stigmas and its rapid ethylene-dependent induction following pollination and data indicating that treatment of the stigma with AVG prior to pollination abolishes pollination-induced ethylene production are consistent with this mechanism of initial pollination-induced ethylene production. Pollination subsequently leads to perianth senescence and other postpollination developmental responses. The interorgan transmission and amplification of the primary pollen signal requires secondary signals that emanate from the stigma. An important component of this signal appears to be ACC. This ethylene precursor has been shown to be mobilized from the stigma to other floral organs where it contributes to ethylene production. In orchid flowers, the role of translocated ACC was especially obvious in that petals and sepals do not accumulate ACC synthase mRNA or enzyme activity, in spite of high levels of ethylene production by these floral organs. This result has led to the proposal that ethylene production in these organs is entirely dependent on translocated ACC. This situation is less pronounced in other flowers, such as petunia, where ACC synthase is induced in petal tissues. However, the petunia petal ACC synthase is ethylene-induced and the initial production of ethylene in petals required to stimulate autocatalytic ethylene production may be derived from translocated ACC. Although it is difficult to test

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POSTPOLLINATION FLOWER DEVELOPMENT

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critically, it would be of interest to determine whether ACC synthase gene expression can be induced in petals by ACC directly. In addition to the coordination of petal senescence, the pollination signal also contributes to developmental events in the ovary (see below). In orchids, there is evidence that both auxin and ethylene participate in regulating events in this organ. Because of the physical connections of the stigma and ovary by the transmitting tract, it is possible that auxin is directly transferred to the ovary following pollination. This is consistent with the report of Strauss and Arditti (1982), who demonstrated that stigma-applied [14C]IAA was transported preferentially to the ovary, and by experiments that demonstrated that exogenous application of NPA, an inhibitor of polar auxin transport, prevented pollination-induced ovary growth (X. S. Zhang and S. D. O'Neill, unpublished). III. POLLINATION REGULATION OF FLORAL PIGMENTATION CHANGES

Many flowers change color in response to pollination or aging to become unattractive or inaccessible to pollinators (Gori 1983). Floral color changes may involve only selective parts of the flower. Pollinators appear to be able to recognize color changes and visit only viable or unpollinated flowers (Gori 1983; Weiss 1991). It was recently reported that at least 74 diverse angiosperm families exhibit floral pigment changes as the flower ages, and specifically following pollination (Weiss 1991). The role of pigmentation changes was proposed to serve as a mechanism to maintain a large floral display from a distance in order to attract pollinators but provide the basis for discrimination between previously pollinated flowers at close range. In addition to this proposed ecological significance of floral pigmentation changes, color fading is an important quality factor in cut flowers and a significant reason for the reduction of vase life (Halevy and Mayak 1979). Pollination can induce diverse patterns of pigmentation changes, including color fading, enhanced pigmentation, or intensification of pigmentation in discrete spots. In Cymbidium orchid flowers, pollination induces anthocyanin formation (Akamine 1963; Arditti and Knauft 1969; Arditti 1971, 1979; Arditti et al. 1971, 1973; Arditti and Flick 1976; Woltering 1989,1990b). Indeed, one of the first visible signs of senescence of Cymbidium flowers is the accumulation of anthocyanins in the labellum or lip. The change in lip coloration

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can be accelerated by pollination, emasculation, or treatment of the stigma with auxin (Arditti et al. 1973). Subsequent studies demonstrated that a small incision at the base of the lip prevented its emasculation-induced coloration and that treatment of completely excised lips with ACC or ethylene promoted anthocyanin accumulation (Woltering 1990b). These data suggested that lip coloration resulted from the translocation of ACC to the lip, which was subsequently converted to ethylene in situ. Pigmentation changes in Vanda orchid flowers are also associated with pollination and ethylene production, but, in contrast to Cymbidium, Vanda flowers undergo rapid color fading (Akamine 1963). As discussed earlier, floral color fading and ethylene production in emasculated Vanda cv. Miss Agnes Joaquim were closely correlated. In lupine flowers, the color of the banner spot changes from yellow to magenta as the flower ages (Stead and Reid 1990). This change in banner spot color appears to be regulated by ethylene and is most likely accelerated by pollination. The change in banner spot color precedes flower wilting by several days, which would presumably maintain the attractiveness of a large floral display, by keeping and not abscising flowers, from a distance but still serve to signal pollinators at close range. Although pigmentation changes are an important ecological and horticultural aspect of postpollination flower development, relatively little is known about the specific biochemical events that underlie this process. Ethylene has been implicated in the control of anthocyanin biosynthesis (Craker et al. 1971; Craker and Wetherbee 1973). Floral organ pigmentation changes can be brought about by carotenoid or anthocyanin biosynthesis (Mohan Ram and Mathur 1984), aging (Yazak 1976), senescence (Pecket 1966), anthocyanin degradation (Proctor and Creasy 1969), or changes in tissue pH (Asen et al. 1977). Indeed, a gene controlling color fading has been reported in Petunia (de Vlaming and van Eekeres 1982). Because of the diversity in patterns of pigmentation changes that are pollination regulated, it is likely that each mechanism may contribute to changes observed in different flowers. Indeed, it has been suggested that pollinationregulated color changes evolved independently in angiosperms many different times and so the underlying biochemical mechanisms are likely to be diverse in different taxa (Weiss 1991). IV. POLLINATION REGULATION OF OVARY DEVELOPMENT Ovary development after pollination depends on a supply of auxin. This was originally demonstrated by studies of natural partheno-

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carpy (Gustafson 1936,1939; Van Overbeek et al. 1941; Crane 1969). Solanaceous species, such as tomato and petunia, could be induced to form mature seedless fruit by treatment of the ovaries with auxin (Gustafson 1936). Extracts of pollen could mimic the effect of auxin, leading to the proposal that pollen contained auxin and that it played a significant role in initiating ovary growth (Laibach 1932, 1933a, b; Gustafson 1937). These observations led to the hypothesis that normal ovary development after pollination was initiated by auxin delivered first with the pollen grain and growing pollen tubes and later by the fertilized ovules (Gustafson 1939). The role of ovule-derived auxin in promoting ovary and fruit development is widely accepted. However, the role of pollination as a primary event in ovary and fruit development is less firmly established except in some species, such as orchid, in which ovary development precedes fertilization by up to approximately 60 or more days (Laibach 1932, 1933b; Gustafson 1937; Duncan and Curtis 1942, 1943; Zhang and O'Neill 1993). Early studies of postpollination development in orchids identified changes in the curvature of the ovary to be one of the earliest postpollination developmental events (Duncan and Curtis 1942). This change in curvature was more recently shown to be accompanied by the formation of hair cells and cell divisions in the placental ridge that eventually participate in expanding the placental cavity (Zhang and O'Neill 1993). These morphological changes that signal the initiation of fruit development occurred approximately 3 days prior to pollen germination and approximately 12 weeks prior to fertilization, thus clearly demonstrating that pollination itself, rather than pollen tube growth or fertilization per se, trigger this developmental event. Experiments using exogenously applied auxin and an inhibitor of ethylene biosynthesis, AVG, demonstrated that the differentiation of ovary wall hair cells, the earliest morphological change, was dependent on the presence of both auxin and ethylene (Zhang and O'Neill 1993). This result suggests that pollen-borne auxin can be translocated to the ovary, which is consistent with the report of Strauss and Arditti (Strauss and Arditti 1982), who reported that [14C]IAA applied to the stigma preferentially accumulated in the ovary. In orchid flowers, ovary development continues for over 3 months prior to fertilization (Zhang and O'Neill 1993), suggesting that a sustained supply of auxin may be provided by developing ovules prior to fertilization or by the ovary itself (Nichols 1971, 1976). Pollination effects on ovary development are not restricted to orchids and have been reported in many other plant species. For example, muskmelon ovaries doubled in size within 48 h after polli-

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nation (Lingle and Dunlap 1991), and ovary growth in Brodiaea (Han et al. 1991) and carnation (Nichols 1971, 1976; Nichols and Ho 1975a, b) was promoted by ethylene treatment that also promoted perianth senescence. Ethylene treatments did not promote extensive fruit development, and it is possible that the growth responses were related to mobilization of carbohydrate from the senescing petals to the ovary. This remobilization of nutrients from the perianth to the ovary is likely to be an important part of the postpollination syndrome of developmental events that coordinates development of the ovary with senescence of the petals, thus recycling components of the spent floral organs to contribute to the success of the overall reproductive process. V. POLLINATION REGULATION OF MALE AND FEMALE REPRODUCTIVE DEVELOPMENT

Following compatible pollination of the stigma in Angiosperms, growing pollen tubes deliver the sperm cells to the female gametophyte. Double fertilization leads to both the development of the zygote (sperm nucleus + egg cell), which develops into the multicellular embryo and represents a return to the sporophytic stage, and endosperm as a result of triple fusion (second sperm nucleus + polar nucleii). Concomitant with these events is the elaboration of ovule structures to form the mature seed, as well as the further growth and development of the fruit. Development of pollen and ovules, as well as the process of fertilization, is thoroughly discussed elsewhere and the reader is referred to these articles for details that are beyond the scope of this review (Maheshwari 1950; Kapil and Bhatnagar 1981; Bouman 1984; Noher de Halac and Harte 1985; Williams et al. 1990; Huang and Russell 1992a; Mogensen 1992; Russell 1993; Reiser and Fischer 1993; Mascarenhas 1993). It is less well recognized that in many cases, development of the male and female gametophyte is incomplete prior to pollination, and final preparation for the fertilization event is accomplished following pollination. Events that are considered here take place within the window of time between pollination and fertilization and are involved in the coordinated development of the male and female gametophytes. A. Female Reproductive Development in Orchids 1. Range of Developmental States Prior to Pollination. One of the most extreme examples of postpollination development of repro duc-

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tive structures is illustrated by many orchids, in which no ovules are present in the ovary prior to pollination. This pattern of development was observed in Cattleya, Sophronitis, Epidendron, Laelia (Duncan and Curtis 1943; Yeung and Law 1989), Phalaenopsis (Duncan and Curtis 1942; Zhang and O'Neill 1993), Dendrobium (Sagawa and Israel 1964; Israel and Sagawa 1965), and Doritis (Yasugi 1983). In these orchid species, pollination triggers proliferation of the placental ridges present in the triloculate ~vary. In many other orchid species, however, ovule primordia are present at anthesis but remain suspended at a stage immediately prior to meiosis until pollination triggers further development. This pattern of development has been observed in Cypripedium, Paphiopedilum, Phragmipedium (Duncan and Curtis 1942), Herminium (Sood and Mohana Rao 1986), Epipactis (Fredrikson 1992), and Platanthera (Fredrikson 1991). The orchid species Dactylorhiza and Calypso appear to represent intermediate cases, because at anthesis ovule primordia are present but have not yet progressed to the stage at which archesporial cell differentiation occurs (Fredrikson et al. 1988; Law and Yeung 1989). The first events in orchid ovule development are cell divisions within the meristematic region of the placental ridges. These ridges elongate and branch dichotomously several times to form thousands of finger-like ovule primordia. At this stage, cells in the dermal and subdermal layers are densely cytoplasmic. Next, a subdermal cell near the apex of the primordia enlarges to form the archesporial cell. The nucellus typically remains uniseriate and is crushed between the integuments and megagametophyte at maturity. The inner integument initiates as a ring of periclinal cell divisions near the tip of the primordia. This is accompanied by assymetric growth and division of cells on one side of the primordia to establish the anatropous orientation of the ovule. The outer integument is initiated somewhat later, after which the cells of the integuments and funiculus enlarge and become vacuolate. The archesporial cell enlarges further to directly form the megasporocyte, which develops a distinctive polar distribution of organelles, enzyme activities, and callose within the wall. Following meiosis of the megasporocyte, four (or in some cases three) products of meiosis are formed and all but the spore closest to the chalaza degenerate and are crushed by the expanding megaspore. Vacuoles begin to coalesce in the surviving megaspore, and subsequent mitotic divisions occur according to Polygonum-type development. After the first division of the megagametophyte, the nucleii migrate to opposite ends of the coenocytic megagametophyte, where they divide twice to form three antipodals at the chalazal end and the egg cell and two synergids at the micro-

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pylar end. The two remaining nucleii converge at the center of the cell to form polar nucleii. After all mitotic divisions are complete, cell walls are formed between the nucleii at both ends of the megagametophyte but not around the polar nucleii, resulting in an 8-nucleate, 7-celled megagametophyte which is wholely contained within the original megaspore wall. 2. Significance of Delayed Female Reproductive Development. The stage of megagametophyte development at the time of pollination has been related to aspects of reproductive ecology in various orchid species. Most researchers agree that it is likely that orchid ovule development is not initiated before pollination because the low probability of pollination of the very specialized orchid reproductive system in the epiphytic environment is compounded by the large investment necessary for ovary maturation. Thus, orchids have evolved a life history strategy that invests in female reproductive development only after fertilization is assured. Moreover, Swamy (1943) has suggested that the variation observed in stage of ovule development at the time of anthesis is adaptively related to the environment in which they typically grow. Thus, epiphytic orchids that typically grow in areas where the growing season is long can "afford" the extra time necessary to complete postpollination ovule development, while terrestrial species that typically grow in temperate regions where the growing season is short must achieve partial female development before anthesis to complete the reproductive cycle within the growing period. Moreover, the readiness for female reproductive development may be fine-tuned within individual orchid genera by specific environmental factors. For example, Fredrikson observed that in Epipactis atrorubens and E. helleborine the megagametophyte is near maturity at the time of anthesis, whereas the related species E. palustris contains ovules only at the megasporocyte stage (Fredrikson 1992). Fredrikson suggests that the precocious nature of ovule development in E. atrorubens and E. helleborine reflects the greater propensity for self-pollination in these species. If self-pollination is common in the breeding system, each ovary is certain to develop into a fruit, making investment by the plant in female reproductive development less risky. This may lead to relaxed selection pressure from "production economy," because a high proportion of the ovules matured by the plant would be fertilized and produce seed.

3. Ultrastructural, Biochemical, and Genetic Studies of Ovule Development. Despite the wealth of descriptive knowledge concern-

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ing ovule development, very little is known about the molecular basis of these events. Clearly, positional or environmental information is interpreted by the developing ovule to establish a program of events that leads ultimately to the sequential development of diffentiated cells of the sporophytic and gametophytic tissues of the ovule. These events undoubtedly include cell-cell signaling mechanisms that allow individual cells to understand their fate and enact the appropriate program of gene expression to achieve structural and biochemical specialization. At this time, these events are almost totally unknown. On the other hand, studies of biochemical events that are regulated during development and lead to specialized cell types are beginning to be understood. During differentiation of the megasporocyte, for example, the ~­ 1,3 polyglucan known as callose is laid down in the cell wall primarily at the micropylar end (Rodkiewicz and Bednara 1976; Willemse and Bednara 1979; Schulz and Jensen 1981; Yeung and Law 1989). Following meiosis, the degenerating megaspores are completely enveloped by callose, while the functional megaspore typically shows callosic deposits only at the micropylar end (Willemse and Bednara 1979), or in diffuse patches (Rodkiewicz and Bednara 1976). This layer of relatively impermeable wall material is thought by some authors to isolate the degenerating megaspores in order to cause their death (Heslop-Harrison 1964; Rodkiewicz 1970) or to isolate the surviving spore from signals that induce degeneration (Law and Yeung 1989). This notion is supported by the fact that plasmodesmatal connections are typically severed in locations where callose accumulates, while they remain intact in the chalazal regions where no callose is observed. On the other hand, genetic studies in Oenothera species suggest that callose does not playa causal role in megaspore selection (Sniezko and Harte 1985). In crosses of O. hookerii, in which the chalazal megaspores degenerate, with O. suaveolens, in which the degenerating megaspores are in variable positions, the progeny examined showed no correlation between the distribution of callose with the position of the degenerating megaspores. It is equally possible that callose deposition is a symptom of a rejection reaction that leads to cell death, rather than the cause of death. For example, callose has been observed to accumulate in stigmatic papillae that come into contact with incompatible pollen grains during pollination (Knox 1973; Elleman et a1. 1992) and in cells that respond to fungal hyphae invasion during pathogen infection (Lukaszewski and Reid 1989). Other investigators have examined the changing structure and location of organelles during megasporogenesis and megagameto-

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genesis. Prior to meiosis in the megasporocyte, cytological changes occur in the appearance of the mitochondria and the plastids, and there is a reduction in the number of ribosomes present in the cell (Israel and Sagawa 1965; Willemse and Bednara 1979; Noher de Halac and Harte 1985; Huang and Russell 199Za). Endoplasmic reticulum has been observed to form concentric cisternae (Noher de Halac and Harte 1985; Huang and Russell 199Za) and sometimes forms autophagic vesicles for the destruction of other organelles (Schulz and Jensen 1981). In some instances, the majority of heritable organelles is localized to the chalazal end of the differentiated megasporocyte prior to meiosis (Huang and Russell 199Za). Following meiosis, the surviving chalazal megaspore inherits the majority of organelles as a result of their premeiotic distribution. Because of the obvious connection between the cytoskeleton and the distribution of organelles and the regulation of meiosis, various investigators have examined the structure of the microtubules in the megasporocyte and megaspores (Bednara et a1. 1988; Webb and Gunning 1990). These investigations have been greatly assisted by the technique of isolation of the megagametophyte from other ovule tissues (Van Went and Kwee 1990; Theunis et a1. 1991), which makes three-dimensional analysis of the megagametophyte structure and biochemistry more feasible. Typically, cytoplasmic microtubules were observed to radiate from the megasporocyte nucleus prior to meiosis, but were not believed to playa role in determining cell shape based on their pattern within the cell (Webb and Gunning 1990). During meiosis, tubulin is located exclusively in the spindle apparatus, but no preprophase band was observed to delineate the plane of meiotic division. Following meiosis, "striking" cytoplasmic arrays of microtubules appear during the one nucleate megagametophyte stage. They most likely serve to organize the location of nucleii and organelles within the coenocytic cell during megagametophyte development (Webb and Gunning 1990). Another exciting possibility is that the microtubule cytoskeleton mediates asymetric distribution of mRNAs during megagametogenesis, as it has been proposed to do during differentiation of the Drosophila oocyte within the syncytial cyst (Theurkauf et a1. 1993). Ultimately, the specialization of cell types within the megagametophyte depend on the biochemical activites that, as a whole, establish the unique functional identities of these cells. To gain some insight into these processes, some investigators have examined the location of proteins, RNA, polysaccharides, and biochemical activities during megasporogenesis and megagametogenesis. Early work employed cytochemistry to infer the activites cell types were en-

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gaged in during development, as well as to assess general metabolic activity. During stages of development immediately prior to and after meiosis, the chalazal end of the nucellus and the megaspore destined to survive show higher levels of proteins, lipids, and such biochemical activities as acid phosphatases and peroxidases (Willemse and Bednara 1979; Schulz and Jensen 1981). Degenerating megaspores, on the other hand, contain "reclamation enzymes," such as esterases and basic phosphatases (Willemse and Bednara 1979), which might be indicative of the process of degeneration. At later stages of development, Zinger and Poddubnaya-Arnoldi (1966) observed high levels of hydrogenase activity in integuments during their elongation phase, and Malik and Vermani (1975) observed high levels of this enzyme during the expansion stage of the megasporocyte. Cytochrome oxidase and peroxidase activities were high in the antipodal cells of the mature megagametophyte, and antipodals were observed to contain large amounts of lipids as well as sulfhydryl compounds, so that the authors concluded that antipodal cells are metabolically active and their role is nutritive (Zinger and Poddubnaya-Arnoldi 1966; Malik and Vermani 1975). An increase in peroxidase and cytochrome oxidase activities in the nucellus near the micropyle was also observed after pollination, leading to the suggestion that signals from the pollen trigger the ovule to prepare for the upcoming fertilization event (Zinger and Poddubnaya-Arnoldi 1966; Malik and Vermani 1975). More recently, Pennell and others have examined the developmental changes in expression of several plasma membrane arabinogalactan proteins during Brassica and Pisum reproductive development using monoconal antibodies (Pennell and Roberts 1990; Pennell et a1. 1991). The antibody JIMB recognizes an epitope in nucellar cells at the two- or four-nucleate embryo sac stage, and later in the egg apparatus but not other cells of the embryo sac. This epitope continues to be present in the developing embryo until the globular stage, when it disappears. Anthers also contain the epitope in microsporocytes and tapetum, and later in the endothecium and two sperm cells of the pollen grain. A different pattern was observed with the antibody MAC207, which stains all cells of the plant except the cells in early reproductive development which are destined to give rise to the tapetum and microspores of the anther or the nucellus and embryo sac of the ovule. Later in reproductive development, the epitope is present in the vegetative cell but not the generative cell of the pollen grain, and the epitope reappears in the heart stage embryo. While the functional significance of these changes in plasma membrane arabinogalactan proteins is not clear, they serve

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as biochemical markers of unique developmental programs that operate during the differentiation of reproductive cells. Finally, other investigators have taken a genetic approach by identifying mutations in genes involved in controlling ovule development. The mutations bell, sinl, ovm2, and ovm3, which cause female sterility, have recently been described in Arabidopsis (Robinson-Beers et al. 1992; Reiser and Fischer 1993). Ovules of bell mutant plants apparently lack the inner integument, and outer integument development is aberrant. The authors suggest that proper integument development may be linked to embryo sac development, because although these mutants undergo meiosis and intitiate megagametogenesis, they do not form normal embryo sacs (RobinsonBeers et al. 1992). Ovules of the mutant sinl, on the other hand, develop similarly to wild-type but morphological development is arrested, though cell division continues to yield ovules with the same cell number and overall size as wild-type, but they retain the immature shape. These ovules undergo meiosis but no functional megaspores are observed (Robinson-Beers et al. 1992). Ovules of the mutant ovm2 undergo megasporogenesis and initiate megagametogenesis, but at anthesis nucellar-like cells have replaced the embryo sac (Reiser and Fischer 1993). Ovules of ovm3 mutant plants never initiate integuments, and megasporogenesis is aberrant after meiosis (Reiser and Fischer 1993). Other genes from Arabidopsis recently shown to be involved in ovule development include ats, which causes abnormal integument development (Leon-Kloosterziel et al. 1994), and sup, which results in defective ovules (Gaiser et al. 1995). A few mutations defective in aspects of megasporogenesis have also been identified, such as msg in wheat (Joppa et al. 1987), sy-2 in Solanum (Parrott and Hanneman 1988), and GfinArabidopsis (Redei 1965). Additionally, Vollbrecht and Hake (1995) examined regions of the maize genome for genes affecting embryo sac development by deficiency analysis and have located several regions that cause specific types of defects in female gametophyte development. Isolation of mutants defective in ovule development and analysis of their genetic interations should lead to hypotheses about the molecular regulation of ovule development. 4. Orchids as a Model System to Study Ovule Development. To

date, it is not clear precisely how orchids suspend development of ovules relative to the other floral organs until the pollination stimulus is received. It is possible that suspension of meiosis finds its analog in animal systems such as Xenopus, where oocytes do not proceed into meiosis until activated by the M-phase promoting fac-

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tor (MPF) of cell cycle control. Recently, it has been shown that hormonal stimulation of de novo translation of mos protein, which converts MPF to the active form, is responsible for the activation of the meiotic program (Yew et al. 1992). It is tempting to speculate that the developmental switch regulating ovule development operates in a similar fashion, to suspend meiosis or ovule elaboration until pollination presents an appropriate hormonal stimulus. Work by Golubskaya and colleagues may provide some insight into the genetic regulation of meiosis in plants. Various mutants defective in male or female meiosis have been examined in order to understand the regulatory cascade involved in normal meiosis in plants (Golubskaya 1989; Golubskaya et al. 1992). Other genes that influence premeiotic, meiotic, and postmeiotic gametophyte development have been discussed by Kaul and Murthy (1985). Many of these genes are candidates for those that function in controlling the arrest and continuance of development in response to the pollination signal. Recent work by our laboratory is beginning to provide insight into how the pollination signal activates development following pollination. It appears likely that in Phalaenopsis orchids, a component of the stimulus triggering ovule development is auxin contained by the pollen (Zhang and O'Neill 1993). Application of NAA to the stigma is sufficient to mimic the effects of pollination by inducing ovary growth and elongation of hair cells from the inner ovary wall, which are the first responses of the naturally pollinated ovary. The role of auxin in promoting ovary growth is also established in parthenocarpy, where auxin can substitute for fertilization by inducing ovary growth; in these cases, however, the fruit already contains ovules (Gustafson 1939; Van Overbeek et al. 1941; Muir 1942; Crane 1969). This hypothesis is further supported by work demonstrating that radioactively labeled auxin applied to the stigma is transported to the ovary within an appropriate timeframe in Cattleya orchids (Strauss and Arditti 1982). Auxin applied in this manner is not adequate, however, to maintain the long-term response necessary to observe the initiation of ovule development, suggesting that other factors from the pollen are also required or that a more sustained supply of auxin is required by the growing ovary. More interestingly, ovary growth and hair cell elongation were partially inhibited following pollination by application of the ACC synthase inhibitor AVG, suggesting that ethylene is also a required component for the ovary response to pollination (Zhang and O'Neill 1993). Because of the precise manner in which the ovule developmental program can be switched on in the mature flowers of orchids, they provide an excellent system in which to study both the regulation of

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ovule development and the intercellular communication events that lead to the activation of this pathway. The Phalaenopsis orchid system is also amenable to biochemical investigations because ovule development is nearly synchronous and thousands of ovules are present in each ovary, making tissue collection feasible. Our group has chosen to assess the mechanisms involved in ovule development by identifying genes that are expressed uniquely in ovules, reasoning that these genes must play some role either in regulating development or in establishing the specialized identities of cells in the ovule. To this end, we have isolated genes specific to developing and mature ovules of Phalaenopsis orchids by differential screening of AZAPII cDNA libraries constructed from mRNA isolated from ovule tissue at two times, which correspond (1) to early events in megasporocyte development and the transition to meiosis, and (2) to megagametophyte development, (Nadeau et a1. 1996). From this work several clones have been identified that are expressed in patterns that suggest that they may playa role in ovule morphogenesis. One clone (039) has been identified from the megasporocyte library that is specific to ovule tissue (it is not detectable in other floral tissues, roots, or leaves). In situ hybridization and RNA blot hybridization experiments show that the expression of this gene begins in ovule primordia at the time of archesporial cell differentiation and continues to be expressed at somewhat lower levels in mature ovules. Sequence analysis of this gene indicates that it is a member of the homeobox family of transcription factors, which bind to DNA in a sequence-specific manner in order to regulate transcription of sets of target genes (Scott et a1. 1989; Gehring 1993). These proteins are highly significant to developmental regulation in both plants and animals and were, in fact, initially identified as the dramatic homeotic mutations in Drosophila that cause such defects as the replacement of antennae with legs (Lewis 1963, 1978; McGinnis et a1. 1984). Evidence from the experimental manipulation of tobacco placentae in tissue culture suggests that the ovule primordium experiences a commitment step at around the time of archesporial cell differentiation (Evans and Malmberg 1989), after which it becomes developmentally determined to form an ovule. We speculate that expression of the 039 gene may be turned on during this commitment step in order to regulate a set of genes that results in the differentiation of the ovule. Another very interesting clone, 040, has been isolated from the megasporocyte library that proved upon inspection by in situ hybridization to be specific to pollen tubes. The expression of this gene

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is downregulated at or near the time of fertilization (11 WAP). Sequence analysis of this clone indicates that it encodes a cytochrome P450 monooxygenase. The cytochrome P450 monooxygenases constitute a large gene superfamily of membrane-bound enzymes present in bacteria, fungi, plants, and animals that catalyze the oxidation of diverse and often overlapping substrates of both endogenous and xenobiotic origin (Nebert and Gonzalez 1987; Donaldson and Luster 1991). We can only speculate about the possible role of this enzyme in the pollen tube, but it may be involved in the biosynthesis of a secondary metabolite necessary for tube growth or in the biosynthesis of a hormone that is involved in growth of the pollen tubes or communication with the ovules. Additionally, several ovule-specific clones isolated from nearmature ovules (0108, 0126, 0141) are also highly stage-specific. Transcript,s corresponding to these clones are undetectable at early stages of development, but are abundant when ovules are completing the mitotic divisions of megagametophyte development and when some fertilization events have occurred. One of these, 0108, is a novel clone that shows no homology to proteins of known function. In situ hybridization demonstrates that 0108 has a very unique pattern of gene expression, however. The 0108 transcripts are present only in the outer layer of the outer integument and in the embryo sac during a narrow window of time prior to fertilization. We speculate that 0108 may be involved in communication and guidance between the ovule and pollen tube. Sequence analysis has shown that clone 0126 is a glycine-rich protein. Many of these proteins are believed to be components of the plant cell wall (Keller et al. 1989; de Oliveira et al. 1990; Condit et al. 1990), so we speculate that 0126, due to its exclusive expression in the mature ovule, may playa role in specialized wall structures in the mature ovule. One interesting candidate is the specialized cell wall associated with the egg apparatus in the female gametophyte. Sequence analysis of 0141 indicates that it is a cysteine proteinase. These proteins play diverse roles in both plants and animals; in plant systems, their proposed functions range from the degradation of seed storage proteins (Rogers et al. 1985; Koehler and Ho 1988) to involvement in developmentally regulated cell death (Koltunow et al. 1990), while in animal systems they have been implicated in the conversion of peptide pro-hormones to the active hormone (Docherty et al. 1982) and developmentally regulated protein turnover (Williams et al. 1985). In situ hybridization shows that expression of 0141

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is limited to the outer integument as it begins to form the seed coat so it is possible that 0141 is involved in this process (Nadeau et al. 1996).

B. Other Examples of Postpollination Female Reproductive Development Postpollination development of the megagametophyte is not confined to the Orchidaceae, but is a significant part of the reproductive systems of many plants. While in most of these species megagametophyte development is almost complete, the final stages of functional differentiation of cells of the megagametophyte may be triggered by pollination. For example, Prunus dulcis (almond) ovules have been shown to be at the megasporocyte stage at anthesis, but the exact stage of ovule development was variable, depending on growth temperatures (Pimienta and Polito 1983). Further development and enlargement of the megagametophyte occurs after pollination. Studies have demonstrated that this development does not occur in ovules of nonpollinated and self-pollinated (incompatible pollination) flowers (Pimienta and Polito 1983). Furthermore, studies of development in other Prunus species in which the megagametophyte is almost mature at anthesis suggest that prior to pollination, polar nucleii have not fused, and that this event is completed only in pollinated flowers (Eaton 1959; Eaton and Jamont 1964; Pimienta and Polito 1983). Experiments with cotton ovule culture have shown that the addition of 5.0 I-lM IAA to the culture media can induce polar nucleii to fuse and begin abnormal endosperm-like proliferation, suggesting that auxin supplied by the pollen may be a natural signal inducing polar nucleii fusion and perhaps other biochemical events in preparation for fertilization (Jensen et al. 1983). Recent work examining the kinetics of double fertilization in Zea mays has revealed the previously unsuspected fact that the egg cell is morphologically immature at the time of pollination in the majority of ovules (Mol et al. 1994). In these cases, the cell has not en1arged to its full extent, vacuolization has not taken place, and the normal polarity of nucleus and cytoplasm within the cell has not been established. These final events are not completed in the majority of cells until well after pollination, but before fertilization, leading to the suggestion that pollen signals are responsible for inducing the final stages of megagametophyte development. Another important event that occurs prior to fertilization is the degeneration of the synergids, which allows the pollen tube to pen-

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etrate the megagametophyte during fertilization. In barley, Mogensen (1984) observed that pollination triggered degeneration before the pollen tubes reached the ovule and concluded that this event was stimulated by pollen tubes in the style. Other investigators have noted that calcium accumulates in the synergids after pollination but prior to fertilization (He and Yang 1992), and this may playa role in establishing a chemotropic gradient to attract the pollen tube and/or causing it to burst after it enters the synergid to release the sperm cells (Chaubal and Reger 1990, 1992). Huang and Russell (1992b) recently examined this question and concluded that a signal from the pollen tube was communicated over a short range to the ovule to stimulate degeneration of the synergid in Nicotiana. The degenerating synergid showed an accompanying increase in membrane bound calcium. A possible candidate for the pollen signal in this case was suggested by experiments using cotton ovule culture in which 0.5 ~M gibberellin supplement in the culture media was shown to induce synergid degeneration in a manner similar to that after pollination, while the synergids of unpollinated ovules did not degenerate (Jensen et al. 1983). Interestingly, in animal cells controlled cell death (apoptosis) has also been linked to the presence of calcium in the cell (Martin et al. 1994). Finally, a number of authors have observed that pollination is required for apomictic embryo development in some species. This process has been referred to as "pseudogamy," because of the apparent requirement for the presence of the male gamete for embryo development (Stebbins 1941). In the orchid Zygopetalum, seed production could be induced by pollination with pollen of the orchid Oncidium, which was unable to complete fertilization (Suessenguth 1923). Hagerup (1944) examined this question in the apomictic orchid species Orchis and concluded that pollination was necessary for apomictic embryos to develop. Observations of this type led Gustafsson (1946) to conclude that chemical substances contained in the pollen induced ovary growth, which indirectly triggered embryo development whether or not fertilization had occurred. This work meshes well with the observations of our own group demonstrating that a chemical substance provided by the pollen can indeed induce ovary, ovule, and gametophyte development in the orchid system (Zhang and O'Neill 1993). Other examples of pseudogamous behavior include horticulturally significant groups such as the grasses and fruit trees. For example, pollination of the apomictic grass Pennisetum setaceum with pollen of P. ciliare has been shown to increase the set of apomictic embryos, though this pollen did not produce hybrid embryos (Simpson and Bashaw 1969;

S. D. O'NEILL AND ]. A. NADEAU

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Bashaw and Hanna 1990). More recently, it has been observed that gamma-ray-treated pollen, which is inefficient at fertilization, can induce a low percentage of haploid, presumably apomictic embryos to develop in apple (Zhang and Lespinasse 1991). In both cases, pollination presents a stimulus that induces development of cells not normally expected to produce embryos.

c.

Male Reproductive Development

In approximately 70% of angiosperm species, the male gametophyte has not completed development at the time at which it is shed from the anther (Yu and Russell 1992). These bicellular pollen grains contain a vegetative cell and a generative cell that is contained entirely within the vegetative cell cytoplasm. Following pollination, both cells move into the growing pollen tube as it passes through the transmitting tract of the stigma and style. The final mitotic division of microgametophyte development in which the generative cell gives rise to two sperm cells takes place when the growing pollen tube tip is in the style or ovary cavity (Hagerup 1944; Zhang and Lespinasse 1991; Yu and Russell 1992; Mogensen 1992). The sperm cells and vegetative nucleus retain a physical connection during postpollination development and this associated unit has been collectively termed the "male germ unit" (Dumas et al. 1984; Mogensen 1992). It has been suggested that the male germ unit plays a functional role in postpollination development, possibly by providing a mechanism to deliver the sperm cells to the egg apparatus simultaneously, or by playing a role in gamete level recognition that controls which sperm fuses with the egg cell (Russell and Cass 1981; Dumas et al. 1984; Mogensen 1992). It has also been suggested that the male germ unit allows the vegetative nucleus to contribute mRNA or other substances to the sperm cells (Shi et al. 1991). This association was observed initially in species such as Plumbago, Brassica, and the grass Alopecurus, which shed fully mature, tricellular pollen (Russell and Cass 1981; Heslop-Harrison and Heslop-Harrison 1984; Dumas et al. 1984), but recently it has also been observed in species such as Cymbidium and Populus in which the microgametophyte is bicellular and immature at anthesis (Rougier et al. 1991; Yu and Russell 1992). The importance of this event is emphasized by species such as Hordeum vulgare, in which tricellular pollen shows no observable association between cells until after pollination when the sperm cells have been observed to reassociate in the pollen grain before proceeding to move down the tube (Mogensen and Wagner 1987).

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A great deal of effort toward understanding the postpollination behavior of the microgametophyte has focused on the pollen recognition event (Knox 1973; Sarker et al. 1988; Villar et al. 1993; Liedl and Andersen 1993; Preuss et al. 1993), the mechanism of pollen tube growth (Polito 1983; Okamoto and Omori 1991; Feij6 et al. 1992), and the interaction between the tube and the stylar tissues it travels through (Sanders and Lord 1992; Wang et al. 1993), though little is known about the molecular events that are responsible for these processes. In early work examining aspects of the hormonal basis of postpollination development, Addicott (1942) examined the role of various substances in promoting pollen germination and tube growth and found 16 substances that influenced one process or the other. Interestingly, inositol, a known component of signal transduction pathways (Berridge 1993), was found to stimulate germination significantly. Auxins, on the other hand, were found to stimulate pollen tube growth. More recently, evidence from work in the Phalaenopsis system seems to indicate that ethylene production by the gynoecium that is triggered by pollination plays a role in stimulating pollen germination and tube growth (Zhang and O'Neill 1993). Pollen germination and tube length was examined in flowers that were pollinated normally as compared to those pretreated with the ACC synthase inhibitor AVG, and it was observed that both germination and tube growth was significantly inhibited by the lack of ethylene production. Moreover, when exogenous ACC was supplied during the pollination to overcome the effects of the inhibitor (and allow the gynoecium to produce ethylene), pollen tube germination and growth was restored to almost normal levels. Other significant information concerning the establishment of the male germ unit and postpollination behavior of the pollen tube may eventually be gleaned from the examination of genes expressed during postpollination development of the microgametophyte. While most researchers thus far have isolated genes expressed during preanthesis development of the microgametophyte rather than postpollination development, it is apparent that a subset of pollen-specific genes encode mRNAs that are stored and/or expressed to be translated following pollen germination (Mascarenhas 1990). Genes that fall into these categories include LAT genes isolated from tomato pollen (Ursin et al. 1989), and the Zmc and Tpc clones isolated from Zea and Tradescantia (Stinson et al. 1987). Little is known about the products encoded by these genes as yet. Other researchers have examined accumulation of new proteins during postpollination development, such as phytases in Lilium pollen (Lin et al. 1987) and an

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unidentified 65-kD protein that accumulates during pollen tube growth in tobacco (Capkova et a1. 1987). It is possible that the genes or proteins examined by these investigators are significant to processes that take place following pollination, including the formation and maintenance of the male germ unit, pollen tube morphogenesis, and the interactive regulatory mechanisms that coordinate development of the pollen tube and the ovule. It is likely, however, that a complete understanding of the molecular regulation of male gametophyte behavior following pollination awaits the isolation of mutants defective in postpollination development. VI. COORDINATE REGULATION OF POSTPOLLINATION GAMETOPHYTE DEVELOPMENT

Appropriate coordinate development of the male and female gametophytes after pollination is essential for the completion of fertilization and involves a series of intercellular and interorgan communication events. Pollen-borne substances and/or the physical interaction of pollen with the stigmatic surface trigger a stigmatic response that communicates to other flower organs that pollination has occurred, as discussed previously. The nature of the pollen signal is as yet unclear, but auxin may be at least one component of the signal mechanism. Work in the Phalaenopsis orchid system has shown that the induction of ethylene biosynthesis is involved in the developmental changes associated with senescence in the perianth, and that it is also necessary for pollen germination and tube growth (O'Neill et a1. 1993; Zhang and O'Neill 1993). At the same time, ethylene and auxin also interact to stimulate ovule development in this species (Zhang and O'Neill 1993). Numerous other plant species also exhibit some degree of postpollination ovule or megagametophyte development that is triggered by compatible or incompatible pollinations, though the nature of the signal that stimulates further development is unknown. The magnitude of the response to pollination is variable, ranging from complete development of the ovule from meristematic tissue to minor changes in structure or biochemistry of nearly mature megagametophytes. In some cases, it is apparent that pollination-induced changes within the ovule will ultimately function to guide the pollen tube to the egg apparatus. For example, degeneration of the synergid in response to pollination may serve to produce a chemotropic signal that attracts the pollen tube to the micropyle by establishing a calcium gradient. Other substances have

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also been observed to be secreted from the filiform apparatus of the synergids, such as polysaccharide material hypothesized to be chemotropic in nature (Chao 1971; Tilton 1981). Alternatively, pollination-induced changes may ready the megagametophyte for the impending fertilization event, as, for example, final development of the egg cell in Zea mays (Mol et al. 1994). It is especially clear that signaling between the micro- and megagametophytes is critical in the orchid system, because the microgametophyte must wait in the ovary for several weeks before the megagametophyte is receptive to fertilization (Zhang and O'Neill 1993). These signals are widespread in plants: recent evidence from Arabidopsis mutants in ovule development shows that there is a long-range signal produced by the normal embryo sac that directs pollen tubes (Hiilskamp et al. 1995). Regardless of the ultimate function of postpollination developmental changes, recent work demonstrates that a complex web of intercommunication occurs between the male and female reproductive organs to coordinate development. VII. CONCLUSION A complex regulatory scheme acts to control the suite of developmental events that occur following pollination. These events include, but are not restricted to, changes in pigmentation, senescence or abscission of floral organs, as well as growth and development of the ovary. Ethylene is known to act to coordinate some of these developmental events, as does auxin. Despite our knowledge, however, several questions remain concerning the identity of the signal molecules that participate in regulating postpollination development of the flower. First, the identity of the primary pollen-borne signal molecule remains undetermined for the majority of plant species. Although auxins appear to playa role in the postpollination response of orchid flowers, ACC appears to be important for species such as petunia. The picture is still incomplete, and it is clear that other factors must be involved in the response as a whole. For example, what accounts for the differential response of petunia flowers to compatible and incompatible pollinations, and what pollen-stigma interactions account for the increase in sensitivity of flowers to ethylene? The role of ethylene is well established as a secondary signal that coordinates responses within the flower, and in all cases of climacteric flowers, ethylene is the direct causative agent involved in peri-

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anth senescence. Ethylene has been shown to be important for male gametophyte development, and auxin participates in ovary growth, but it remains to be understood exactly how these substance interact to regulate development. Our understanding of the physiology of these processes is far from complete, and nothing is known about the molecular regulation of these events. The mechanism by which ovule and gametophyte development is regulated by pollination is not understood. Clearly, signaling between the male gametophyte and the gynoecium of the flower occurs, but nothing is known about the mechanism of these interactions. A thorough understanding of these processes awaits testing in a model genetic system such as Arabidopsis in which the function of specific genes can be determined using mutant analysis and "reverse genetic" approaches. LITERATURE CITED Adams, D.O., and S. F. Yang. 1979. Ethylene biosynthesis: identification of 1aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. (USA) 83:7755-7759. Addicott, F. T. 1942. Pollen germination and pollen tube growth, as influenced by pure growth substances. Plant Physiol. 18:270-279. Akamine, E. K. 1963. Ethylene production in fading Vanda orchid blossoms. Science 140:1217-1218. Anderson, J. D., A. K. Mattoo, and M. Lieberman. 1982. Induction of ethylene biosynthesis in tomato leaf disks by cell wall-digesting enzymes. Biochem. Biophys. Res. Commun. 107:588-596. Arditti, J. 1971. Orchids and the discovery of auxin. Am. Orchid Soc. Bull. 40:211214. Arditti, J. 1979. Aspects of the physiology of orchids. Adv. Bot. Res. 7: 421-655. Arditti, J., and B. H. Flick. 1976. Post-pollination phenomena in orchid flowers, VI: excised floral segments of Cymbidium. Am. J. Bot. 63: 201-211. Arditti, J., and C. R. Harrison. 1979. Postpollination phenomena in orchid flowers, VIII: water and dry weight relations. Bot. Gaz. 140:133-137. Arditti, J., and R. L. Knauft. 1969. The effects of auxin, actinomycin D, ethionine and puromycin on post-pollination behavior in Cymbidium (Orchidaceae) flowers. Am. J. Bot. 56:620-628. Arditti, J., D. C. Jeffrey, and B. H. Flick. 1971. Post-pollination phenomena in orchid flowers, III: effects and interactions of auxin, kinetin or gibberellin. New Phytol. 70:1125-1141. Arditti, J., N. M. Hogan, and A. V. Chadwick. 1973. Post-pollination phenomena in orchid flowers, IV: effects of ethylene. Am. J. Bot. 60: 883-888. Asen, S., R. N. Stewart, and K. H. Norris. 1977. Anthocyanin and pH involved in the color of 'Heavenly Blue' morning glory. Phytochemistry 16:1118-1119. Bashaw, E. c., and W. W. Hanna. 1990. Apomictic reproduction. p. 100-130. In: G. P. Chapman (ed.), Reproductive versatility in the grasses. Cambridge Univ. Press, Cambridge, UK.

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2 Specialty Mushrooms and Their Cultivation Daniel J. Royse Mushroom Research Center, Department of Plant Pathology Pennsylvania State University University Park, Pennsylvania, USA

I. II.

III.

IV. V.

Introduction Spawn A. Spawn Making B. Breeding and Selection C. Culture Maintenance Mushroom Production Technology A. Auricularia spp. B. Flammulina velutipes C. Ganoderma lucidum D. Grifola frondosa E. Hericium erinaceus F. Hypsizygus marmoreus G. Lentinula edodes 1. Natural Log Production 2. Synthetic Log Production H. Morchella esculenta I. Pleurotus spp. J. Pholiota nameko K. Tremella fuciformis L. Volvariella spp. Marketing Future Prospects Literature Cited

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I. INTRODUCTION

Mushrooms are considered macroscopic fungi with a fruiting body (also called a sporocarp) large enough to be seen with the naked eye and to be picked up by hand. Mushrooms may grow underground, have a nonfleshy texture, and need not be edible. It has been estimated that over 1,500,000 species of fungi exist on the earth; yet only about 69,000 species have been described to date (Hawksworth 1991). Approximately 14% (10,000) of the described species of fungi are considered mushrooms (Kendrick 1985). Mushrooms may be cultivated on a wide variety of substrates. The most commonly used materials include straw (wheat, oat, and rice), wood chips and bark, cotton waste (seed hulls and screenings), hay, banana leaves, corn stalks, and other agricultural waste products. Successful cultivation of some mushroom species requires a composting process to produce a selective medium. Agaricus bisporus is the most widely cultivated species requiring a composted, selective medium. Other species more usually are cultivated on a noncomposted substrate that has been heat-treated to remove unwanted competitive microorganisms. Total commercial mushroom production worldwide has increased more than 10-fold in the last 25 years from about 350,000 t in 1965 to about 4,300,000 t in 1991 (Chang 1993). The bulk of this increase has occurred during the last 10 years. A considerable shift has occurred in the composite of genera that constitute the mushroom supply. During the 1979 production year, the button mushroom, Agaricus bisporus (=A. brunnescens Peck) accounted for over 70% of the world's supply. By 1991, only 37% of world production was A. bisparus. The People's Republic of China is the major producer (2.2 million tonnes-or about 50% of the total) of edible mushrooms. In 1994-95, the United States produced 360,035 t (or about 8% of the total world supply) of mushrooms. Agaricus bisporus accounted for over 90% of total mushroom production value, while Lentinula, Flammulina, Pleuratus, Hypsizygus, Hericium, March ella, and Grifala were the main specialty genera cultivated. The value of the 1994-95 specialty mushroom crop in the United States amounted to $28.3 million-a 73% increase over the 1993-94 season (USDA 1995). Average annual increases for specialty mushroom production in the United States has averaged over 20% since 1987 (USDA 1995). This review covers the cultivation of specialty mushrooms, i.e., economically significant mushrooms with the exception of Agaricus bisparus (A. brunnescens). The culture of A. bisparus has been widely reviewed (San Antonio 1984; Flegg et al. 1985; van Griensven

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1988). The literature on other "specialty" mushrooms is scattered or included in sources unavailable to the English-speaking community. Truffles will not be covered herein, but these mushrooms were reviewed recently by Giovannetti et al. (1994). This review includes general production statistics and cultural practices. Traditional food and medicinal uses of various species are included where information is available. Many Oriental cultures make extensive use of the mushrooms mentioned herein for medicinal purposes, but the scientific literature is not uniform in support of these claims. It is not the intent of this review to refute or support the claims in the literature, but rather to point the readers to further information.

II. SPAWN A. Spawn Making Mushrooms are grown from mycelium (thread-like filaments that become interwoven) propagated on a base of steam-sterilized cereal grain (usually rye or millet). This cereal grain/mycelium mixture is called spawn and spawn is used to seed mushroom substrate. Most spawn is made with mycelium from a stored culture, rather than mycelium whose parent was a spore. This is because each spore is likely to yield a new strain and its performance would be unpredictable. Spawn making is a rather complex task and is not practicable for the common mushroom grower. Spawn of various mushroom species may be purchased from commercial spawnmakers who usually provide instructions for its use. Spawn frequently is shipped from the manufacturer to growers in the same aseptic containers used for spawn production. Inoculum for spawn production frequently is produced in glass containers of 4- to 5-L capacity. In recent years, polypropylene or polyethylene bags containing a microporous breather strip for gas exchange have become popular final spawn production containers. Most commercial spawn production companies produce spawn only from inoculum that has met strict quality control standards. These standards include verification of inoculum production performance before it is used to produce spawn, and insurance of the spawn's biological purity and vigor. B. Breeding and Selection A thorough understanding of the breeding system of a mushroom is of major importance to commercial breeding programs. Without such understanding, crosses and heterokaryons are difficult to make in a

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controlled manner. Breeding systems genetics have been extensively reviewed by Raper (1978, 1985) and Chang et a1. (1993). The breeding systems of commercially important mushrooms have been summarized by Wu (1990). Mushroom breeding and selection, Le., the development, evaluation, and maintenance of new genotypes, have progressed at variable rates according to species. Breeding and selection programs are designed to bring together desired traits possessed by two or more individuals. Extensive testing or selection of numerous new genotypes is necessary to identify high-quality, productive candidates. Beginning with small-scale trials, hybrids are evaluated for the production traits of interest. Promising candidates are moved to increasingly larger scale tests. Only a few candidates are eventually released as new cultivars. C. Culture Maintenance

Prior to 1970, cultivars used for commercial spawn production were maintained on various agars or cereal grains with periodic subculturing of growing mycelium to fresh medium. This method, for the most part, was reliable, although cases of culture degeneration were reported periodically by spawnmakers and researchers. Then, in 1970, San Antonio and Hwang (1970) reported successful preservation and maintenance of stability of spawn stocks of Agaricus bisporus stored in liquid nitrogen. Several subsequent reports (San Antonio 1978; Jong 1978; Jodon et a1. 1982) for culture maintenance using this method have verified the suitability of cryogenic preservation. These developments fundamentally changed the way spawnmakers handled the cultures used for commercial spawn production. In practice, cryogenic preservation is used to ensure use of superior spawn-starter cultures. Many vials (perhaps as many as 200 to 300) containing spawn or mycelium from cultures of promising spawn lines are stored in liquid nitrogen. Following successful testing of the spawn lines at both pilot plant and commercial testing facilities, the spawnmaker is able to easily reproduce the superior lines many times during subsequent years. III. MUSHROOM PRODUCTION TECHNOLOGY A list of cultivated species and their common English and Japanese names is given in Table 2.1. Twelve genera comprise the bulk of cultivated mushrooms as outlined below.

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Table 2.1. Scientific and English and Japanese names for some cultivated specialty mushrooms. Scientific Name

Agrocybe cylindracea (DC.: Fr.) Maire Armillaria mellea (Vahl:Far.) Kummer Auricularia auricula (Hook) Underw. Auricularia polyticha (Mont.) Sacco Coprinus comatus (Mull.:Fr.) S.F.Gray Cordyceps sinensis (Berk.) Sacco Creolophus pergamenus Karsten Dictyophora indusiata (Vent.:Pers.) Fisch. Flammulina velutipes (Curt.:Fr) Karst. Ganoderma lucidum (Leyss.:Fr.) Karst. Grifola frondosa (Dicks.:Fr.) S.F.Gray Hericium erinaceus (Bull.:Fr.) Pers. Hypsizygus marmoreus (Peck) Bigelow Lentinula edodes (Berk.) Pegler Lepista nuda (Bull.:Fr.) Cook Lyophyllum decastes (Fr.:Fr.) Sing Morchella esculenta Pers. ex St. Amans Naematoloma sublateritivum Karsten Pleurotus abalonus Han et a1. Pleurotus cornucopiae (PauL) Roll. Pleurotus cystidiosis O.K. Miller Pleurotus djmour (Fr.) Boedijn Pleurotus ostreatus (Jacq.:Fr.) Kumm Pleurotus pulmonarius (Fr.) Quel. Panellus serotinus (Fr.) Kuh. Pholiota nameko (T.Ito) S.Ito et Imai Pholiota adiposa (Fr.) Quel Tremella fuciformis Berk. Tricholoma matsutake (Ito et Iman) Sing. Tuber aestivum Vitt. Tuber magnatum Pico ex Vitt. Tuber melanosporum Vitt. Volvariella diplasia (Berk & Br.) Sing. Volvariella volvacea (Bull.:Fr.) Sing.

English Name South popular Chiodini, honey Black ear, wood ear Cloud ear, tree ear, wood ear Shaggy ink cap, lawyer's wig Chinese caterpillar fungus Bear's head Bamboo sprouts, collared stinkhorn Winter, velvet stem, golden, snow puff Ling-zhi, reishi Hen of the woods Monkeyhead, bear's head Shimeji Black forest, black, oak Blewit Fried chicken Morel Bricktop, chestnut Abalone Golden oyster, horn of plenty Ohritake Rose, pink oyster Oyster, white oyster, gray oyster Phoenix-tail Green oyster, late fall oyster Vicid, nameko Fat pholiota Snow fungus, silver ear, white jelly Pine

Japanese Name Yangimatusutake Naratake Kikurage Angekikurage

Banshariake Enokitake Reishi Maitake Bunashimeji Shiitake Hatakeshimeji Kuritake Kuroawabitake Tamogitake

Hiratake Mukitake Nameko Numerisugtake Shirokikurage Matsutake

Summer truffle Piedmont white truffle Perigord black truffle Banana, straw Fukurotake Straw, paddy straw

A. Auricularia spp. Commonly known as wood ear, Auricularia auricula is the first recorded cultivated mushroom (Chang 1993). Total production of Auricularia spp. in 1991 exceeded 465,000 t fresh wt (Table 2.2). This value is an increase of 346,000 t or 290% over 1986 levels (Chang 1993). Auricularia spp. production in 1991 represented about 11 % of the total cultivated mushroom supply worldwide. The health effects of high dietary fiber content and hypocholesterolemic properties of Auricularia spp. have been promoted by the Japanese and Chinese for many years (Misaki and Kakuta 1995). Among edible mushrooms, Auricularia spp. are nearly the highest in dietary fiber because of the high proportion of indigestible polysaccharides, such as (1-3)-p-glucans and acidic polysaccharides (Kurasawa et al. 1982). The addition of 1 to 5% crude, powdered Auricularia auricula polysaccharide to a high-cholesterol rat diet, lowered serum cholesterol levels after 2 weeks. The hypocholesterolemic effects exhibited by these polysaccharides may be attributable to the high-molecular comb-type anionic charged polysaccharides, involving the suppression of absorption of cholesterol from the digestive tract (Misaki and Kakuta, 1995). Members of the genus Auricularia are distributed worldwide (Lowy 1951, 1952). Some authors have proposed as many as 50 species (Saccardo 1882-1931), while others suggest that all tropical auri-

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cularias belong to single, widely distributed, and highly variable species (Holterman 1898; Moller 1895). Many authors (Quimio 1982a; Duncan and Macdonald 1967; Duncan 1972) now accept the recognition of 10 species according to those described by Lowy (1952). Auricularia auricula and A. polytricha commonly are produced on a synthetic medium consisting of sawdust, cottonseed hulls, bran, and other cereal grains or on natural logs of broad-leaf trees (Quimio 1982b; Chang and Quimio 1982; Oei 1991). For cultivation on naturallogs, members of the oak family (Fagaceae) are preferred, but many other species of both hard and softwoods can be used. In general, the species of Auricularia are very diverse in terms of their ability to grow on a wide variety of tree species (Oei 1991). For synthetic medium production of Auricularia, the substrate may be composted for up to 5 days or used directly after mixing. In either case, the mixed substrate (about 2.5 kg wet wt) is filled into heatresistant polypropylene bags containing a microbial-resistant breather strip and sterilized (substrate temperature 121°C) for 60 min. Composted substrate is prepared by mixing and watering ingredients (sawdust [78%]:bran [20%]:CaC0 3 [1 %]:sucrose [%] ) in a large pile. The pile then is covered with plastic and turned (remixed) two times total at 2-day intervals. For direct use of substrate, a mixture of cotton seed hulls (93%), wheat bran (5%), sucrose (1 %) and CaC0 3 (1 %) is moistened to about 60% moisture and then filled into polypropylene bags. Sterilization is the same as outlined for composted substrate. After the substrate has cooled, it is inoculated with either grain or sawdust spawn, either mechanically or by hand. After inoculation, the mycelium is allowed to colonize the substrate (spawn run). Temperatures for spawn run are maintained at about Z5 ± ZOC for about 28 to 30 days. Light intensity of more than 500 lux during the spawn run may result in premature formation of primordia. To promote formation of primordia at the end of spawn run, light intensity may be increased to about 2000 lux and the temperature lowered to 20 ± 2°C. Six to eight holes of about 2.5 em are cut in the bags to allow emergence and maturation of the fruitbodies. Temperature, light intensity, and relative humidity (RH) all interact to influence the nature and quality of the basidocarps. In Fujian, China, a system has been worked out to intercrop Auricularia spp. with sugar cane. Bags containing colonized substrate are suspended in mid-air on a rope stretched between rows of sugar cane. The bags then are covered with a thin layer of plastic to help

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regulate RH. Carbon dioxide generated from the growing mycelium apparently stimulates the growth of the sugar cane (Oei 1991).

B. Flammulina velutipes

Worldwide production of F. velutipes (enokitake) has increased from about 100,000 tin 1986 to about 187,000 tin 1991 (an 87% increase). Japan is the main producer of enokitake (Furukawa 1987). In 1986, Japan produced 74,387 t-by 1991, production had risen to 95,123 t (Royse 1995). In 1993, Japan produced 103,357 t-an increase of about 8%. From these data, a faster growth in total production is occurring in other countries. In the United States, for example, enokitake production has increased at an estimated rate of about 25% per year for the last 4 years (D. J. Royse, unpublished). Production of most enokitake in Japan is based on synthetic substrate contained in polypropylene bottles (Fig. 2.1). Substrates (primarily sawdust and rice bran; 4:1 ratio) are mechanically mixed and filled into heat resistant bottles with a capacity of 800 to 1000 mL. Sawdust consisting primarily of Cryptomeria japonica, Chamaecyparis obtusa, or aged (9-12 months) Pinus spp. appears to produce the best yields. In the United States, ground corn cobs supplemented with bran serve as the primary medium. After being filled into bottles, the substrate is sterilized in a two-step process (4 h at 95°C and 1 h at 120°C), mechanically inoculated, and incubated at 18 to 20°C for 20 to 25 days. When the substrate is fully colonized, the original inoculum is removed mechanically from the surface of the substrate and the bottles may be placed upside down for a few days. At the time of original inoculum removal, the air temperature is lowered to 10 to 12°C for 10 to 14 days. To improve quality during fruiting, temperatures are lowered to 3 to 8°C until harvest. As the mushrooms begin to elongate above the lip of the bottle, a plastic collar is placed around the neck and secured with a Velcro strip. This collar serves to hold the mushrooms in place so that they are long and straight. When the mushrooms are 13 to 14 cm long, the collars are removed and the mushrooms are pulled as a bunch from the substrate. The mushrooms then are vacuum packed and placed into boxes for shipment to market. Usually only one flush of mushrooms is harvested prior to mechanical removal of the "spent" substrate from the bottles. In some cases, a second flush may be harvested but the quality of the mushrooms is inferior to that of the first flush.

~

Fig.2.1. Production of enokitake (Flammulina velutipes) on synthetic substrate contained in polypropylene bottles: (a) applying collars to maintain straight mushroom stipes; (b) collar removed to show maturing mushrooms; (c) vacuum packaging of mushroom clusters; and (d) packing mushrooms for delivery to market.

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C. Ganoderma lucidum

Known as reishi or mannentake to the Japanese and Ling Zhi to the Chinese, G. lucidum is renowned for its medicinal properties (for an extensive review see Willard [1990]). Traditionally, reishi is associated with health and recuperation and longevity (Stamets 1990, 1993). It is believed that certain triterpenes and polysaccharides may ac-

Fig. 2.2. Production of reishi (Ganoderma lucidum) in (a) bottles and (b) plastic bags. (Photo courtesy of Chow-Chin Tong, University of Pertanian, Malaysia.)

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count for the multiple activities of reshi. Considerable time and effort has gone into the isolation and characterization of these compounds. About 100 triterpenes have been isolated from either the sporocarps or mycelium but few have been tested for bioactivity (Mizuno et a1. 1995). Most cultivation of G. lucidum is on supplemented sawdust contained in heat-resistant polypropylene bottles (Fig 2.2a) or bags (Fig 2.2b). Sawdust of hardwoods is supplemented with rice bran (10%) and CaC0 3 (3%), moistened with water, and filled (700 g) into plastic bags. A plastic collar then is fitted onto each bag and stoppered with a cotton plug. After heat treatment (95-100°C for 5 h), the substrate is allowed to cool overnight and then inoculated with grain or sawdust spawn. The inoculated substrate then is incubated for 3 to 4 weeks or until the spawn has fully colonized the substrate. Mushroom production is initiated by stacking the colonized substrate horizontally (Fig. 2.2b). Air temperatures are maintained at about 28°C, with relative humidity in the range of 85-90%. Spray nozzles operated automatically can be used to mist the exposed ends of the bagged substrate. Basidiocarps begin to appear about 1 to 2 weeks after the start of misting. Approximately 2 to 3 months after the appearance of primordia, mushrooms are ready to harvest. A mushroom is considered mature when the whitish margin around the edge of the basidiocarp has turned red. The substrate may yield another harvest of mushrooms after removal of the first flush. Biological efficiencies (ratio of fresh mushrooms harvested to oven-dry substrate) average around 30% for this system. Another system, recently adapted in the United States, involves the use of colonized "wafers" (2.5 em thick, 10 em diameter) of oak wood. The wafers are placed in plastic bags, autoclaved, and inoculated with G. lucidium spawn. After the wafers are colonized, they are removed from the plastic bags, placed in horticultural plastic pots, and cased (covered) with a layer of moistened peat moss. Sporocarps then emerge from the casing layer and are harvested and processed into tablets or powder.

D. Grifola frondosa

Japan is the major producer and consumer of G. frondosa (Maitake). Commercial production of maitake in Japan (325 t) began in 1981 (Takama et a1. 1981). By 1986, production was 2203 t and, by 1991, production reached 7950 t, a 261 % increase (Chang 1993). Japanese production of maitake reached 9617 t in 1993 (Table 2.3) and was

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Japanese production of Grifola frondosa (Maitake), Hypsizygus marmoreus (Bunashimeji), Flammulina velutipes (Enokitake), and Pholiota nameko (Nameko) from 1981 through 1993.

Table 2.3.

Production (t fresh wt) Year

H. marmoreus

G. frondosa

1981 1983 1985 1986 1987 1989 1991 1993

1,885 4,666 9,157 11,439 13,688 22,349 36,623 48,479

325 699 1,501 2,203 3,015 6,167 7,950 9,617

F. velutipes

53,282 55,769 69,530 74,378 78,129 83,200 95,123 103,357

P. nameko 16,348 18,141 19,793 20,079 21,054 21,125 21,738 22,613

Source. Ohmasa (1994).

produced primarily in the provinces of Niigata, Nagano, Gunnma, and Shizuoka. Most Maitake is marketed as food. However, Maitake has been shown to have both anti-tumor and anti-viral properties (Jong and Birmingham 1990; Jong et al. 1991; Mizuno and Zhuang 1995). Powdered fruitbodies are used in the production of many health foods such as maitake tea, whole powder, granules, drinks and tablets. Maitake also is believed to lower blood pressure, reduce cholesterol, and reduce the symptoms of chronic fatigue syndrome (Jong and Birmingham 1990; Mizuno and Zhuang 1995). Commercial production of most G. frondosa is on synthetic substrate contained in polypropylene bottles or bags (Fig 2.3). A common substrate used for production is composed of sawdust supplemented with rice bran or wheat bran in a 5:1 ratio, respectively (Takama et al. 1981). For bottle production, the containers are filled with moistened substrate and sterilized or pasteurized prior to inoculation. Most growers use automated inoculation equipment, thereby saving on labor costs. For production in bags, the moistened substrate (2.5 kg) is filled into microfiltered polypropylene bags and sterilized to kill unwanted competitive microorganisms. After cooling (16 to 20 h), the substrate is inoculated and the bags are heat-sealed and shaken to uniformly distribute the spawn throughout the substrate. Spawn run lasts about 30 to 60 days, depending on strain and substrate formulation. Temperatures then are lowered from about 22 to 14°C to induce fruiting and fruitbody maturation.

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Fig. 2.3. Maitake (Grifola frondosa) fruiting on substrate contained in plastic bags.

E. Hericium erinaceus In the wild, H. erinaceus occurs on old logs and stumps and on wounds of living trees, especially maple, beech, oak and hickory. The sporocarp is formed as a large white mass (5 to 30 cm across) that is toothed in many small tufts. The Chinese were the first to domesticate the fungus and, in 1991, production reached 66,000 t (Table 2.1). In Japan, the mushroom is cultivated on synthetic substrate in bags and bottles and on logs. It is sold for $10 to $15 per kilogram. Polysaccharides in Hericium spp. are believed to inhibit a variety of cancers by enhancing immune functions (Mizuno 1995b). It also has been suggested that the phenol-analogous compounds hericenone-C, -D, -E, and Y-A-8-c, which induce the synthesis of nerve growth factor, might be effective in treating patients suffering from Alzheimer's disease (Mizuno 1995b).

F. Hypsizygus marmoreus The Japanese are the main producers and consumers of H. marmoreus. Bunashimeji production has increased steadily over the last few years although not as fast as some other types of mushrooms (Royse 1995). In 1986, production of Bunashimeji was 11,439 t in

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Japan; by 1991 production reached 36,623 t, an increase of 220% (Table 2.3). Production of H. marmoreus increased 38% in the 2 year period 1991-1993. The antitumor polysaccharide, f3-(1-3)-D-glucan, isolated from H. marmoreus showed very high activity (Ikekawa 1995). The water solubility of the polysaccharide was much higher (and therefore more biologically active) than the same polysaccharide isolated from other fungi. Dried mushroom powder from this mushroom is believed to stimulate radical-trapping activity of blood (Ikekawa, 1995). Excessive free radicals in the bloodstream are believed to hasten the aging process. Bunashimeji usually is produced in polypropylene bottles contained in plastic trays. After the completion of vegetative mycelial growth, bottle lids are removed and the colonized substrate is subjected to environmental conditions known to stimulate fruiting. When the mushrooms are mature, entire clusters of sporocarps are removed from the bottles. The mushrooms are packaged by placing an entire cluster (or multiple clusters) into each overwrapped package. Only one flush of mushrooms is harvested prior to mechanical removal of the "spent" substrate from the bottles. The bottles then are refilled with fresh substrate consisting of supplemented sawdust of cottonwood, willow, oak, alder, beech or elm (Stamets 1993) and the process is repeated. G. Lentinula edodes

The cultivation of L. edodes (shiitake) first began in China about 1100 AD (Nakamura 1983; Royse et a1. 1985; Chang and Miles 1987, 1989). It is believed that shiitake cultivation techniques were probably introduced to Japanese farmers by Chinese growers (Ito 1978). Shiitake is one of the best-known and best-characterized mushroom used for medicinal purposes. Medicinal properties include antitumor polysaccharides and glycoproteins, antiviral nucleic acids, platelet agglutination inhibitive substances, and anti-cholesterol active substances (Tokuda et a1. 1974; Fujii et a1. 1978; Suzuki et a1. 1979; Tokuda and Kaneda 1978; Ying et a1. 1987; Mizuno 1995a). Lentinan, a f3-D-glucan (polysaccharide), has shown antitumor activity (Breene 1990; Mizuno 1995a). Antitumor activity of lentinan is thought to be due to activation of immune functions. It is believed that lentinan binds to the surface layer of lymphocytes or to a specific serum protein. This binding process activates macrophage, T cells, NK cells, and other effector cells and increases production of antibodies, interleukins, and interferon.

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1. Natural Log Production. Shiitake is traditionally cultivated on various species of trees (San Antonio 1981). One of the primary species used in one area of Japan in past years was the shii tree (Castanopsis cuspidata)-thus the derivation of the name shii-take (take = fungus of) (Singer 1961). Most production today, however, is on various species of oak (Ito 1978; Stamets and Chilton 1982; Harris 1986). Natural logs usually are cut in the fall (after leaf drop) and may be inoculated within 15 to 30 days of felling. Trees that are cut in the fall also may be left intact through winter and, just before inoculation, cut into lengths of about 1 m. Trees that are cut in the summer tend to have bark that is more loosely bound and sugar contents usually are lowest during this time. If trees are cut during the summer, the bark may strip off more easily, increasing the chances of contamination of the wood by competitive organisms. The most efficient log diameter appears to be in the 7 -to 15-cm range (Ito 1978). Logs greater than 25 em in diameter are often cut in half (length wise) prior to inoculation (Royse et al. 1985). Once logs are cut to the desired length, they are ready for inoculation. Growers who use wood-piece spawn (Fig 2.4a) drill holes in the logs with high-speed drills to correspond to the diameter and length of the wood-piece spawn. Enough holes are drilled in the log to provide spacing of about one hole per 500 cm 2 • The wood spawn is driven into the holes with a hammer and usually covered with hot wax to prevent excessive drying of the spawn. Sawdust spawn is sometimes used instead of wood-piece spawn. Spawn run (vegetative mycelial growth) may last from 6 to 9 months, depending on the tree species, log size, spawn cultivar, moisture, temperature, and other variables (Leatham 1982). After the spawn run period, logs often are transferred to a "raising" yard (Fig. 2.4b,c). Raising yards usually are cooler and moister than the spawn run area. The change in conditions provides an optimum environment for the growth and development of mushrooms. In addition, in a "shocking treatment" to stimulate primordial formation, logs may be banged with a hammer or dropped on end (Chang and Miles 1989). In the raising yard, the logs are arranged to provide for convenient harvesting of the mushrooms (Fig. 2.4d). Most production occurs in the spring and fall when conditions are most favorable and thus, prices are usually lowest during these periods. Growers may use greenhouses for winter production of mushrooms (Przybylowicz and Donoghue 1988). More overall production is possible, and prices for fresh mushrooms are considerably higher in winter than during the rest of the year. In the greenhouse method,

'.:J

~

Fig. 2.4. Shiitake (Lentinula edodes) production on natural logs: (a) mushroom spawn; (b, c) inoculated logs in raising yards used for production of basidiomes, and (d) shiitake fruiting on oak logs.

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logs usually are soaked in water (usually less than 48 h) and vibrated mechanically for various periods prior to placement in the greenhouse. Soaking allows water to displace COz contained in air spaces, and provides enough moisture for one flush of mushrooms. After the mushrooms are harvested, the logs are incubated further (up to 3 months) and the process is repeated up to five times. Once the logs no longer are productive they may be cut up and used as firewood to provide heat to the greenhouse in winter. 2. Synthetic Log Production. Sawdust is the most popular basal ingredient in synthetic formulations of substrate used to produce shiitake in the United States (Miller and Jong 1987), but other basal ingredients may include straw, corn cobs, or their mixtures. Regardless of the main ingredient used, starch-based supplements (10-35% dry wt), such as wheat bran, rice bran, millet, rye, and maize, are added to the mix. These supplements serve as nutrients to provide an optimum growing medium. Other supplements, added in lesser quantities, include CaC0 3 , gypsum, and table sugar (Royse et al. 1990). Once the proper ratio of ingredients is selected, they are combined in a mixer and water is added to raise the moisture content of the mix to around 60%. On large farms, the mix then is augured to a machine that fills and weighs the substrate so that a uniform amount is filled into each bag (Fig. 2.5a). The filled bags are stacked on racks, loaded into a industrial-sized autoclave (Fig. 2.5b), sterilized for 2 h at 121°C, cooled, and inoculated with shiitake spawn. After a 20- to 25-day spawn run (Fig. 2.5c), the bags are cut off and the substrate blocks are exposed to an environment conducive for browning of the exterior log surfaces (oxidation of surface mycelium, also called artificial bark formation or curing by growers) (Fig. 2.5d). During the browning period (4 weeks) logs are maintained at a temperature of 19°C while COzlevels are maintained at 2200 to 3000 ppm. Logs may be watered lightly once per day to maintain continuous surface moisture, which helps to facilitate the browning process. As the browning process nears completion, primordia begin to form about 2 mm under the surface of the log, indicating that the log is ready to produce mushrooms. Once the logs are ready to fruit, primordia maturation is stimulated by soaking the logs in water (12°C) for 3 to 4 h (Fig. 2.6). Soaking allows water to rapidly displace carbon dioxide contained in air spaces, and provides enough moisture for one flush of mushrooms. After soaking, logs are placed on shelves and mushrooms begin to

'1

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Fig. 2.5. Preparation of synthetic logs for shiitake cluture: (a) filling polypropylene bags with nutrient-supplemented sawdust, (b) autoclave used for sterilizing synthetic medium; (c) spawn run in plastic bags; and (d) browning process prior to induction of basidiomes.

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Fig. 2.6. Placement of synthetic logs of shiitake in water tank for soaking to induce production of basidomes.

enlarge. Approximately 9 to 11 days after soaking, mushrooms are ready to harvest (Fig. 2.7). Mushrooms are twisted from the surface and the residual substrate is removed with a knife or scissors. After all mushrooms have been harvested from the substrate, the logs again are soaked in water. The second soaking may require up to 12 h to replace the water lost through production of mushroom tissue and through evaporation. The average time from the peak harvest of one flush to the peak of the next flush is about 18 days. In Japan, shiitake are now being produced on colonized substrate first incubated then removed from polypropylene bottles (Fig. 2.8) In The Netherlands, shiitake are produced on shelves (Fig. 2.9). These

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Fig. 2.7. Shiitake fruiting from synthetic logs in production room.

developments are an adaptation of previous technology used for producing other edible mushrooms. The main advantages of using synthetic medium over natural logs is reduced time and increased efficiency. The cycle for synthetic medium cultivation lasts approximately 4 months, from inoculation to cleanout. Biological efficiencies for this method average from 75 to 125 %. In contrast, the natural log cultivation cycle usually lasts about 6 years, with maximum efficiencies around 33%. The time required on synthetic substrate is about 6% of the natural system, with about 3 times the yield efficiency. As a result of these developments, shiitake production in the United States has increased dramatically in the last 9 years (Fig. 2.10). H. Morchella esculenta

Morels (Fig. 2.11) are some of the most highly prized mushrooms found in the wild. Researchers have long sought to consistently cultivate the morel, but until recently this was not possible. Ower (1982) described the first successful production of ascocarps of March ell a esculenta under laboratory conditions. Since that first report, several patents (Ower et al. 1986, 1988) have been issued describing a process for the commercial cultivation of these fungi.

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Fig. 2.8. Production of shiitake: (a) vegetative growth on synthetic medium contained in polypropylene bottles; and (b) shiitake fruiting on colonized medium removed from bottles.

At present, one company in the United States (Terry Farms, Auburn, Alabama) is producing morels on a commercial scale. Commercialcultivation involves the production of sclerotia, an early overwintering stage of the mushroom (Fig. 2.12). "Nutrient-primed"

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Fig. 2.9. Production of shiitake in the Netherlands on synthetic medium: (a) vegetative growth of mycelium into substrate covered with plastic sheeting; and (b) basidiomes developing on colonized substrate contained on shelves.

sclerotia are produced in soil placed on a layer of sterilized wheat or rye grain (Ower et al. 1986). The production of nutrient-primed sclerotia requires about 18 to 21 days under optimum conditions. The sclerotia are harvested, soaked in clean water for 24 h, and distributed into a thin layer of pasteurized bark/soil mix. The sclerotia

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SPECIALTY MUSHROOMS AND THEIR CULTIVATION

2500

III II

81

Pleurotus Shiitake

'Ci) £:

£

o

2000 -t---t---t---+---+---+---+---+--ffll+---ml-I

'i:

Q)

S

1500 -t--+--f------1f----+--+--+---+--rnH--mt-l

£:

:u:so

1000 - t - - - + - - + - - + - -

"C

o

0:

500

o

1987 1988 1989 1990 1991 1992 1993 1994 1995 Year

Fig. 2.10. Shiitake and Pleurotus spp. production in the United States from 1987 to 1995 (USDA 1995).

germinate via the production of mycelium. After the mycelium has spread throughout the soil mix, a continuous (12 to 36 h) fine mist of clean water stimulates the formation of ascocarps. Several problems have yet to be solved in the commercial production of morels, including consistent fruiting, control of competitive weed molds, poor yields, and small mushroom size. However, research to further understand the environmental and genetic factors involved in the regulation of fruiting should result in more consistent production, which should lower the cost of commercially produced morels. I. Pleurotus spp.

Oyster mushroom production has increased at a rapid rate worldwide during the last few years (Table 2.2). From 1986 to 1991, oyster mushroom production increased from 169,000 to 917,000 t (442%). China was responsible for most of the production increase. In the United States, production of oyster mushrooms was 881.8 tin 1995, up 94% from the previous year (USDA 1995). Pleurotus spp. (P. ostreatus and P. cornucopiae) production in Japan peaked in 1989 at about 36,000 1. Production was 24,000 t in 1993, a decrease of 33% in 4 years (Royse 1995).

D.

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Fig. 2.11.

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Morcbella esculenta (morel) fruiting in the wild.

In the United States, the primary ingredients used for Pleurotus spp. production is chopped wheat straw or cottonseed hulls or mixtures thereof. For production on wheat straw, the material is milled to a length of about 2 to 6 em. The pH of the material is adjusted with limestone to about 7.5 or higher to provide selectivity against Trichoderma spp. green mold (Stolzer and Grabbe 1991). On some farms, a short (12 to 24 h) fermentation process is used to increase the pH to 8 or higher. After completion of pasteurization (60°C for 1 to 2 h) the substrate is cooled and spawned with the desired strain (Fig. 2.13). At time of spawning, a commercial delayed release supplement consisting of formaldehyde-denatured soybean or corn gluten may be

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SPECIALTY MUSHROOMS AND THEIR CULTIVATION

Fig. 2.12.

83

Sclerotium of morel used to produce ascocarps under controlled

conditions.

added (rates of 3 to 10% of dry substrate wt) to increase yield and size of the mushroom (Royse and Schisler 1987; Royse et al. 1991; Royse and Zaki 1991). Use of supplements, however, may cause overheating of the substrate if growers are unable to anticipate and control air temperatures to maintain a steady substrate temperature. Additional cooling capacity is required when higher levels of supplement are used. Production of Pleurotus spp. on cottonseed hulls has an advantage over straw-based production systems in that chopping of the hulls is not required (Royse 1995). The pasteurized, supplemented hulls are spawned and filled (12 to 15 kg) into clear or black perforated polyethylene bags and then incubated at 23 to 25°C for 12 to 14 days. Similar overheating problems (as with straw-based systems) may occur if higher rates of supplement are used. Upon completion of spawn run, slashes are made in the polyethylene bags to allow for mushroom maturation. Mushrooms are harvested from the substrate approximately 3 to 4 weeks after spawning, depending on strain, amount of supplement used, and temperature of spawn run. In Japan, bottle production of oyster mushrooms is most common (Fig. 2.14). Substrate is filled into bottles, sterilized, and inoculated with Pleurotus spp. spawn. Upon completion of spawn run, bottle lids are removed and mushrooms emerge from the surface of the sub-

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(a) Pleurotus ostreatus spawn (colonizing chopped wheat straw contained in black plastic bag; (b) close up of mycelium colonizing straw.

Fig. 2.13.

strate. After the mushrooms are harvested, they are weighed (Fig. 2.14d) and packaged for shipment to market. Worker exposure to airborne spores is of concern on most farms. In the United States, masks are worn to filter out spores that are released from the maturing mushrooms. Inhaled spores can cause an allergic reaction in some workers. Exposure can be minimized by

co CJl

Fig. 2.14. Production of Pleurotus ostreatus: (a) vegetative mycelial growth in polypropylene bottles; (b) clusters of basidiomes emerging; (c) collected basidomes; and (d) weighing and packaging.

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introducing higher volumes of fresh air 1 to 2 h before harvesting and by wearing a proper mask.

J. Pholiota nameko Nameko (Fig. 2.15a) means "viscid mushroom" in Japanese. This mushroom is prized for its gelatinous viscosity and its flavor and is generally used in miso soup, cooked fresh with grated radish, or steamed in a pipkin. Japan produced 21,738 t of.R nameko in 1991, an increase of 1700 t (8%) from 1986 levels (Tables 2.2,2.3). Worldwide production increases averaged 60% over the same time period. In 1991, Japan produced about 54% of the total world production ofnameko compared to 80% of total production in 1986. Production of nameko rapidly is gaining popularity in other Asian countries. Preparation of the medium for nameko production is similar to that for enokitake except that a higher moisture content of the substrate is desirable. A substrate of broadleaf tree sawdust is preferred but sawdust from conifers such Pinus spp. and Cryptomeriajaponica is suitable for growth (Arita 1978). Rice bran usually is added as a supplement in the ratio of 15% for conifer sawdust and 10% for broad-leaf sawdust. Mushrooms are harvested from the substrate by cutting the stems (Fig. 2.15b) near the base with scissors. The harvested mushrooms are washed (Fig. 2.15c) and packed for shipment to market. K. Tremella fudformis

Known as the white jelly fungus or silver ear, T. fuciformis has been used as a delicacy food in China for many years. This mushroom can be cultivated on natural logs or on synthetic medium (Quimio et a1. 1990). The cultivation technique used to produce the mushroom on natural logs is similar to that used for shiitake production. In recent years, most production of T. fuciformis has been on synthetic substrate using a mixed culture inoculum technique developed in Fujian, China (Huang 1982). The mixed culture technique involves the use of "helper" mycelium of Hypoxylon archeri, an ascomycete commonly associated in nature with decaying wood. Hypoxylon archeri increases the ability of T. fuciformis to digest the substrate thereby increasing the yields. Exploitation of this mycelial association is accomplished through use of dual cultures to make mother spawn (Quimio et a1. 1990). Tremella mycelium is subcultured into a number of test tubes that

CXl '1

Fig. 2.15. Production of nameko (Pholiota nameko): (a) basidomes emerging from substrate contained in bottles; (b) harvesting; (c) washing; and (d) packaging.

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are incubated until the colony diameter has reached 1 cm. The mycelium of H. archeri is then inoculated into each tube and allowed to grow into the mycelium of Tremella. Spawn (hardwood sawdust and rice or wheat bran, 5:1 ratio) may be produced from the dual culture (mother culture). Substrate used for mushroom production is the same as that used for spawn production. The supplemented substrate is packed into plastic bags (50 cm long; 9 cm diam) and ends of the bags are tied with cotton string. Six holes (1 cm diam) then are punched in the filled bags and covered with a breathable fabric. The substrate is sterilized for 6 to 8 h, cooled, and inoculated with the mother culture. After about 30 days of vegetative mycelial growth, the hole covers are removed and the exposed substrate is exposed to conditions favorable for primordia formation (Huang 1982). If optimum conditions are maintained in the growing houses, clusters of jelly fungus should be ready for harvest within 12 to 15 days. Yield for each bag of substrate is in the range of 350 to 500 g fresh weight (35 to 50 g dry wt). L. Volvariella spp.

The straw mushroom derives its name from the substrate on which it originally was grown (San Antonio and Fordyce 1972). Cultivation of Volvariella was believed to have begun in China as early as 1822 (Chang 1977). In the 1930s, straw mushroom cultivation began in the Philippines, Malaysia, and other Southeast Asian countries (Chang 1982). Production of the straw mushroom increased from 178,000 t in 1986 to about 253,000 t in 1991, a 42% increase. Volvariella accounts for approximately 6% of the total worldwide production of edible mushrooms (Table 2.2). Many agricultural by-products and waste materials have been used to produce the straw mushroom. These include paddy straw, water hyacinth, oil palm bunch, oil palm pericarp waste, banana leaves and sawdust, cotton waste, and sugarcane waste (Chang 1982; Ho 1985). Volvariella is well suited for cultivation in the tropics because of its requirement for higher production temperatures. In addition, the mushroom can be grown on nonpasteurized substratemore desirable for low input agricultural practices. In recent years, cotton wastes (discarded after sorting in textile mills) have become popular as substrates for straw mushroom production (Chang 1982). Cotton waste produces higher and more stable biological efficiencies (30 to 45%), and earlier fructification (4 days

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after spawning) and harvesting (first 9 days after spawning) than that obtained using straw as a substrate. Semi-industrialization of paddy straw cultivation on cotton wastes (Fig. 2.16) has occurred in Hong Kong, Taiwan, and Indonesia as a result of the introduction of this method (Chang 1979).

Fig. 2.16. Cultivation of paddy straw mushroom (Volvariella volvacea) on cotton waste near Hong Kong: (a) primordia forming on substrate; and (b) basidiomes emerging.

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IV. MARKETING

Marketing of specialty mushrooms in the United States is a relatively new enterprise. Since 1984, some farms have seen their production rise as prices have fallen. For example, Donovan (1991) indicated that production of shiitake on their farm has increased from slightly less than 1 t per week in 1984 to over 7 t per week in 1990, while the price decreased from US$12.50/kg to about $8.80/kg. In the 1994-95 growing season, the price growers received for shiitake was about $8.43/kg (USDA 1995). Over the past 7 years (1987-1995) the price of shiitake has declined an average of $0.17/kg per year (USDA 1995). In recent years, the trend for specialty mushroom sales has been toward the retail market (Gunn 1992; Sorenson 1992). This trend is driven partly by an increased interest in specialty mushrooms and by the convenience packaged products offer to the consumer. In some retail markets, only 10% of the customers buy 90% of the specialty types (Gunn 1992). Specialty mushrooms such as shiitake and oyster mushrooms typically are packaged and sold at retail in units of 100 g (3.5 oz). Often specialty mushrooms are used to highlight the common cultivated mushroom, which may be sold packaged (whole or sliced) or in bulk. In fact, some purveyors insist that specialty mushrooms should not be banished to a specialty section but should be kept aligned with the mushroom section next to other best-selling produce (Gunn 1992). Some merchandisers have projected a steady growth in consumption of specialty mushrooms. As consumers become more aware of specialty mushrooms, demand is expected to increase and aggressive marketing should help to find new markets for these relatively new products. Specialty mushroom producers seeking new outlets for their mushrooms may want to check sources listing reputable produce industry firms (Anon. 1996a, b). Specialty mushrooms are sold fresh, dried (Fig. 2.17), or processed (Fig. 2.18) in Japan and China. In Japan, most fresh shiitake is collected and shipped to central wholesale markets, where brokers and other participants buy the mushrooms through a bidding process (Hara 1988), and then distributed to retailers. Mushrooms, such as Pleurotus, may be packaged at the farm and shipped directly to brokers or to retailers. In Japan, dried shiitake is distributed through specialized traders (Rara 1988). In 1988, about 400 traders purchased shiitake at special bidding markets and then distributed the product to retailers for in country consumption or to trading firms for overseas export. In re-

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Fig. 2.17. Handling of dried shiitake in China: (a) sorting and grading; (b) transporting to market.

cent years, however, exports of shiitake from Japan have declined as the number of shiitake producers have declined and shiitake production has decreased (Anon. 1992; Royse 1995). Chinese production of shiitake and exportation of the product to Japan has increased dramatically in the last five years.

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Fig. 2.18. Processed products (nutritional and medicinal) made from specialty mushrooms.

~

FUTURE PROSPECTS

Based on recent and historical trends, it is expected that diversification of the mushroom industry will continue in the United States and many other western countries. As consumers become more aware of the additional culinary characteristics offered by a variety of mushrooms, demand for specialty mushrooms will increase. The development of improved technology to cultivate each species more efficiently will allow the consumer price to decline. At the same time, product quality should increase, furthering demand. At present, it appears that shiitake would hold the greatest future potential for production increases in the United States and perhaps other western countries. This mushroom has a relatively long shelf life, is becoming widely accepted by consumers, and is available at a reasonable price. The pace of additional price reductions for consumers is expected to slow, however, in the future. A temporary plateau may have been reached in production efficiencies for this mushroom. Additional research is needed to find ways to reduce the time of spawn run and browning and to shorten the cycle between breaks. It is anticipated thatPleurotus spp. production in the United States will continue to increase due to their relative ease of production.

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However, research is needed to extend the shelf life of these mushrooms and to improve management of sporulation during cultivation. Production of various species of Pleurotus allows growers to take advantage of the many colors of the basidiome for marketing purposes. In the United States, some growers currently are marketing overwrapped packages containing several species of mushrooms. To date, these have been well received by consumers. Growth in the production of maitake is also expected to accelerate in future years, particularly in the United States. Consumers appear to have received enthusiastically the introduction of this mushroom to the marketplace. Research is need, however, to increase biological efficiency and productivity. Such increases would reduce grower costs and ultimately lower the price of mushrooms for consumers. Enoki production has increased substantially in the United States over the past 5 years. This trend is expected to continue and perhaps to accelerate in the years to come. These mushrooms most likely will be produced using technology developed in Japan and adapted for local conditions and availability of raw materials. It will be difficult for small growers to enter this market because of the large capital requirements needed to establish a production facility. Therefore, it is anticipated that a few large companies will dominate the production of this species in the future. The other mushrooms that consumers may find increasingly available on grocery store shelves are morels. As additional research is conducted to increase mushroom yield and mushroom size, it is anticipated that the cost of production will decrease. Production cost savings then could be passed on to consumers in the form of reduced costs. Finally, as economies improve in Central and South America and in Eastern Europe, production of specialty mushrooms could increase at an even faster rate than in the United States. The culinary advantages offered by specialty mushrooms bode well for the continued growth and development of the specialty mushroom industry worldwide.

LITERATURE CITED Anon. 1992. Discussion of fresh shiitake imports (in Japanese). Special Produce Information 13(10):16-27. Anon. 1996a. The red book, Vol. 107. Vance, Overland Park, KS.

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Anon. 1996b. The red book produce industry handbook Vance, Overland Park, KS. Arita, I. 1978. Pholiota nameko. In: S. T. Chang and W. A. Hayes (eds.), The biology and cultivation of edible mushrooms. Academic, New York. Breene, W. M. 1990. Nutritional and medicinal value of specialty mushrooms. Food Protection 53 :883-894. Chang, S. T. 1977. The origin and early development of straw mushroom cultivation. Econ. Bot. 31:374-376. Chang, S. T. 1979. Cultivation of Volvariella volvacea from cotton-waste composts. Mushroom Sci. 10:609-618. Chang, S. T. 1982. Cultivation of Volvariella mushrooms in Southeast Asia. In: S. T. Chang and T. H. Quimio (eds.), Tropical mushrooms: biological nature and cultivation methods. Chinese Univ. Press, Hong Kong. Chang, S. T. 1993. Mushroom biology: the impact on mushroom production and mushroom products. In: S. T. Chang et a1. (ed.), Mushroom biology and mushroom products. Chinese Univ. Press, Hong Kong. Chang, S. T., and P. G. Miles. 1987. Historical record of the early cultivation of Lentinus in China. Mush. J. Tropics 7:31-37. Chang,S. T., and P. G. Miles. 1989. Edible mushrooms and their cultivation. CRC, Boca Raton, FL. Chang, S. T., and T. H. Quimio (eds.) 1982. Tropical mushrooms: biological nature and cultivation methods. Chinese Univ. Press, Hong Kong. Chang, S. T., J. A. Buswell, and P. G. Miles. 1993. Genetics and breeding of edible mushrooms. Gordon & Breach Science, Philadelphia. Donovan, K. 1991. Marketing specialty mushrooms. Mushroom News 39(8):9-10. Duncan, E. G. 1972. Microevolution in Auricularia polytricha. Mycologia 64:394404. Duncan, E. G., and J. A. Macdonald. 1967. Microevolution in Auricularia auricula. Mycologia 59:803-818. Farr, D. F. 1983. Mushroom industry: diversification with additional species in the United States. Mycologia 75:351-360. Flegg, P. B., D. M. Spencer, and D. A. Wood. 1985. The biology and technology of the cultivated mushroom. Wiley, New York. Fujii, T., H. Maeda, F. Suzuki, and N. Ishida. 1978. Isolation and characterization of a new antitumor polysaccharide, KS-2, extracted from culture mycelia of Lentinus edodes. J. Antibiot. 31:1079-1090. Furukawa, H. 1987. Mushroom production in Japan. Farming Jpn. 22(6):12-23. Giovannetti, G., N. Roth-Bejerano, E. Zanini, and V. Kagan-Zur. 1994. Truffles and their cultivation. Hort. Rev. 16:71-107. Gunn, J. 1992. From specialty to mainstream. Mushroom News 40(5):18-22. Hara, Y. 1988. Trends of the mushroom market. Farming Jpn 22(6):28-37. Harris, B. 1986. Growing shiitake commercially. Science Tech, Madison, WI. Hawksworth, D. L. 1991. The fungal dimension of biodiversity: magnitude, significance, and conservation. Myco1. Res. 95:641-655. Ho, K. Y. 1985. Indoor cultivation of straw mushroom in Hong Kong. Mushroom Newsletter Tropics 6(2):4-9. Holtermann, D. 1898. Mykologische Untersuchungen aus den Tropen. Borntaeger. Berlin. Huang, N. L. 1982. Cultivation of Tremella fuciformis in Fujian, China. Mushroom Newsletter for the Tropics 2(3):2-5. Ikekawa, T. 1995. Bunashimeji, Hypsizigus marmoreus antitumor activity of extracts and polysaccharides. Food Rev. Int. 11:207-209.

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Ito, T. 1978. Cultivation of Lentinus edodes. In: S. T. Chang and W. A. Hayes (eds.), The biology and cultivation of edible mushrooms, Academic, New York. Jodon, M. H., and D. J. Royse, and S. C. Jong. 1982. Productivity of Agaricus brunnescens stock cultures following 5-, 7-, and 10-year storage periods in liquid nitrogen. Cryobiology 19:602-606. Jong, S. C. 1978. Conservation of the cultures. In: S. T. Chang and W. A. Hayes (eds.), The biology and cultivation of edible mushrooms. Academic, New York. Jong, S. C., and J. M. Birmingham. 1990. The medicinal value of the mushroom Grifola. World J. Microbiol. Biotechnol. 6:227-235. Jong, S. C., J. M. Birmingham, and S. H. Pai. 1991. Immunomodulatory substances of fungal origin. EOS - J. Immunol. Immunopharmacol. 11:115-122. Kendrick, B. 1985. The fifth kingdom. Mycologue, Waterloo, Ontario, Canada. Kurasawa, S., T. Sugahara, and J. Hayashi. 1982. Studies on dietary fiber of mushrooms and edible wild plants. Nutr. Rep. Int. 26:167-173. Leatham, G. F. 1982. Cultivation of shiitake, the Japanese forest mushroom, on logs: a potential industry for the United States. Forest Prod. J. 32:29-35. Lowy, B. 1951. A morphological basis of classifying the species of Auricularia. Mycologia 43:315-358. Lowy, B. 1952. The genus Auricularia. Mycologia 44:656-691. Miller, M., and S. C. Jong. 1987. Commercial cultivation of shiitake in sawdust filled plastic bags. Developments in Crop Science, Cultivating Edible Fungi 10:421426. Misaki, A., and M. Kakuta. 1995. Kikurage (tree-ear) and shirokikurage (white jellyleaf): Auricularia auricula and Tremella fuciformis. Food Rev. Int. 11:211-218. Mizuno, T. 1995a. Shiitake, Lentinus edodes: functional properties for medicinal and food purposes. Food Rev. Int. 11:111-128. Mizuno, T. 1995b. Yamabushitake, Hericium erinaceum: bioactive substances and medicinal utilization. Food Rev. Int. 11:173-178. Mizuno, T., and C. Zhuang. 1995. Maitake, Grifola frondosa: pharmacological effects. Food Rev. Int. 11:135-149. Mizuno, T., G. Wang, J. Zhang, H. Kawagishi, T. Nishitoba, and J. Li. 1995. Reishi, Ganoderma lucidum and Ganoderma tsugae: bioactive substances and medicinal effects. Food Rev. Int. 11:151-166. Moller, Z. 1895. Protobasidiomyceten. Fischer, Jena. Nakamura, N. 1983. An historical study in shiitake (mushroom) culture. Tosen Shuppon, Tokyo. Oei, P. 1991. Manual on mushroom cultivation: techniques, species and opportunities for commercial application in developing countries. Tool, Amsterdam. Ohmasa, M. 1994. Mushroom production in Japan. Forestry and Forest Products Research Institute, Danchi-Nai, Ibaraki, Japan. Ower, R. 1982. Notes on the development of the morel ascocarp: Morchella esculenta. Mycologia 74: 142-144. Ower, R. D., G. 1. Mills, and J. A. Malachowski. 1986. Cultivation of Morchella. U.S. Patent 4,594,809. Ower, R. D., G. 1. Mills, and J. A. Malachowski. 1988. Cultivation of Morchella. U.S. Patent 4,757,640. Przybylowicz, P., and J. Donoghue. 1988. Shiitake growers handbook. Kendall/Hunt, Dubuque, IA.

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Quimio, T. H. 1982a. Taxonomy and basidiocarp development of Auricularia mushrooms. p. 383-396. In: S. T. Chang and T. H. Quimio (eds.), Tropical mushrooms: biological nature and cultivation methods. Chinese Univ. Press, Hong Kong. Quimio, T. H. 1982b. Physiological considerations of Auricularia spp. In: S. T. Chang and T. H. Quimio (eds.), Tropical mushrooms: biological nature and cultivation methods. Chinese Univ. Press, Hong Kong. Quimio, T. H., S. T. Chang, and D. J. Royse. 1990. Technical guidelines for mushroom growing in the tropics. FAO Plant Production and Protection Paper 106. FAO United Nations, Rome. Raper, C. A. 1978. Sexuality and breeding. In: S. T. Chang and W. A. Hayes (eds.), The biology and cultivation of edible mushrooms. Academic, New York. Raper, C. A. 1985. Strategies for mushroom breeding. p. 513-528. In: D. Moore, L. A. Casselton, D. A. Wood and J. c. Frankland (eds.), Developmental biology of higher fungi. Cambridge Univ. Press, Cambridge, UK. Royse, D. J. 1985. Effect of spawn run time and substrate nutrition on yield and size of the shiitake mushroom. Mycologia 77:756-762. Royse, D. J. 1995. Specialty mushrooms: cultivation on synthetic substrate in the USA and Japan. Interdisciplin. Sci. Rev. 20:205-214. Royse, D. J., and L. C. Schisler. 1987. Yield and size of Pleurotus ostreatus and Pleurotus sajor-caju as effected by delayed-release nutrient. Appl. Microbiol. Biotechnol. 26:191-194. Royse, D. J., and S. A. Zaki. 1991. Yield stimulation of Pleurotus flabellatus by dual nutrient supplementation of pasteurized wheat straw. Mushroom Sci. 13:545547. Royse, D. J., L. C. Schisler, and D. A. Diehle. 1985. Shiitake mushrooms: consumption, production and cultivation. Interdisciplin. Sci. Rev. 10:329-335. Royse, D. J., B. D. Bahler, and C. C. Bahler. 1990. Enhanced yield of shiitake by saccharide amendment of the synthetic substrate. Appl. Environ. Microbiol. 56:479-482. Royse, D. J., S. L. Fales, and K. Karunanandaa. 1991. Influence of formaldehydetreated soybean and commercial nutrient supplementation on mushroom (Pleurotus sajor-caju) yield and in vitro dry matter digestibility of spent substrate. Appl. Microbiol. Biotechnol. 36:425-429. Saccardo, P. O. 1882-1931. Sylloge fungorum 6, 11, 15. San Antonio, J. P. 1978. Stability of spawn stocks of the cultivated mushroom stored for nine years in liquid nitrogen (-160 to -196°C). Mushroom Sci. 10:103-112. San Antonio, J. P. 1981. Cultivation of the shiitake mushroom (Lentinus edodes (Berk.) Sing.). HortScience 16:151-156. San Antonio, J. P. 1984. Origin and improvement of spawn of the cultivated mushroom Agaricus brunnescens Peck. Hort. Rev. 6:85-119. San Antonio, J. P., and C. Fordyce. 1972. Cultivation of the paddy straw mushroom Volvariella volvacea (Bull. ex. Fr.) Sing. HortScience 7:461-464. San Antonio, J. P., and S. W. Hwang. 1970. Liquid nitrogen preservation of spawn stocks of the cultivated mushroom, Agaricus bisporus (Lange) Sing. J. Am. Soc. Hort. Sci. 95:565-569. Singer, R. 1961. Mushrooms and truffles, Leonard Hill, London. Sorenson, R. 1992. Marketing of specialty mushrooms in California. Mushroom News 40(10):14-15. Stamets, P. 1990. A discussion on the cultivation of Ganoderma lucidum (Curtis:Fr.) Kar.: the reishi or ling zhi mushroom of immortality. McIlvainea 9(2):40-50.

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Stamets, P. 1993. Growing gourmet and medicinal mushrooms. Ten Speed, Berkeley, CA. Stamets, P., and J. S. Chilton. 1982. The mushroom cultivator: a practical guide to growing mushrooms at home. Agarikon, Olympia, WA. Stolzer, S., and K. Grabbe. 1991. Mechanisms of substrate selectivity in the cultivation of edible fungi. Mushroom Sci. 13:141-146. Suzuki, F., C. Suzuki, E. Shimomura, H. Maeda, T. Fujii, and N. Ishida. 1979. Antiviral and interferon-inducing activities of a new peptidomannan, KS-2, extracted from culture mycelia of Lentinus edodes. J. Antibiot. 32:1336-1345. Takama, F., S. Ninomiya, R. Yoda, H. Ishii, and S. Muraki. 1981. Parenchyma cells, chemical components of maitake mushroom (Grifola frondosa S.F. Gray) cultured artificially, and their changes by storage and boiling. Mushroom Sci. 11:767-779. Tokuda, S., and T. Kaneda. 1978. Effect of shiitake mushroom on plasma cholesterol levels in rats. Mushroom Sci. 10:793-796. Tokuda, S., A. Tagiri, E. Kano, Y. Sugawara, S. Suzuki, H. Sato, and T. Kaneda. 1974. Reducing mechanism of plasma cholesterol by shiitake. Mushroom Sci. 9:445-462. U.S. Department of Agriculture. 1995. Mushrooms. Agricultural Statistics Board, Washington, DC. van Griensven, L. F. L. D. 1988. The cultivation of mushrooms. Mushroom Experimental Station, Horst, The Netherlands. Willard, T. 1990. Reishi mushroom: herb of spiritual potency and medical wonder. Sylvan, Issaquah, WA. Wu, L. C. 1990. Mushroom genetics and breeding. Plant Breeding Rev. 8:189-215. Ying, J., S. Mao, Q. Ma, Y. Zong, and H. Wen. 1987. leones of medicinal fungi from China. Science, Beijing.

3

Glucosinolates in Crop Plants E. A. S. Rosa Horticulture Section Universidade de Tras-os-Montes e Alto Douro Vila Real, Portugal R. K. Heaney and G. R. Fenwick Institute of Food Research Norwich Laboratory Norwich Research Park, United Kingdom C. A. M. Portas Horticulture Section Instituto Superior de AgronomHl Lisbon, Portugal

I. II.

III.

Introduction Biodistribution, Chemistry, and Biochemistry A. Occurrence B. Chemistry 1. Structure of Intact Glucosinolates 2. Glucosinolate-Degrading Enzymes C. Biosynthesis 1. Amino Acid Modification 2. Aldoxime Formation 3. Glucosinolate Formation D. Biochemical Genetics of Glucosinolates E. Myrosinase F. Factors Affecting the Composition and Amount of Myrosinase-Induced Breakdown Products Analysis A. Quantitative Methods 1. Preparation and Cleanup of Glucosinolate Extracts 2. Enzymes Used in Glucosinolate Analysis

Horticultural Reviews, Volume 19, Edited by Jules Janick ISBN 0-471-16529-8 © 1997 John Wiley & Sons, Inc. 99

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E. A. S. ROSA, R. K. HEANEY, G. R. FENWICK, AND C. A. M. PORTAS

3.

Reference Glucosinolates and Internal Standards Methods that Measure Total Glucosinolate Content Methods that Measure Individual Glucosinolates 6. Other Methods IV. Levels of Glucosinolates in Plants V. Factors Associated With Glucosinolate Variation A. Genetics B. Morphology and Development C. Environment D. Cultural Practices E. Pests and Diseases F. Soils and Plant Nutrients 1. Soil Type 2. Organic Matter 3. Sulfur 4. Nitrogen 5. Micronutrients G. Water Stress H. Growth Regulators I. Processing 1. Cooking and Pulping 2. Freezing and Refrigerating 3. Dehydrating and Lyophilization 4. Fermenting 5. Others VI. Biological Effects A. Humans and Other Animals 1. Cancer 2. Rapeseed B. Effects of Glucosinolates on Insects and Mites C. Effects of Glucosinolates and Breakdown Products on Microorganisms and Viruses D. Glucosinolates and Plasmodiophora brassicae E. Allelopathic Effects F. Nematodes G. Milk and Meat VII. Flavor VIII. Concluding Remarks/Prospects Literature Cited 4. 5.

I. INTRODUCTION

Reviewing glucosinolate research over a decade ago, Fenwick et al. (1983a) identified three stages of investigative activity, stretching back over a century: (1) the classical structural studies of Gadamer at the end of the last century; (2) the natural product chemical investigations of the groups of O. E. Schulz and Anders Kjaer over the period ca. 1950-70; and (3) interdisciplinary studies responding to

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the importance of rapeseed as an oilseed of commerce and of its defatted meal as a protein-rich animal feedstuff. Today a fourth stage of investigation may be identified, emanating from the role and mechanisms of glucosinolates and their products in protecting plants against fungal and insect attack and in protecting animals and humans against biological processes associated with cellular damage and cancer development. Glucosinolate research, therefore, is expanding into areas and disciplines undreamed of even a few years ago. Coordinated and targeted multisciplinary researches will increasingly be necessary if the compositions of glucosinolate-containing crops are to be most effectively manipulated by conventional selection procedures or through the application of biotechnology so as to exploit current and future market opportunities. Against this complex background of scientific development, market opportunities, and consumer interest, a detailed review of glucosinolates is timely. It is appropriate that this be undertaken from a horticultural perspective, since addressing opportunities for enhancing postharvest quality (nutritional and organoleptic) and exploiting "nutriceutical or functional food" market niches ultimately depend on the success of the crop scientist and primary producer. A number of reviews have been written about glucosinolates and their products, many addressing specific agricultural, food, or biochemical aspects, such as Fenwick et al. (1983a), McGregor et al. (1983), Mawson et al. (1993a,b; 1994a,b,c), Duncan (1991), S0rensen (1990) and Heaney and Fenwick (1995). Our aim is to critically assess the results of recent researches and to highlight areas deserving of future scientific study. II. BIODISTRIBUTION, CHEMISTRY, AND BIOCHEMISTRY A. Occurrence

Authenticated occurrences of glucosinolates are limited to dicotyledonous angiosperms within which the distribution appears to be discontinuous and restricted to a few loci, including the order Capparales, comprising the Capparaceae, Brassicaceae (Cruciferae), Koeberliniaceae, Moringaceae, Resedaceae, and Tovariaceae (Kjaer 1974). Glucosinolates are preponderant in the Brassicaceae, plants of which are cultivated as vegetables, condiments, oilseeds, and forage. Within this family, generally accepted as a natural taxon com-

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E. A. S. ROSA, R. K. HEANEY, G. R. FENWICK, AND C. A. M. PORTAS

prising some 350 genera and 2500 species, a significant minority of these species have been studied for their contents of glucosinolates (for example, Kjaer [1976] refers to over 300). In the other large family listed, Euphorbiacea, glucosinolates are known from only two species and appear to be absent, along with myrosinase (see below), from many more. In general, various parts of a given crucifer (roots, stems, leaves, inflorescences, fruits, and seeds) qualitatively display the same pattern, but changes occur in each glucosinolate from the roots to green organs and seeds (Kjaer 1980). B. Chemistry The chemistry of glucosinolates will be considered from the perspective of structure, cleavage under the influence of enzymes or chemical agents, and biosynthesis. Over the past few years there have been a number of significant developments in the latter area and in the inheritance of individual glucosinolates. 1. Structure of Intact Glucosinolates. The common structural features of intact glucosinolates are shown in Fig.3.1, with details of side chain (R) presented in Table 3.1. Following degradative and physicochemical investigations (see Fenwick et al. 1983a), glucosinolates are considered to be (Z)-cis-N-hydroximinosulfate esters, possessing a side chain R and sulfur-linked D-glucopyranose moiety. While natural glucosinolates appear to contain exclusively a f3-D-glucopyranosyllinkage, Blanc-Muesser et al. (1990) have reported the synthesis of the "pseudo" a-glucoside. The basis of the structural heterogeneity within glucosinolates (and of the biological activities of their chemical and/or enzymatic cleavage products-see below) is the structural diversity and, hence, chemical reactivity associated with variation in the side-chain structure. Such variation, involving aliphatic, aromatic, and heteroaromatic chains, is similar to that found within the protein amino acids, although additional complexity is introduced through biosynthetic chain elongation, oxidation, or glycosylation processes.

R-C- S-I3-D-Glucose

II

N-OSO; Fig. 3.1.

General structure of glucosinolates.

3.

GLUCOSINOLATES IN CROP PLANTS

Table 3.1.

103

Structure of the side chain R of the most common glucosinolates.

Structure of R

Chemical name

Trivial name

Methyl glucosinolate 2-Propenyl or allyl glucosinolate But-3-enyl glucosinolate Pent-4-enyl glucosinolate 2-Hydroxybut-3-enyl glucosinolate 2-Hydroxypent-4-enyl glucosinolate 3-Methylthiopropy 1 glucosinolate 4-Methylthiobutyl glucosinolate 3-Methylsulfinylpropyl glucosinolate 4-Methylsulfinylbutyl glucosinolate 4-Methylsulfinylbut-3enyl glucosinolate 5-Methylsulfinylpentyl glucosinolate 3-Methylsulfonylpropyl glucosinolate 4-Methylsulfonylbutyl glucosinolate

Glucocapparin Sinigrin

Aliphatic glucosinolates CH s CH, =CH-CH,CH, =CH-CH,-CH 2CH, ""CH-CH 2-CH,-CH 2CH, =CH-~H-CH2OH CH, =CH-CH 2-~H-CH,­ OH CH s -S-CH 2-CH 2-CH,CH s -S-CH,-CH 2-CH 2-CH,CH s -SO-CH, -CH,-CH,CH s-SO-CH,-CH, -CH,-CH,CH s -SO-CH-CH -CH,-CH,CH s -SO-CH 2-CH, -CH,-CH,-CH,CH s -SO,-CH 2-CH,-CH,CH s -SO,-CH, -CH, -CH,-CH,-

Gluconapin Glucobrassicanapin Progoitrin Gluconapoleiferin Glucoiberverin Glucoerucin Glucoiberin Glucoraphanin Glucoraphenin Glucoalyssin Glucocheirolin Glucoerysolin

Aromatic glucosinolates OCHzOCHz-CH 2Q,CH--CH zOH HOOCH2-

Indole glucosinolates R CHOJ N 4

z

R1

Benzyl glucosinolate Glucotropaeolin 2-Phenethyl glucosinolate G1uconasturtiin 2-Hydroxy-2-phenylethyl Glucobarbarin glucosinolate p-Hydroxybenzyl Glucosinalbin glucosinolate Indol-3-ylmethyl glucosinolate

(R = R = H) 1-M~tho~yindol-3­

ylmethyl glucosinolate (R = OCH . R4 = H) 4-Hydroxyin'dol- 3ylmethyl glucosinolate (R = H; R = OH) 4- M~thoxyin4dol-3­ ylmethyl· glucosinolate (R 1 = H ; R4 = OCH 3 ) N-Sulfoindol-3-ylmethyl glucosinolate (R 1

= SO-3;

Source. From Bjerg and S0rensen (1987)

R4

= H)

Glucobrassicin Neoglucobrassicin 4-Hydroxyglucobrassicin 4-Methoxyglucobrassicin Sulfoglucobrassicin

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E. A. S. ROSA, R. K. HEANEY, G. R. FENWICK, AND C. A. M. PORTAS

Extensive studies by S0rensen and coworkers in Copenhagen suggest that acyl-substituted glucosinolates, possessing sinapic, caffeic, and malic acids esterified to the sugar moiety, may be more widely distributed than was previously thought. However, the biological and biosynthetic consequences of such ubiquity remain to be assessed (S0rensen 1990). 2. Glucosinolate-Degrading Enzymes. Glucosinolates may be degraded by sulfatase and myrosinase (thioglucoside glucohydrolasesee below) enzymes. The former is not known to occur in glucosinolate-containing plants (S0rensen 1990), although desulfoglucosinolates are the penultimate intermediates in glucosinolate biosynthesis. Since many methods of quantitative and qualitative analysis, including cleanup procedures, involve desulfoglucosinolates, the action of sulfatase is discussed. Myrosinases catalyse the cleavage of a glucosinolate molecule to an unstable thiohydroxamate-O-sulfonate and other aglucones (Figs. 3.2-3.4). The further, chemical, breakdown of this intermediate is the basis of the formation of a host of reactive chemicals that possess wide-ranging properties relevant not only to the developing plant, its bacterial and fungal pathogens, and insect and herbivore pests, but also to farm animals and humans consuming significant amounts of glucosinolate-containing feedstuffs and foods. In passing, it is noteworthy that myrosinases have only a limited effect on the cleavage of desulfoglucosinolates and acylated glucosinolates. Given the importance of the myrosinases as key intermediates in the formation of these industrially exploitable, biologically active

R-C

/S-C6 H ll 0

5

~NOSO-3

Thioglucosidase

Alkenyl glucosinolate

Fig. 3.2.

...

./ S [ R-C

~N-

J

+ D-Glucose

+HSO~

Thiohydroxamate-O-sulfonate

R-N=C=S

R-C:=N

R-S-C:=N

Isothiocyanate

Nitrile + Sulfur

Thiocyanate

Hydrolysis of alkenyl glucosinolates.

3.

GLUCOSINOLATES IN CROP PLANTS

105

/ s- 13- D -

CH-CH-- CH z- C ~ I NOSO-

OH

Glucose

3

2-Hydroxybut-3-enyl glucosinolate H20

r

/S""

CH -CH -CH-CH C=N

I

z

z

OH

[

~ Thioglucosidase

CH-CH-CHzC~SH I

OH

~ NOSO-3

= CH-CH-CHzC=N

CH z

I

OH

]

+ Glucose

CH-CH-- CH z

I

I

0......

. . . . C,/ II S

Epithionitrile

Fig. 3.3.

Nitrile

Oxazolidine-2-thione

Hydrolysis of 2-hydroxybut-3-enyl glucosinolate.

compounds, it is surprising that they remain comparatively so little studied. Techniques such as fast-polymer liquid chromatography (Buchwaldt et al. 1986), affinity chromatography, and chromatofocussing (Palmieri et al. 1986) have been employed to produce highly purified fractions. Crude myrosinase extracts from seeds of Brassica, Sinapis, and Raphanus species were separated into active tetramers, active dimers and inactive monomers, possessing molecular weights of 260,000 140,000 and 78,000, respectively, with the polymeric species comprising isoenzymic species.

c. Biosynthesis The initial stages of the biosynthesis of glucosinolates involve the modification of amino acids or chain extended derivatives thereof, via an aldoxime intermediate. These modifications are also involved in the biosynthesis of a second class of natural toxicant, the cyanogenic glycosides, although examples of plants containing both

NH

E. A. S. ROSA, R. K. HEANEY, G. R. FENWICK, AND C. A. M. PORTAS

106

/' 5--[3- 0- Glucose ~CH1-C~

~NjJ R

~NOS03

Hp Thioglucosidase

[ OyfCH NCS J

O=Y

2

~

I

N R

CH1 CN

I

+S +

Glucose

3-Indolacetonitrile

Unstable isothiocyanate

J I I O=Y ~

I I O=Y l:O

CH 20H

::V

~

N R

Indole-3-earbinol

I

I

CH2

~

N

N.o-

R

R

3-Diindolylmethane

R= HorOCH 3

Fig. 3.4.

Hydrolysis of indole glucosinolates.

classes-such as 'Carica papaya-are very infrequent. Recent investigations into the: biosynthesis of cyanogenic glycosides (Halkier et al. 1991; Koch etal. 1992) have further elucidated the mechanisms for these earlier biosynthetic stages (see Larsen 1981). Following formation of the aldoxime, glucosinolate biosynthesis is continued in a second stage via S-insertion to the thio-hydroximic acid, glycosylation, and sulfation. A third modification of the side chain R may then occur as a result of, for example, oxidation and elimination reactions. Th~ above three stages are considered in turn. 1. Amino Acid Modification. The similarities between the carbon

skeletons of the side chains of a number of amino acids and some glucosinolates, referred to above, led to the early suggestion that amino acids may be natural progenitors of the glucosinolate aglucon moiety (see Ettlinger and Kjaer 1968), and this has subsequently been confirmed. In the first published report on the biosynthesis of a glucosinolate, Kutacek et al. (1962) demonstrated that tryptophan was converted into indol-3-ylmethyl glucosinolate (glucobrassicin). Independently, Underhill et al. (1962) and Benn (1962) reported the

3.

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107

biosynthesis of benzylglucosinolate (glucotropaeolin) and showed phenylalanine to be incorporated with high efficiency. Shortly thereafter, Kindl (1964,1965) found that labeledp-coumaric acid was incorporated into p-hydroxybenzyl glucosinolate (glucosinalbin) by Sinapis alba leaves much more efficiently than either tyrosine or phenylalanine. It was later found that S. alba effectively metabolized p-coumaric acid into tyrosine. These and other biosynthetic studies revealed that most glucosinolates are formed by a common biosynthetic pathway (Underhill 1980; Larsen 1981). In this brief review a distinction is made between those glucosinolates derived directly from protein amino acids and the majority, derived from such compounds following initial skeletal modification, most commonly involving chain elongation. The latter occurs via acetate addition to the a-keto acid and decarboxylation. Many aspects remain unclear, such as the exact location(s) of such structural manipulations, the mechanisms ofbiosynthetic regulation, and the factors controlling their accumulation and interconversion (Heaney and Fenwick 1987).

Glucosinolates derived directly from common protein amino acids. The parent glucosinolate (R ::;: H), derived from glycine, has not been isolated as natural product most probably because of its chemical instability (Kjaer 1976). Methyl glucosinolate (R::;: Me) from alanine is apparently absent from brassicacious plants (Gmelin and Kjaer 1970), but constitutes the most widely distributed glucosinolate within the Capparaceae (Kjaer 1976). Isopropyl and sec-butyl glucosinolates are widely distributed within Brassicaceae, and arise from the incorporation of valine and L-isoleucine units, respectively. The leucine counterpart is in isobutyl glucosinolate. Phenylalanine and tyrosine have their amino acid counterparts in benzyl- and phydroxybenzylglucosinolates, respectively. The analogue of tryptophan, indol-3-ylmethyl glucosinolate, stands out as a component whose occurrence is seemingly limited to seedlings and young vegetative tissue of many species from a number of families, including Brassicaceae. Glucosinolates derived from modified protein amino acids. Though experimentally tested in only a few cases, it is considered that the sequence set out in Fig. 3.5 is a general one, which can be cycled repeatedly to produce greater chain elongation (Kjaer 1976). Several enzymes, of unknown substrate specificity, are involved in the various homologization steps. As an example of this metabolic transfor-

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E. A. S. ROSA, R. K. HEANEY, G. R. FENWICK, AND C. A. M. PORTAS COOH

•

* R-CH-COOH

R-C-COOH

I

NH z

o"

Fen ylalanine

Acetate

C H 3 -COOH

-------1..-_

I

R- C- OH

I

CH z- COOH 2-Benzyl malic acid

COOH

I

R-CH

I

HO-CH-COOH 3-Benzyl malic acid

R-CH z

I

O=C-COOH

•

..-

*

•

R- CHz- CH- COOH

I

NH z 2-Amino-4-fenyl butyric acid

Fig. 3.5. Side-chain elongation of amino acids, precursors of glucosinolates (Underhill 1980).

mation, alanine may be incorporated into ethyl glucosinolate. Such homoamino acid formation has reached its culmination in the family of glucosinolates derived from methionine that occur in brassicacious plants (Dawson et al. 1993b). This family contains side chains that can be collectively expressed as MeS[CHz]n' with n ranging from 3 to 11 (Kjaer 1976); additional complexity is introduced via later-stage oxidations, which produce the corresponding sulfoxides or sulfones. Aromatic amino acids may also undergo homologization, for example, phenylalanine being metabolized into 2phenethyl glucosinolate (Dawson et al. 1993b). 2. Aldoxime Formation. The aldoxime-forming enzyme system involved in cyanogenic glycoside biosynthesis in cassava and sorghum (Halkier and Lindberg-M011er 1991; Koch et al. 1992) and the different system implicated in the formation of indole-3-aldoxime in chinese cabbage (Ludwig-Muller and Hilgenberg 1988) are both membrane bound. The proposed pathway for aldoxime biosynthesis described by Larsen (1981) has recently been amended following the researches of Halkier et al. (1991) and Koch et al. (1992), and may be summarized as shown in Fig. 3.6. The aldoxime is envisaged to undergo oxidation to yield the aci tautomer of the nitro compound. Studies by Dawson et al. (1993b) have shown homophenylalanine to be effectively converted to 3-phenylpropanaldoxime when added to rapeseed (Brassica napus) leaf microsomal preparations. The aldoxime formed was characterized as a mixture of (E)- and (Z)-iso-

3.

GLUCOSINOLATES IN CROP PLANTS R-CH-COOH

109

---.... R- CH - COOH I NHOH

I

NH z Fenylalanine

---....

Fenylacetaldoxime

N-Hydroxyfenylalanine

R-CH z -NO z

~

[

R-CH II HO-N-O-

R-C-SR' /I NOH

+

-L]

Cysteine (?)

I-Nitro-2-fenylethane

(Not clarified)

Aci tautomer (thiol aceptor)

UDPGlc

R-C-S/I NOH

R-CH /I NOH

UDP

'>,.J

PAPS

R-C- SGlc

PAP

_\.......:::::_.J:::.....--1... ~

II

NOH

Fenylacetothiohydroximic acid

Desulfoglucosinolate

R - C- SGlc II N-OS0 3 Benzylglucosinolate

[S-(~-D-Glucopyranosyl)fenylacetothiohydroximic acid]

Fig. 3.6.

Glucosinolate biosynthetic pathway with aldoxime formation.

mers; given that it would be the (E)-aldoxime isomer that would yield the glucosinolate with the generally accepted (Z) -conformation, it is probable that the (Z)-3-phenylpropanaldoxime isomer is formed following chemical equilibration. In contrast, when the same authors fed labeled dihomomethionine to the same microsomal preparation, it proved impossible to unequivocally identify the involvement of the putative 5-methylthiopentaldoxime. This may be explained by the rapid turnover of this metabolite via oxidation, elimination, or glucosinolate-forming reactions. Lykkesfeldt and Lindberg-M0ller (1993) have observed extracts of Tropaeolum majus to inhibit glucosinolate formation in active microsomal systems and also to inhibit cyanogenic glucoside formation in a sorghum microsomal preparation, and have suggested benzyl isothiocyanate to be responsible, at least in part. Given the wide-ranging biological activities of isothiocyanates, and the particular chemical reactivity of the benzyl compound, this would appear to be a reasonable conclusion. 3. Glucosinolate Formation. Formation of the aldoxime is followed by conjugation reactions which successively introduce sulfur (to form

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E. A. S. ROSA, R. K. HEANEY, G. R. FENWICK, AND C. A. M. PORTAS

a thiohydroxamic acid), glucose, and sulfate. Plant feeding studies have shown that these conjugation reactions are independent of the structure of the side chain, R (GrootWassink et a1. 1987, 1990). In studies reported by Underhill et a1. (1973), cysteine-S was most effectively incorporated to produce the intermediate thiohydroxamic acid. Subsequent S-glucosylation is effected by the enzyme thiohydroximate S-glucosyltransferase; initially, the problem of separating this enzyme from ubiqitous flavonol glucosyltransferase activity meant that demonstration of activity and specificity was difficult. However, GrootWassink et a1. (1994) have separated and purified the former enzyme immunologically and have shown it to be specific for thiohydroximates; and Reed et a1. (1993) have shown it to be without activity on oxygen-containing analogues. Sulfation of the penultimate desulfoglucosinolate has been shown to be effected by 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Important modifications of the side-chain occur after the overall glucosinolate skeleton has been elaborated; these include stereo-specific insertion of oxygen and elimination of methylthiol (probably in an oxidized form) from w-methylthioalk(en)ylglucosinolates and their sulfoxides and sulfones. D. Biochemical Genetics of Glucosinolates The possibility of producing Brassica crops with higher protective effects against cancer, less susceptible or resistant to diseases, less attractive to some specific pests, with lower levels of antinutrient factors, and with desirable, agronomic, storage and sensory characteristics, generally with higher commercial value, by manipulating the levels of glucosinolates, has generated interest in understanding the biochemistry and genetics of glucosinolate biosynthesis. Although there have been several biochemical investigations into the biosynthesis of glucosinolates, which is far from being clear in many details, little information is available in the genetic regulation of these compounds. The glucosinolate profile of B. napus is characterized by the aliphatic glucosinolates, but-3-enyl and pent-4-enyl and their hydroxylated analogues. In contrast, B. oleracea may contain 2-propenyl and/ or but-3-enyl glucosinolates, whereas B. rapa contains but-3-enyl and often pent-4-enyl glucosinolates. Both species may also possess significant quantities of methylthioalkyl and methylsulfinylalkyl homologues (Magrath et a1. 1993). It is supposed that the alkenyl glucosinolates, 2-hydroxybut-3-enyl and 2-hydroxypent-4-enyl, may be produced by hydroxylation of but-

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3-enyl and pent-4-enyl glucosinolates, respectively. In the synthesis of indole glucosinolates, indol-3-ylmethyl is generally accepted to be the precursor of the hydroxylated, methoxylated, and sulfated analogues (McDanell et al. 1988). Further genetic evidence is necessary to prove those pathways, B. napus being the primary species under study. Breeding programs had resulted in production of rapeseed cultivars with low erucic content (so-called "single-low" or "single-zero" cultivars). However, the presence of hydrolytic products from hydroxyalkenyl and from aliphatic glucosinolates has detrimental effects on rapeseed quality (see below), leading to breeding programs to reduce levels of glucosinolates. The reduction in oilseed glucosinolates has been almost entirely due to a reduction in alkenyl glucosinolates, which are under complex poligenic control (Rucker and Rudloff 1992), but the reduction has been restricted to the propagative tissues (Milford et al. 1989a, Mithen 1992) and no major differences can be noted in total and individual glucosinolate content throughout the growth period between leaves of single-low and double-low (also called "double-zero" cultivars-Iow in erucic acid and glucosinolates) rapeseed cultivars (Porter et al. 1991). The identification of alleles responsible for a low alkenyl glucosinolate content in the Polish rapeseed cultivar Bronowski (total glucosinolate content of 10%, i.e., 12 llmollg) and the successful transfer of the alleles into the double-low cultivars confirmed a genetic involvement in the synthesis of glucosinolates. Since the glucosinolate profile of F 1 hybrid Brussels sprouts was generally similar to the mean value of the patterns of both parents, Heaney et al. (1983) suggested that breeding of selected parent lines might produce a desirable pattern of glucosinolates in the F 1 hybrid. Similar breeding programs might also be applied to other species. Studies on the inheritance of aliphatic glucosinolates in B. napus (Magrath et al. 1993) suggested that the profile of this group of compounds is determined by a simple genetic system with two distinct sets of genes: one to determine the side chain length and the other to modify the structure of the side-chain regardless of its length. The interaction between these two sets of genes results in the profiles frequently observed in Brassica and other genera of Brassicaceae. A genetic model for the biosynthesis of aliphatic glucosinolates in B. napus was presented by Magrath et al. (1993) in which eight loci were described. The expression of propyl and pentyl glucosinolates was regulated by one locus each; two loci are involved in the production of alkenyl glucosinolates: One regulates the oxidation of methylthioalkyl to methylsulfinylalkyl glucosinolates, while the

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E. A. S. ROSA, R. K. HEANEY, G. R. FENWICK, AND C. A. M. PORTAS

other controls the oxidation of methylsulfinylalkyl to methylsulfonylalkyl glucosinolates. Two other loci regulate the hydroxylation of both butenyl and pentenyl glucosinolates and another two loci determine the expression of butyl glucosinolates. A general model for the biosynthesis of aliphatic glucosinolates with reference to the genetic regulation of side chain was proposed by Magrath et al. (1994) and Mithen et al. (1995), and the same working group (Parkin et al. 1994) has also reported the genetic regulation of hydroxylation of but-3-enyl and pent-4-enyl glucosinolates. The proposed genetic models are good tools to explain the origin of some Brassica species as in the case of B. napus and to confirm the biochemical models that have been proposed for the biosynthesis of glucosinolates. An understanding of the regulation of glucosinolates in the different parts of the plant is important to control the potential biological effects of each part. Magrath and Mithen (1993) established the aliphatic glucosinolate profile of seed, cotyledons, and leaf tissue of B. napus and showed that in the developing seed there was a transference of fully formed glucosinolates from maternal tissue, in agreement with findings by Haughn et al. (1991) and Love et al. (1990), whereas in cotyledons there was no glucosinolate biosynthesis but existing glucosinolates undergo modification. Within true leaves there is de novo glucosinolate biosynthesis and so the profiles of the reciprocal F 1 hybrids were identical to the F 1 genotype. Further attempts to change the glucosinolate profile of Brassica crops might involve genetic modification. Cloning of genes that regulate specific steps in glucosinolate biosynthesis, supported by mapbased strategies for instance from Arabidopsis, was suggested as an alternative to isolating these genes (Mithen and Toroser 1995). These clones may be then used as probes to identify Brassica homologues or used directly in transformation experiments. Antisense technique for regulating specific steps in the biosynthesis pathway is another tool with potential application in glucosinolate transformation. However, the introduction or elimination of a particular glucosinolate or group of glucosinolates through genetic engineering must be followed by extensive field studies to understand the different interactions and to assess agronomic performance. E. Myrosinase Almost without exception glucosinolates occur in the plant in conjunction with the hydrolytic enzyme myrosinase, a thioglucosidase

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(thioglucoside glucohydrolase, E.C. 3.2.3.1.). This enzyme is released when the plant tissues are crushed or when autolysis occurs within the plant, but some enzymic hydrolysis may occur at a low rate in normal plant tissues (Rosa, unpublished). Glucosinolates, although considered nontoxic themselves, are important because of the multiplicity of physiologically active products derived from them as a result of enzymic cleavage. As will be shown below, the glucosinolate/myrosinase system plays a major role in plant defence against fungal diseases and pest infestation. Hydrolytic activity similar to plant myrosinase has been found in the fungi Aspergillus niger and Aspergillus sydowi (Reese et al. 1958; Ohtsuru and Hata 1973), in bacteria of the lower mammalian intestine (Escherichia coli, Paracolobactrum aerogenoides, Enterobacter cloacae, and Bacteroides vulgatus) (Oginski et al. 1965; Tani et al. 1974; Nugon-Baudon et al. 1988; Rabot et al. 1993a,b), in the distal gastrointestinal tract (Otte et a1. 1994), in mammalian tissues (Goodman et al. 1959), in Lactobacillus spp (Nugon-Baudon et al. 1990a; Smits et al. 1993), in the cabbage aphid Brevicoryne brassicae (MacGibbon and Allison 1968), and in the mustard aphid Lipaphis erysimi (MacGibbon and Beuzenberg 1978). A low recovery of ingested glucosinolates in excreta from poultry (Slominski et a1. 1987; 1988) and in the lower parts of the digestive system from rats, pigs, and young bulls (Eggurn et al. 1985a) suggested a considerable absorption and/or degradation of intact glucosinolates in the digestive tract of these animals. Myrosinase has been found in seed, leaf, stem, and roots of glucosinolate-containing plants. The accepted belief that myrosinases occur in myrosin cells has been confirmed by Thangstad et al. (1990) using immunocytochemical techniques. Pocock et a1. (1987) suggested that myrosinase is produced by the extensive rough endoplasmic reticulum in myrosin cells and subsequently stored in the vacuole, a view that was supported by Thangstad et al. (1991), who showed the subcellular location inside the myrosin cells and its association with the protein bodies/vacuoles of idioblasts or myrosin cells using immunogold-EM techniques. An improved method for myrosinase purification was reported by Pessina et al. (1990). The activity of myrosinase appears to be higher in young tissues of the plant, making preparation of glucosinolate extracts of these plant parts more critical. Iversen and Baggerud (1980) showed that myrosinase is synthesized at a higher rate in hypocotyls and primary roots. Bones (1987) confirmed hypocotyls to have higher myrosinase activity followed by cotyledons and also reported (1990)

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E. A. S. ROSA, R. K. HEANEY, G. R. FENWICK, AND C. A. M. PORTAS

that specific myrosinase activity in different developmental stages and organs of B. napus decreased gradually from a maximum observed at 2 days seedling growth. Pocock et al. (1987) suggested that myrosin cells are produced very early in leaf development and that after this time no new myrosin cells are differentiated. Pihakaski and Pihakaski (1978) and Iversen and Baggerud (1980) suggested that one function of myrosinase in vivo is regulation of plant growth, based on the reports of indole glucosinolate metabolism to indolyl-3-acetonitrile and eventually to indolacetic acid (IAA) (Wightman 1962). This has been confirmed by Bones (1987) who found the myrosinase activity in calli with shoots to be much higher than in control calli without shoots. Myrosinase does occur in multiple forms in plants, but as isoenzymes. The reason for the presence of isoenzymes is not yet understood (Buchwaldt et al. 1986), but the number of isoenzymes present depends on plant species, genotype, and tissue type. Buchwaldt et al. (1986) using fast polymer liquid chromatography (FPLC) for purification and stability of plant myrosinase isoenzymes, confirmed the presence of 14 isoenzymes in white mustard and found a mixture of isoenzymes in B. nigra, B. napus, and Sinapis alba. The activity of isoenzymes varies with type of glucosinolate used as substrate. MacLeod and Rossiter (1986) suggested that the nature of the glucosinolate side chain might determine the rate of enzyme hydrolysis, and in glucosinolates with more hydrophilic side chains, such as those with a hydroxyl group present, hydrolysis occurs at a slower rate than in those with less hydrophilic side chains. In a study covering several brassicacious vegetables, Wilkinson et al. (1984) reported the highest myrosinase activity in Raphanus sativus and the lowest in B. campestris and Nasturtium officinalis. Of the subspecies of B. oleracea studied, the groups Capitata and Gemmifera had the highest activities, whereas the Sabauda, Gongylodes, and Botrytis groups had similar activities to that of the B. napus Napobrassica group and B. chinensis Pekinensis group (Yen and Wei 1993). Myrosinase activity is generally modified by processing conditions of the raw materials (see below). Ascorbate and some ions are known to affect the myrosinase reaction and MacLeod and Rossiter (1987) concluded that the majority of the thioglucosidases of plants so far studied in this respect are activated by ascorbate. Metal ions Fe 2 + and Cu 2 +have been studied to determine how they affect both the course of the reaction and the ratio of products formed (MacLeod and Rossiter 1987; Uda et al. 1986a). In the degradation of 2-hydroxybut-3-enyl glucosinolate, MacLeod and Rossiter (1987) reported that Fe 2 +is not simply an acti-

3.

GLUCOSINOLATES IN CROP PLANTS

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vator of thioglucosidase as is ascorbate. Instead, it actually directs the course of the degradation, while Cu 2 + drastically inhibits the reaction. Sulfate was also shown to induce differential expression of myrosinases (Bones et al. 1994). Searle et al. (1984) studied the effects of pH, iron, copper, and manganese on the degradation of indol-3-ylmethyl glucosinolate in the presence and absence of myrosinase and concluded that at pH 4.0 cupric ion was the most effective cofactor, followed by ferric and ferrous ions, whereas manganese ions were ineffective. pH had two effects on the enzymic degradation of indolylmethyl glucosinolate. First, in the presence of ferrous, cupric, and cuprous ions, low pH values increase the proportion of IAN at the expense of 3diindolylmethane (DIM) and ascorbigen (ASC). Second, the relative amounts of DIM and ASC, also depend on pH and on the presence or absence of metallic ions. At pH 4.0 the yield of ASC was 80% of the theoretical maximum, while increasing the pH to 7.0 results in an increase of DIM at the expense of ASC. Uda et al. (1986b) reported that some thiol compounds (L-cystein, reduced glutathione, cysteamine, thiobenzoic acid, thiomalic acid, and thiophenol) greatly accelerate the formation of nitriles from 2propenyl glucosinolate in the presence of ferrous ion, even though the reaction medium was maintained at a neutral pH of 6.5. The thioglucosidase isoenzymes have been found to have different properties, particularly with regard to their pH, optimum temperature, and response to ascorbate, but a range of pH from 4.0 to 9.0 was found to be optimal for various thioglucosidases. F. Factors Affecting the Composition and Amount of MyrosinaseInduced Breakdown Products Myrosinase-induced hydrolysis of glucosinolates yields a wide variety of products, the exact nature of which is determined by a variety of factors, including pH, the presence of certain cofactors, and, most clearly, the structure of the parent glucosinolate and, more specifically, the structure of the side chain R. As indicated in Table 3.1, most glucosinolates can conveniently be placed within three categories according to the nature of their side chains. The largest category contains aliphatic (saturated and unsaturated) groups, which form isothiocyanates at pH 5-7 (Fig. 3.2). At more acid pH, nitriles, rather than isothiocyanates, are formed. Since the latter contain much more extensive organoleptic, biological, and plant-protective properties than the former, this pH dependence has notable consequences. The second category, small in number, but

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E. A. S. ROSA, R. K. HEANEY, G. R. FENWICK, AND C. A. M. PORTAS

significant because of their ubiquity in major commercial crops, such as Brassica vegetables and rapeseed, are glucosinolates possessing a side-chain containing a p- (and occasionally an y-) hydroxyl substituent (Fig. 3.3). Such substitution renders unstable the hydroxyalkyl isothiocyanate initially formed at pH 5-7, and results in spontaneous cyclization. In the case of the P-substituted glucosinolates, the result is an oxazolidine-2-thione, which possesses irreversible anti-thyroid activity. The third category possesses an indolic or substituted indolic side chain (Fig. 3.4), and yields, via an unstable isothiocyanate, the corresponding indole-3-carbinol and thiocyanate ion (Hanley et al. 1990); the former can undergo secondary reaction to form a diindolylmethane. Although Hanley and Parsley (1990) were unable to detect the instable isothiocyanate from indol-3-ylmethyl glucosinolate, they identified for the first time the 1-methoxyindol-3-ylmethyl isothiocyanate from hydrolysis of 1methoxyindol-3-ylmethyl glucosinolate. Under more acid conditions (ca. pH 3) all three glucosinolate types produce increasing amounts of nitriles. These compounds are also formed during autolysis, even when the pH apparently favors the formation of isothiocyanates. According to Uda et al. (19S6a,b), the explanation for this phenomenon lies in the inhibitory effect of ferrous ion and perhaps endogenous thiols on isothiocyanate formation. The involvement of thiols may also explain the reduction in nitrile production that is evident after heating glucosinolate-containing tissue, which was attributed by Tookey et al. (19S0a) to the destruction of a thermolabile "nitrile-forming factor." Ferrous ion is also a cofactor, together with an inactive epithiospecifier protein (ESP), for the formation of episulfides. Epithiospecifier protein is not present in all brassicacious plants, and Macleod and Rossiter (1 9S5) have speculated that it is absent from those species lacking glucosinolates possessing terminally unsaturated side chains. Episulfide formation has been rationalized through a variety of mechanisms. Petroski and Kwolek (19S5) concluded from kinetic studies that ESP is a noncompetitive inhibitor of myrosinase, and the sulfur atom is transferred to the terminal site by a substantially intramolecular mechanism (Brocker and Benn 19S3). III. ANALYSIS

It would be inappropriate in this review to detail the many methods for the analysis of glucosinolates that have been described during

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the last three decades. Development of new methods has paralleled the advent of new analytical procedures and reflects the increased awareness of the diversity of glucosinolates, their role in the plant, and the biological effects of these compounds and their breakdown products. Analytical methodology has received added impetus due to the considerable interest shown by the animal feed industry and regulatory bodies in the levels of glucosinolates and products derived therefrom in feed formulations based on rapeseed. Regulation of glucosinolate levels in rapeseed called for the development of acceptable methods for their analysis, and national governments and the European Commission offered financial support for the development of Official Methods. In parallel with this rapeseed- motivated research, methods have been modified or new methods developed to meet the needs of researchers investigating the much wider range of glucosinolates that occur in other brassicacious plants. Methods have been developed for the analysis of the total glucosinolate content of plant material, for the determination of the level of each individual glucosinolate, and for the measurement of the large number of breakdown products that result from the action of myrosinase. Such methods have been the subject of several critical reviews (McGregor et al. 1983; Fenwick et al. 1983a; Bjerg et al. 1987a; Dietz and Harris 1990; Mawson et al. 1993a; Heaney and Fenwick 1993; S0rensen 1990). A study of the literature in order to identify a method of analysis suited to a particular purpose is at first confusing. Ideally, the method should be rapid, simple, sensitive, and accurate and should discriminate between all compounds of interest. Rarely are such attributes found together in one method and selection of a suitable method must often be based on the sacrifice of one or other of these features with due regard to the specific needs of the analyst. In summarizing this complex picture, little attention will be given to methods like paper and thin-layer chromatography. Such qualitative techniques have a place in the identification of glucosinolates, particularly in conjunction with other methods, and they have been used to good effect in some laboratories (Rodman 1980, 1981). However, such methods reveal no more qualitative information than good quantitative methods, which, without doubt, are now the techniques of choice. It will be clear from the foregoing that the choice of method for the analysis of glucosinolates is a complex matter and is determined by a number of factors. However, the study of this subject has reached a stage where it can be said with confidence that a method or combination of methods exists to meet all needs.

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A. Quantitative Methods Quantitative methods may be divided into several types and it is convenient to think of them in terms of those that provide an estimate of the total amount of glucosinolates in the plant material and those that give a measure of the level of individual glucosinolates. Although extraction of glucosinolates and cleanup of the extract are usually critical stages in most procedures, they are by no means a prerequisite for all methods. Nevertheless, these techniques, together with methods for the isolation of the enzymes myrosinase and sulfatase, are briefly discussed here and their relevance to particular methods will later become more apparent. Similarly, many methods for the determination of individual glucosinolates rely on the availability of suitable reference standards, most of which are not commercially available, and methods for the isolation or synthesis of such compounds are also summarized. 1. Preparation and Cleanup of Glucosinolate Extracts.

Glucosinolates coexist with myrosinase in the plant, and any process such as cutting or grinding of fresh tissue will initiate a rapid hydrolysis of these compounds. Consequently, the importance of carefully selected conditions for reduction of sample size and extraction of glucosinolates cannot be overemphasized. Before disruption of the cellular integrity, samples should be completely dry (oven- or freezedried) or frozen in liquid nitrogen. The use of aqueous methanol for extraction, in combination with high temperatures, inhibits myrosinase activity (Heaney and Fenwick 1993). The anionic nature of glucosinolates facilitates cleanup procedures based on initial adsorption onto ion-exchange media and many methods include this preliminary step. Thereafter, procedures differ, with glucosinolates being eluted intact or after treatment with enzymes, either sulfatase to liberate the neutral desulfoglucosinolates or myrosinase hydrolysis to liberate glucose. 2. Enzymes Used in Glucosinolate Analysis. Many of the methods for glucosinolate analysis make use of enzymes to generate a range of products that are more amenable to quantification. Myrosinase may be obtained commercially or prepared from mustard seed (Appelqvist and Josefsson 1967). Commercially available sulfatase, used in many gas chromatography (GC) and high-performance liquid chromatography (HPLC) methods, may contain some glucosidase activity (resulting in a "loss" of desulfoglucosinolates) and should be purified before use (EC 1990).

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3. Reference Glucosinolates and Internal Standards. One of the major problems in the analysis of glucosinolates has been the dearth of suitable standards. The commercially available 2-propenyl glucosinolate is not a suitable internal standard for certain brassicacious plants due to their high content of this compound. Brassicaceae-derived glucosinolates, not normally present in Brassica, have been used as internal standards for brassicacious plant analysis, and methods for the isolation and purification of these and other glucosinolates have been described (Bjerg and S0rensen 1987; Hanley et al. 1983; Zhong et al. 1989 ).

4. Methods that Measure Total Glucosinolate Content. Several meth-

ods have been described for the measurement of total glucosinolate content, although consideration of the very different biological properties of individual glucosinolates and their breakdown products has led some authors to question the relevance of this approach (S0rensen 1990). In the simplest procedure, glucosinolates can form a complex with palladium to produce a chromophore easily measured by colorimetry, a method that today finds little use. The enzymic hydrolysis of glucosinolates releases a stoichiometric amount of glucose (Fig. 3.2-3.4). This is true for almost all glucosinolates, and methods based on the measurement of enzymically released glucose have proved to be relatively rapid and simple to apply. In its simplest form, the released glucose is measured after autolysis of the sample, but this takes no account of endogenous glucose. More sophisticated approaches involve the extraction of glucosinolates followed by selective cleanup procedures that eliminate free glucose and other interfering compounds, followed by the controlled enzymic release of bound glucose. Various procedures are available for measuring glucose in solution, ranging from simple test papers impregnated with enzymes and a chromagen, to clinical test kits and glucose-specific electrodes. In another approach, glucosinolates are hydrolyzed by strong sulfuric acid in the presence of thymol, resulting in a suitable chromogen. The method is sensitive to impurities, however. Many of these methods were developed for rapeseed analysis and for that purpose they have proved adequate, but certain glucosinolates occurring in other Brassicaceae material cannot be measured satisfactorily by this approach. For example, the glucosinolates of radish seed, containing a sinapoyl substituent in the glucose moiety, represent a significant proportion of the seed glucosinolates and present problems in analysis (S0rensen 1990).

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E. A. S. ROSA, R. K. HEANEY, G. R. FENWICK, AND C. A. M. PORTAS

Myrosinase hydrolysis of glucosinolates gives rise to an unstable aglucone, which then undergoes a Lossen rearrangement to produce an equimolar amount of bisulfate (Fig. 3.2). Several methods have been described for the quantitation of this ion using titrimetric and gravimetric procedures. Schnug (1987) has described a method in which the bisulfate liberated by sulfatase enzyme is precipitated with barium chloride and residual barium is measured by X-ray emission spectroscopy. The same author reported an indirect X-ray fluorescence method based on the relation between total sulfur and total glucosinolate content (Schnug and Haneklaus 1987a,b). A good correlation with gas chromatography and high-performance liquid chromatography (see below) was found after correction for the contribution from sulfur in the protein (assumed to be 22%) and the method was adopted by the European Commission and was used as a German national method. The importance of correcting for real rather than assumed protein content was emphasized by Zhao et a1. (1992). The problem of protein sulfur variability is eliminated by the use of wavelength dispersive X-ray spectrometry to measure fully oxidized sulfur (S6+) and covalently bound sulfur (Pinkerton et a1. 1993). All of these methods depend on satisfactory calibration procedures. Near infrared reflectance using scanning instruments showed early promise, particularly for the nondestructive analysis of rapeseed, but suffered from the disadvantage that extensive calibration sets were needed and instruments in use by industry were mostly of the fixed wavelength type. 5. Methods that Measure Individual Glucosinolates. The biological activity of glucosinolates is determined by the nature of the R group (Table 3.1) and the methods described above give little information about individual compounds. Such information is essential for plant breeders, agronomists, animal nutritionists, feed processors, clinicians, etc., and many methods have been described for the quantitative assay of individual glucosinolates. Based largely on GC or HPLC, the methods have in common the need to effect a satisfactory extraction of the compounds of interest. Extracted glucosinolates, after cleanup, may then be analyzed without further modification or they may first be subjected to hydrolysis either with the enzyme myrosinase, yielding a range of so-called "split products," or, in later methods, with sulfatase enzyme to yield characteristic desulfogl ucosinolates. Gas chromatography (GC). Gas chromatography was the earliest technique used to measure individual glucosinolates and many methods

3.

GLUCOSINOLATES IN CROP PLANTS

121

have been published for the separation of split products, intact glucosinolates, and desulfoglucosinolates. The split products were extracted into dichloromethane and injected onto the GLC column, whilst in the case of intact and desulfoglucosinolates it was first necessary to render the compounds volatile by derivatisation. Early methods for the analysis of split products suffered from their inability to analyze all compounds of interest and the difficulty of controlling the nature of the hydrolysis products. However, this approach is still regularly used and recently, in a study of the role of glucosinolates in the suppression of soil-borne plant pests, Brown et al. (1994) developed a method using capillary GC to analyze the products of glucosinolate degradation in the soil. The method gives recoveries greater than 90%, including oxazolidinethione, hitherto difficult to measure. Isothermal GC failed to separate indolyl glucosinolates, compounds that ultimately assumed great significance (see below), and only after the introduction of temperatureprogramming was this possible. Despite the thermal instability of 4-hydroxyindol-3-ylmethyl glucosinolate and the development of multiple peaks for compounds with terminal methylsulfinyl groups, this approach was adopted by the Canadian Grain Commission and (until replacement by HPLC) by the European Commission, for the analysis of glucosinolates in rapeseed. Several workers have sought to improve the GC methods, including Hase et al. (1988) who found desulfoglucosinolates to be unstable in the presence of even a trace of moisture. Slominski and Campbell (1987) reexamined the problems of indolyl glucosinolate stability in the analysis of Canadian rapeseed. They noted that heating to inactivate the endogenous myrosinase was detrimental to these compounds and suggested a modification of this step, concluding that the modified method gave satisfactory results for routine studies.

High-performance liquid chromatography (HPLCj. The development of high-performance liquid chromatography for the analysis of glucosinolates or their breakdown products started somewhat later than GC but followed a similar course. The catalyst was again the need of the rapeseed industry for comprehensive, reliable, and rapid methods. The application of HPLC to the investigation of glucosinolate decomposition products has been limited due to the volatility of many such compounds and the fact that some products, particularly thiocyanates and nitriles, are not detectable spectrometrically. Several authors have described HPLC methods for oxazolidinethiones

122

E. A. S. ROSA, R. K. HEANEY, G. R. FENWICK, AND C. A. M. PORTAS

(Maheshwari et al. 1979; Josefsson and Akerstrom 1979; McLeod et al. 1978; Benns et al. 1979), and Quinsac et al. (1992) used complexation/extraction procedures prior to HPLC analysis of oxazolidinethiones in biological fluids with a high degree of selectivity, accuracy, and sensitivity. However HPLC finds most use in the analysis of intact glucosinolates or desulfoglucosinolates. Reverse-phase HPLC with ion pairing has been used for the separation and identification of intact glucosinolates (Helboe et al. 1980). The method separates p-hydroxybenzyl and indol-3-ylmethyl glucosinolates, although overlap of but-3-enyl and 2-phenylethyl glucosinolates can occur (Fenwick et al. 1983a). Betz and Page (1990) used C18 solid-phase cleanup and tetrabutyl ammonium sulfate to achieve separations that avoid this problem. The procedure has the benefit that potentially all types of glucosinolate are amenable, although sometimes limited by their baseline separation. The alternative approach of prior desulfation in exactly the same manner as used for GC offers a degree of selectivity, but occasionally the presence of flavonoid sulfates might confuse interpretation, and compounds with acyl substituents may not be desulfated (S0rensen 1990). Nevertheless, for some purposes, HPLC of desulfoglucosinolates is to be preferred and Wathelet et al. (1987) concluded that this approach offered better separation, constant retention times, and longer column life. The use of HPLC for the separation of desulfoglucosinolates, using reverse-phase C18 columns and avoiding the need for buffer solutions or ion-pairing reagents, was first described by Minchinton et al. (1982). Since that time, several authors have reported the use of HPLC for the analysis of desulfated glucosinolates prepared from seed, root, and leaf tissue of brassicacious plants (Spinks et al. 1983, 1984; Sang and Truscott 1984). The method compares favorably with GC, and most desulfoglucosinolate standards are sharply separated, including all four indole glucosinolates. A comparison of 91 samples analyzed by GC and HPLC gave a very high correlation coefficient for the major glucosinolates present in the plant material (Spinks et al. 1983). Desulfation, normally performed on small ion-exchange columns (minicolumns), which also serve for the sample cleanup step, takes at least 12 h at room temperature. Quinsac and Ribaillier (1987) added sulfatase to a crude aqueous extract, reducing the desulfation time to 20 min. The method gave higher values for 4-hydroxyindol-3ylmethyl glucosinolate without further sample cleanup. Quinsac et al. (1991) proposed a rapid isocratic method for desulfoglucosinolates using a water mobile phase.

3.

GLUCOSINOLATES IN CROP PLANTS

123

The HPLC analysis of desulfoglucosinolates was adopted by the European Commission as an official method (EC 1990) and was later adopted as a British Standard method (British Standards Institute 1993). Shortly afterward, Fiebig (1991) recommended improvements to the EC method after showing that the concentration of the internal standard, together with sulfatase activity and incubation time, was of vital significance. The lack of a commercial source of suitable compounds for use as internal standards and as internal markers has always been a problem in all GC and HPLC methods. Studies describing the isolation and purification of glucosinolates from natural sources are referred to in the following section. Alternative artificial desulfoglucosinolates not present in Brassicaceae have been recommended as standards (Elfakir et a1. 1992). 6. Other Methods

Capillary electrophoresis (CE). Capillary electrophoresis is attracting much interest for the analysis of glucosinolates and desulfoglucosinolates, being rapid and inexpensive to operate. Separations are good and micellar electrokinetic capillary chromatography (MECC) has been applied to both classes (Michaelsen et a1. 1992; Morin et a1. 1992). More recently, factors affecting separation of compounds using CE and MECC were studied by Morin and Dreux (1993), and Feldl et a1. (1994) have used MECC to separate a range of indolyl glucosinolates and related compounds. It still remains for this technique to be made fully quantitative. Mass spectrometry (MS). Mass spectrometry has proved to be an invaluable tool in the identification and structural elucidation of glucosinolates and their breakdown products. The technique gives direct information relating to the structures of ionic species generated in the gas or liquid phase by measuring their mass-to-charge ratio. Early techniques using electron impact (EI) or chemical ionization (CI) techniques were best suited to relatively volatile samples or mixtures. However, the advent of the newer desorption ionization techniques has enabled intact glucosinolates to be examined directly. Positive ion fast atom bombardment (FAB) (Fenwick et a1. 1982) has yielded mass spectra characterized by abundant protonated and cationized molecular ions with relatively little fragmentation. In the negative ion mode, FAB produces an abundant molecular ion (of the glucosinolate anion) (McGregor et al. 1983). This proved especially advantageous in the analysis of crude plant extracts and mixtures of purified glucosinolates.

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E. A. S. ROSA, R. K. HEANEY, G. R. FENWICK, AND C. A. M. PORTAS

The use of MS coupled to either GC or HPLC permits the characterization of individual peaks as they elute from the column. Recent papers describing the use of GC/MS include those of Eagles et a1. (1981), Christensen et a1. (1982), Heeremans et a1. (1989), Shaw et al. (1990), and Kore et a1. (1993b). Collision-induced dissociation of the [M-H]- anion produced by FAB has been tested on five glucosinolates of known structure by Bojesen and Larsen (1991), who concluded that the method was potentially useful. Kokkonen et al. (1991) have used this approach coupled to HPLC and found that daughter ions yielded much useful data based upon group and compound-specific fragmentation. This approach offers improvement on the thermospray LCMS techniques as described by Mellon et a1. (1987) and Lange and Petrzika (1991). Electrospray LC/MS is a promising approach to analysis of glucosinolates in that it is sufficiently sensitive to be coupled to high-resolution capillary electrophoresis (Smith et a1. 1988), but the technique has not yet been applied to glucosinolates. Although originally perceived as expensive, mass spectrometric techniques are becoming more popular with the advent of relatively inexpensive bench-top GC/MS and LC/MS equipment. Nuclear magnetic resonance (NMR). In conjunction with other information (e.g., mass spectra, IR, and UV spectra), NMR provides much useful information in the identification of glucosinolates and their products. Both lH- and 13C-NMR have been reported by Olsen and S0rensen (1979, 1980) for intact glucosinolates. The same authors (1981) reported 13C chemical shifts for a range of glucosinolate pyridinium salts. Visentin et a1. (1992) confirmed the nature of the main glucosinolate of radish root, and Kore et a1. (1993b) have used NMR to verify the structures of isothiocyanates and nitriles isolated from broccoli and other seed. Enzyme-linked Immunosorbent Assay (ELISA). ELISA was originally perceived as offering great potential for the rapid analysis of large numbers of samples. Although successfully applied to the analysis of mustard seed (Hassan et a1. 1988), ELISA has not found much support in other areas. IV. LEVELS OF GLUCOSINOLATES IN PLANTS The accurate assessment of glucosinolate levels in plant foods and feedstuffs has been prompted by the possible physiological conse-

3.

GLUCOSINOLATES IN CROP PLANTS

125

quences to humans and animals of a high dietary intake of glucosinolates. As a consequence, a considerable amount of data on levels of total and individual glucosinolates is now available. Generally, a plant species contains more than one glucosinolate and often there are both qualitative and quantitative differences between the roots, leaves, and seeds of a plant. The highest concentrations occur in seeds, except for indol-3-ylmethyl and Nmethoxyindol-3-ylmethyl glucosinolates, which are rarely reported in seeds (Tookey et al. 1980a). The roots of B. napus were found to have higher glucosinolate content than the leaves, the major compound being 2-phenethyl glucosinolate (Clossais-Besnard and Larher 1991). Generally, the same glucosinolates occur in a particular subspecies regardless of genetic origin and in most species only between one and four glucosinolates are found in relatively high concentrations. Brassica crops, either fresh, cooked, or otherwise processed, are widely consumed in the human diet and constitute the major source of glucosinolates, but they are also commonly grown as forage in certain parts of the world. In the following paragraphs a general consideration of the nature and amounts of glucosinolates in plant foods is summarized, referring mainly to the major glucosinolates. In the late 1970s and 1980s, several studies examining the glucosinolate composition of cabbage were conducted, mainly in the United States and United Kingdom. In 1991, Daxenbichler et al. reported the seed glucosinolate content of 259 Brassicaceae out of 297 lesser-known species, and a glucosinolate spectrum of some Algerian Brassicaceae has been produced by Lockwood and Belkhiri (1991). VanEtten et al. (1976) found no significant differences in total and individual glucosinolate content between open-pollinated and hybrid cultivars of cabbage, but head-to-head variation, with both large and small heads, was greater for the open-pollinated cultivars. Within each cultivar, the total glucosinolate content tended to be inversely proportional to head size. Bible et al. (1980) have also shown that head SCN- content was negatively correlated with marketable head fresh weight and with total top weight. No significant differences were noted for total glucosinolate content between cabbage c~lti­ vars produced for kraut, storage, or fresh market, although kraut and storage types were lower in "indole" and 4-methylsulfonylbutyl glucosinolates. VanEtten et al. (1980) in a study with 79 cultivars (67 white cabbage, 4 savoy cabbage, and 8 red cabbage) confirmed these findings. The glucosinolates found in most common Brassica are shown in Tables 3.2 and 3.3, and for comparison purposes, data

126

E. A. S. ROSA, R. K. HEANEY, G. R. FENWICK, AND C. A. M. PORTAS

Table 3.2. Principal glucosinolates occurring in the main Brassica. Concentration (p.mol/l00 g of fresh wt) Species

Glucosinolate

White cabbage (B. oleracea L., Capitata group) 2-Propenyl

3-Methylsulfinylpropyl

Indole glucosinolates Indol-3-ylmethyl Total

Average

Range

36.3 26.4 57.2 66.2 34.7 28.3 72.7 97.6 49.4 31.2 39.3 60.7 143.8 117.3 68.6

4.3-147.4 8.8-148.6 18.6-104.3 18.6-162.7 13.0-70.9 10.0-58.6 5.0-193.1 5.0-279.8 28.0-106.4 10.5-104.9 9.3-129.8 9.3-200.0 66.4-236.7 57.5-234.5 17.7-112.8

200.9 238.3

93.8-348.2 78.8-602.6

VanEtten et al. (1976) VanEtten et al. (1980) Sones et al. (1984a) Sones et al. (1984c)z VanEtten et al. (1976) VanEtten et al. (1980) Sones et al. (1984a) Sones et al. (1984c)z VanEtten et al. (1976)Y VanEtten et al. (1980)Y Sones et al. (1984a) Sones et al. (1984c)z VanEtten et al. (1976) VanEtten et al. (1980) Mullin and Sahasrabudhe (1977) Sones et al. (1984a)x Sones et al. (1984c)z

35.8 0.1-39.7 31.5-162.7 100.7 15.2-91.1 72.5-279.8 111.3 61.7-108.2 70.2-199.8 1.6 0.0-1.3 5.6-29.5 275.6 100.4-265.0 267.1-653.4

VanEtten et al. (1976) VanEtten et al. (1980) Sones et al. (1984a) VanEtten et al. (1976) VanEtten et al. (1980) Sones et al. (1984a) VanEtten et al. (1976)Y VanEtten et al. (1980)Y Sones et al. (1984a) VanEtten et al. (1976) VanEtten et al. (1980) Sones et al. (1984a) VanEtten et al. (1976) VanEtten et al. (1980) Sones et al. (1984a)x

11.1-14.1 1.5-25.7 12.4-19.7 4.8-31.0 46.7-66.9 31.6-82.1 42.6-102.9 31.9-67.9 13.9-16.3

VanEtten VanEtten VanEtten VanEtten VanEtten VanEtten VanEtten VanEtten VanEtten

Savoy cabbage (B. oleracea L., Sabauda group) 2-Propenyl 14.2 93.2 3-Methylsulfinylpropyl 46.7 169.8 Indole glucosinolates Indol-3-ylmethyl 2-Hydroxybut-3-enyl

80.5 123.0 0.5 13.8

Total Red cabbage (B. oleracea L., Capitata group) 2-Propenyl

164.5 461.3

Indole glucosinolates

12.6 10.5 16.1 14.5 56.8 52.3 72.8

But-3-enyl

15.1

3-Methylsulfinylpropyl 4-Methylsulfinylbutyl

Reference

et al. et al. et al. et al. et al. et al. et al. et al. et al.

(1976) (1980) (1976) (1980) (1976) (1980) (1976)Y (1980)Y (1976)

3.

GLUCOSINOLATES IN CROP PLANTS

Table 3.2.

127

(continued). Concentration (llmol!100 g of fresh wt) Glucosinolate

Average

Range

But-3-enyl (cant.) 2-Hydroxybut-3-enyl-

9.9 12.2 8.3 204.3 163.4 68.8

4.6-15.6 10.1-14.3 4.4-5.5 150.5-258.1 88.2-234.4 34.4-98.9

VanEtten et al. (1980) VanEtten et al. (1976) VanEtten et al. (1980) VanEtten et al. (1976) VanEtten et al. (1980) Mullin and Sahasrabudhe (1977)

Brussels sprouts (B. oleracea L., Gemmifera group) 2-Propenyl 136.0

27.7-392.9

10.7 112.1 76.6 11.8 8.2 113.2

3.9-22.7 4.0-280.6 0.0-154.2 2.4-18.9 0.4-22.6 45.3-228.4

128.4 391.8 21.3

54.3-326.3 327.8-469.4 1.9-34.3

Heaney and Fenwick (1980a) Carlson et al. (1987a) Sones et al. (1984c) Sones et al. (1984c) Carlson et al. (1987a) Carlson et al. (1987a) Heaney and Fenwick (1980a) Sones et al. (1984c) Carlson et al. (1987a)Y Sones et al. (1984c)

Species

Total

3-Methylsulfiny 1propy1 4-Methylsulfinylbutyl Indol-3-ylmethyl

1-Methoxyindol3-ylmethyl But-3-enyl

2-Hydroxybut-3-enyl

Total

36.5

7.3-121.7

61.3 4.2 67.9

6.1-221.2 0.5-12.2 93.7-231.9

111.9 8.3 367.2

29.3-303.5 1.0-25.4 330.3-406.5

461.9

138.6-900.7

495.0 553.0

318.4-861.9 465.6-600.6

Collards (B. oleracea L., Acephala group) 2-Propenyl 20.7 3-Methylsulfinylpropyl 38.6 Indol-3-ylmethyl 55.5 47.3

12.6-28.7 8.4-69.3 44.2-69.5

Reference

Heaney and Fenwick (1980a) Sones et al. (1984c) Carlson et al. (1987a) Heaney and Fenwick (1980a) Sones et al. (1984c) Carlson et al. (1987a) Mullin and Sahasrabudhe (1977) Heaney and Fenwick (1980a) Sones et al. (l984c) Carlson et al. (1987a)w

220.4

64.4-306.7

Carlson et al. (1987a) Carlson et al. (1987a) Carlson et al. (1987a)Y VanEtten and Tookey (1979) Carlson et al. (l987a)w

Kale (B. oleracea L., Acephala group) 2-Propenyl 97.0

62.5-197.3

Carlson et al. (1987a)

Total

128

E. A. S. ROSA, R. K. HEANEY, G. R. FENWICK, AND C. A. M. PORTAS

Table 3.2.

(continued). Concentration (llmol!100 g of fresh wt) Glucosinolate

Species

Kale (cont.) 3-Methylsulfinylpropyl Indol-3-ylmethyl But-3-enyl 2-Hydroxybut-3-enyl Total Broccoli (B. oleracea L., Italica group) 3-Methylsulfinylpropyl 4-Methylsulfinylbutyl Indol-3-ylmethyl 1-Methoxyindol3-ylmethyl Total

Average

Range

11.7 107.5 21.3 70.1 439.1

0.0-49.9 67.2-165.3 5.8-38.1 16.8-130.3 316.1-600.0

Carlson Carlson Carlson Carlson Carlson

74.1 63.9 97.5 59.4 56.0 8.6

0-327.2 28.9-88.3 54.0-190.2 42.2-71.7 22.8-101.0 2.4-18.4

Lewis et al. (1991) Carlson et al. (1987a) Lewis et al. (1991) Carlson et al. (1987a)Y Lewis et al. (1991) Lewis et al. (1991)

161.9

98.5-323.9

188.2 248.4

102.2-262.7 152.2-448.6

Mullin and Sahasrabudhe (1977) Carlson et al. (1987a)w Lewis et al. (1991)

37.8 35.8 10.0 63.8 41.0 37.5 5.2 51.0 50.0 46.7 60.6 42.1 10.0

1.3-157.9 1.3-157.9 2.9-16.5 1.8-190.1 0.0-90.9 1.3-90.9 0.0-22.8 0.0-327.2 14.8-162.3 13.6-162.3 18.8-104.7 21.0-101.0 1.1-32.0

Sones et al. (1984b) Sones et al. (1984c) Carlson et al. (1987a) Lewis et al. (1991) Sones et al. (1984b) Sones et al. (1984c) Carlson et al. (1987a) Lewis et al. (1991) Sones et al. (1984b) Sones et al. (1984c) Carlson et al. (1987a)Y Lewis et al. (1991) Sones et al. (1984b)

9.3 7.3 105.0

1.2-32.0 2.3-17.4 59.1-180.6

161.9 135.7 94.6 178.2

30.2-520.4 30.2-455.8 41.1-160.6 57.1-448.6

Sones et al. (1984c) Lewis et al. (1991) Mullin and Sahasrabudhe (1977) Sones et al. (1984b) Sones et al. (1984c) Carlson et al. (1987a)w Lewis et al. (1991)

Cauliflower

Reference et et et et et

al. al. al. al. al.

(1987a) (1987a)Y (1987a) (1987a) (1987a)w

(B. oleracea 1., Botrytis group)

2-Propenyl

4-Methylsulfinylbutyl 3Methylsulfinylpropyl

Indol-3-ylmethyl

1-Methoxyindol3-ylmethyl

Total

Turnip tops

(B. campestris L. and B. rapa L., Rapifera group)

But-3-enyl 103.0

294.0 38.0-181.0

Carlson et al. (1981) Carlson et al. (1987b)

3.

129

GLUCOSINOLATES IN CROP PLANTS

Table 3.2.

(continued). Concentration (pmol/100 g of fresh wt) Glucosinolate

Species

Average

Pent-4-enyl 58.0 Total 186.0

Range 151.0 20.0-112.0 586.0 80.0-292.0

Reference Carlson Carlson Carlson Carlson

et et et et

al. al. al. al.

(1981) (1987b) (1981)w (1987b)w

Rapeseed

(B. napus L.)

3,187 But-3-enyl 2-Hydroxybut-3-eny 1 10,937 Pent-4-enyl 824 2-Hydroxypent-4-enyl 522 Total (Summer rape) (Spring rape)

8,031 2,175

Fenwick Fenwick Fenwick Fenwick 8,425-17,002 8,140-12,582 1,000-2,700

et et et et

al. al. al. al.

(1983a) (1983a) (1983a) (1983a)

Fenwick et al. (1983a) Fenwick et al. (1983a) Sang and Salisbury (1988)

Rapeseed (B. campestris L.)

But-3-enyl

13,455 3,863 14,207

2-Hydroxybut-3-enyl

23,450 1,836 209

Pent-4-enyl

250 1,704 240

2-Hydroxypent-4-enyl

322 161

4-Hydroxyindol-3ylmethyl

396 261

10,706-16,107 Fenwick et al. (1983a) 1,302-10,281 Sang and Salisbury (1988) 11,960-15,698 Sang and Salisbury (1988) Davis et al. (1991) 1,050-2,387 Sang and Salisbury (1988) 0.0-520 Sang and Salisbury (1988) Davis et al. (1991) 1,092-2,941 Sang and Salisbury (1988) 0-334 Sang and Salisbury (1988) 234-385 Sang and Salisbury (1988) 0-334 Sang and Salisbury (1988) 294-475 Sang and Salisbury (1988) 0-474 Sang and Salisbury (1988)

Include spring and summer cabbage as well as savoy cabbage. Indole glucosinolate content based on thiocyanate levels. x Total glucosinolates by GC method. w Total glucosinolates calculated by the glucose released method (average molecular weight 457). Z

Y

130

E. A. S. ROSA, R. K. HEANEY, G. R. FENWICK, AND C. A. M. PORTAS

Table 3.3. Other glucosinolates found in lower amounts « 10 vmol/l00 g FW). Species Glucosinolate Z White cabbage Savoy cabbage Red cabbage Brussels sprouts Collards Kale Broccoli Cauliflower Turnip tops

1,3,7,10,11 1,3,7,10,11 11,12 10 3,5 5,10 3,4,5,6,7,8,12,13,14 3,4,7,9,10,12,13,14 2,3,4,10,12,15,16

Source. See Table 3.2. 1 = l-Methoxyindol-3-ylmethyl 2 = l-Methylpropyl 3 = 2-Hydroxybut-3-enyl 4 = 2-Hydroxypent-4-enyl 5 = 2-Phenethyl 6 = 2-Propenyl 7 = But-3-enyl 8 = 3-Methylsulfinylpropyl

9 = 3-Methylthiopropyl 10 = 4-Methylsulfinylbutyl 11 = 4-Methylsulfonylbutyl 12 = 4-Methylthiobutyl 13 = 4-Hydroxyindol-3-ylmethyl 14 = 4-Methoxyindol-3-ylmethyl 15 = 5-Methylsulfinylpentyl 16 = 5-Methylthiopentyl

Z

for B. napus and B. campestris, are also reported in Table 3.2. But-3enyl and 2-hydroxybut-3-enyl glucosinolates predominate in high glucosinolate ("single-low") cultivars (± 109 llmol/g defatted meal), but indole glucosinolates, detected as thiocyanate ion for the first time by McGregor (1978), constitute an increasingly important proportion of the total glucosinolates in cultivars with low (20-30 llmol/ g defatted meal) glucosinolates ("double-low" cultivars). Pent-4-enylcan also be a prominent glucosinolate in oilseed rape (Bilsborrow et a1. 1993b). Glucosinolates with a 3-carbon chain (2-propenyl and 3methylsulfinylpropyl) predominate over 4-carbon glucosinolates in white and savoy cabbages, whereas in red cabbages, 4-methylsulfinylbutyl glucosinolate is the major compound. Indole glucosinolates (particularly indol-3-ylmethyl) are also major components of these three cabbage types. Methylsulfinylpropyl glucosinolate was confirmed as one of the major glucosinolates in several cabbage types (Rosa 1992) and similar results were reported in a study of British white and savoy cabbages (Sones et a1. (1984a,c). Although the qualitative findings in this study were similar to those reported by VanEtten et a1. (1976; 1980), quantitative findings (expressed as total glucosinolate contents) of the UK white and savoy

3.

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cabbage cultivars were much higher than those reported by the American workers (VanEtten et al.1980), due mainly to higher levels of 2propenyl, 3-methylsulfinylpropyl, and indole glucosinolates in the UK cabbage. Large differences were noted in the levels of the major glucosinolates in white and savoy cabbages (Sones et al. 1984a,c), probably due to varieties, growing season, site, and sampling time. In Brussel sprouts the major glucosinolates are 2-propenyl, indol-3ylmethyl, and 2-hydroxybut-3-enyl, but larges differences were noted between English and American studies (Table 3.2), which, despite the variation of SCN- ion precursors throughout the growing season, may be explained by a different genetic base among varieties (Carlson et al. 1987a). Collard leaves were analyzed by Carlson et al. (1987a) who found the predominant glucosinolates to be indol-3-ylmethyl, 3methylsulfinylpropyl, and 2-propenyl, which is in agreement with findings by Rosa and Heaney (1996) for Portuguese kale. However, according to Carlson et al. (1987a), kale has generally higher levels of glucosinolates than collards, particularly 2-hydroxybut-3-enyl, the third major glucosinolate that was found in lower amounts in the material studied by Rosa and Heaney. Bradshaw et al. (1983) also found indol-3-ylmethyl and 2-propenyl to be the major glucosinolates of leaf kales for fodder. In kales, glucosinolate variation might be larger than in other cole crops as due to the rather diverse origins of a number of cultivars that are classified as kale. In cauliflower, indol-3-ylmethyl, 3-methylsulfinylpropyl, and 2propenyl were the major glucosinolates (Table 3.2), however, Lewis et al. (1991) also reported 4-methylsulfinylbutyl as a major glucosinolate in green pyramidal cauliflower (Romanesco). The level of total glucosinolates was comparable with that previously reported for cabbage, but much lower than the levels found in Brussels sprouts and broccoli. Large variations in SCN- content have been noted in cauliflower varieties (Mullin and Sahasrabudhe 1977; Ju et al. 1980a). Carlson et al. (1987a), studying 6 cultivars of broccoli, reported 4methylsulfinylbutyl and indol-3-ylmethyl as the major glucosinolates and first reported the presence of 4-methylthiobutyl glucosinolate in broccoli heads. In purple-headed broccoli, 3-methylsulfinylpropyl glucosinolate is a major compound (Lewis et al. 1991) and in comparison with cauliflower curds, broccoli heads, have less 2-propenyl and higher levels of 4-methylsulfinylbutyl glucosinolates. Several other studies have reported the glucosinolate content of minor brassicacious crops. In turnip tops, the major compounds were but-3-enyl and pent-4-enyl glucosinolates (Carlson et al. 1981,1987b)

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and the total mean glucosinolate content tended to be slightly higher than in cabbage. Davik and Heneen (1993) reported the glucosinolate profiles of 60 cultivars of oilseed turnip (B. rapa Oleifera group), identifying but-3-enyl and pent-4-enyl as the major glucosinolates in the seed, with mean levels of 41.6 and 10.3 llmol/ g dried meal. In Japanese rapeseed the total glucosinolate content was reported to be between 72 and 112 llmol/g dry defatted meal (Amarowicz et al. 1991). In swedes (B. napus Rapifera group) 2-hydroxybut-3-enyl, 2phenethyl, and 4-methoxyindol-3-ylmethyl are the major glucosinolates (Griffiths et al. 1991). In kohlrabi the major glucosinolates were 4-methylthiobutyl, 3-methylthiopropyl, and 2phenethyl (MacLeod and MacLeod 1990), while in rutabaga 2hydroxybut-3-enyl and 4-methylthiobutyl glucosinolates were the main compounds (Shattuck et al. 1991). From 12 cuItivars of Chinese cabbage total glucosinolate levels were between 69 and 465 llmol/ 100 g of fresh weight (FW) (Bajaj et al. 1991). In 20 cultivars of Japanese radishes the variation in total glucosinolates was between 151 and 369 llmol/l00 g of FW (Ishii 1991), lower than the range (90 to 214 llmol/l00 g FW) for 6 cuItivars reported by Shim et al (1993). Methyl glucosinolate, the precursor of the active flavor methyl isothiocyanate in capers (Capparis spinosa 1.) was found at levels between 10 and 67 llmol/l00 g (Sannino et al. 1991). The glucosinolate content in young etiolated sprouts of sea kale (Crambe maritima 1.), used in human food, was reported by Quinsac et al. (1994), who found epiprogoitrin and 4-hydroxyindol-3-ylmethyl representing around 90 and 10%, respectively, of the total glucosinolate content, which ranged between 541 and 728 llmoll 100 g FW, much higher than for the common brassicacious species reported in Table 3.2. V. FACTORS ASSOCIATED WITH GLUCOSINOLATE VARIATION A. Genetics

The variation in both total and individual glucosinolate content in a plant is an effect of genetic factors as well as stage of growth, environment, and their interaction. Rodman (1980) demonstrated that glucosinolate profile characteristics segregated independently from other characters so that selection can be effective for a particular glucosinolate phenotype. Glucosinolate diversity varies widely in families, suggesting that diversification has accompanied speciation (Rodman 1981). Some taxonomic problems have been solved by

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studying the glucosinolate profile among species (Rodman 1980; Luning et al. 1992). Since glucosinolates are stable, genetically determined characters that can be variable within genera, the compounds are potentially useful genetic markers in biosystematic studies (Rodman 1980). Heaney and Fenwick (1980b) have shown that despite quantitative differences, similarities in the glucosinolate profile may be used to characterize different cultivars of Brussels sprouts. However, Davik and Heneen (1993), characterizing cultivars based on glucosinolate profiles, failed to discriminate among oilseed turnip cultivars (B. rapa Oleifera group), probably due to the rather uniform glucosinolate profile between cultivars. The consistently lower thiocyanate content of rapekale (B. napus) as compared to kale (B. oleracea) has been related to genetic background (Paxman and Hill 1974a), and variation in indole glucosinolate content in marrow stem kale has been reported as genetic component (Josefsson et al. 1972). Glucosinolate levels of vegetative tissues and the seed sometimes vary independently of each other and are therefore probably controlled by different genetic and physiological mechanisms, as shown in "single-low" and "double-low" rapeseed cultivars, where glucosinolate concentration differs in seed but not foliage (Inglis et al. 1992). Tookey et al. (1980b) demonstrated that in cabbage, the glucosinolate patterns of the seed have some predictive value for patterns in the leafy heads. This is especially true for the but-3-enyl, 2-hydroxybut-3-enyl, and 4-methylsulfinylbutyl glucosinolates, which provide a convenient means of comparing new and standard cultivars. Similar findings were reported by Jurges (1982) for B. napus Napobrassica group.

B. Morphology and Development There is a marked variation in the absolute amount of glucosinolates between Brassica species, including Chinese cabbage (Daxenbichler et al. 1979), rutabaga, and turnips (Mullin et al. 1980) (Table 3.2), the differences between cultivars being reflected in the wide variation in levels of specific glucosinolates. The glucosinolate content of the growing plant shows significant changes, particularly in the early stages of growth (Rosa 1992) and during bolting, when increased levels of indole glucosinolates occur (Fenwick et al. 1983a). During vegetative growth, from seed germination to the appearance of flower buds, glucosinolate concentrations decline, the change being greater in stem than in leaf tissues (Milford et al. 1989a; Clossais-Besnard

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and Larher 1991), and most noticeable when flowers and seeds were produced (Booth et al. (1991). In rapeseed, glucosinolate concentration was higher in floral tissues than in vegetative tissues and increased from green buds, through yellow buds, to open flowers. Zhao et al. (l993a) and Bilsborrow et al. (1993b) suggested that in both "single" and "double low" oilseed rape cultivars, pod walls are the major site for the biosynthesis of seed glucosinolates, supporting previous findings of an inverse relationship between glucosinolate concentration in pods and seeds (Booth et al. 1991). Variation in thiocyanate ion (as an indication of glucosinolate content) and glucosinolates during ontogeny has been studied in radish plants (Neil and Bible 1972; Chong and Bible 1974a), cauliflower and broccoli (Ju et al. 1980a), forage rape (B. napus and B. campestris), marrow stem kale (Josefsson 1967b), rapeseed (Milford et al. 1989a; Clossais-Besnard and Larher 1991), swedes (Griffiths et al. 1991), and cabbages and kale (Rosa 1992). In the development of root radishes, the highest SCN- content occurred at the cotyledon stage, decreasing until the 6-leaf stage and subsequently to flowering stage (Neil and Bible 1972; Chong and Bible 1974a). Decreasing SCN- content has been observed in roots of kale and rape species during early growth of plants, although trends in SCN- content varied among the species at later stages of growth (Josefsson 1967b). In the foliage of radish, a decreasing content of SCN- during the rosette period preceded large increases during reproductive growth, with the highest amount found at early bolting, followed by a decrease at flowering. Large differences in SCN- content between cultivars have been observed in the foliage (Chong and Bible 1974a). In the foliage of forage rape and of marrow-stem kale variable trends in the SCN- content have been observed, with kale plants showing the highest content of SCN- at the time of maximum growth, with a subsequent decrease to a low value throughout later growth (Josefsson 1967b). In leaves and stems of cauliflower and broccoli, the highest SCNcontent has been observed in 15-day seedlings at the cotyledon stage of development followed by a rapid decrease. A similar trend has been found in the roots of rutabaga (B. napobrassica Mill) and turnip (B. rapa 1.) (Ju et al. 1980b). In cauliflower curds and broccoli heads the highest quantity of SCN- was found prior to the flowering stage. The thiocyanate content of small young leaves of kale can be up to five times higher than levels found in large fully formed leaves and about twice the amount present in leaves of intermediate size (Paxman and Hill 1974b). Studies indicate that young leaves or other

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young photosynthetic tissues are perhaps major sites of synthesis or storage of the parent indole glucosinolates, which have some role to play in auxin metabolism Uu et a1. 1980a; Chong and Bible 1974a,b). Jurges (1978) estimated indole glucosinolates (by analysis of thiocyanate ion) in addition to a group of aliphatic glucosinolates (2propenyl, but-3-enyl, pent-4-enyl, 2-hydroxybut-3-enyl, and 2hydroxypent-4-enyl) in B. napus and B. campestris. The aliphatic glucosinolates showed relatively high concentrations at the beginning of the vegetation period, with a permanent reduction during ontogeny. In a particular variety of B. napus, similar indoleglucosinolate concentrations were found in flower buds and in vegetative material at the beginning of the vegetative period. Subsequently, this concentration decreased in vegetative material throughout plant development, while in the generative parts, 4 weeks after the start of flowering (development of siliques), levels started rising. The volatile isothiocyanates tend to decrease from early stages of growth to maturity, as shown for turnip and rutabaga by Ju et a1. (1980b), although, in turnip, an increase occurred during bolting. Total glucosinolates increased during the first 5 days of germination of B. napus, B. campestris, B. juncea, and Eruca sativa, with higher accumulation in the epicotyl than in the hypocotyl (Sukhija et a1. 1985). In rapeseed there was a gradual increase in total glucosinolates following the lowest concentration, which occurred 76 h after imbibition (Kim et a1. 1988). The fact that glucosinolate levels change during the growth of the plants suggests that insect attraction and infestation might be favored at certain growing stages of the plant. Plant morphology can be associated with glucosinolate content. According to Josefsson et a1. (1972), the indole glucosinolate content of marrow stem kale was significantly higher in plants with thin stems (average diam 20 mm), whereas plants with thick stems (average diam 35 mm) were higher in glucosinolates producing isothiocyanates and oxazolidinethiones. Glucosinolate content depends on plant part, and levels in stems and petioles were shown to be lower than those of roots and heads in marrow stem kale, broccoli, cauliflower, rutabaga, and savoy cabbage (Josefsson 1967a). In white cabbage, the highest glucosinolate content was in roots and stems, followed by heads and petioles. Higher levels of glucosinolates were found in roots than in leaves of cabbage seedlings (E. A. S. Rosa and R. K. Heaney, unpublished) and although, in general, roots have higher glucosinolate levels than other organs, the glucosinolate content was lower in roots of marrow stem

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kale than in the lamina and in heads of Brussels sprouts and broccoli (Sang et al. 1984). Bradshaw et al. (1984) found glucosinolate content of fodder kale and rape to be higher in stem than in leaves, except for indole glucosinolates. Carlson et al. (1981) demonstrated quantitative differences in the distribution of glucosinolates in shoots, peeled roots, and peelings of turnips (B. campestris Rapifera group) and peelings and peeled roots of rutabagas (B. napus Napobrassica group). Besides changes between plant organs, total and individual glucosinolate concentration showed a dynamic variation during ontogeny in leaves and heads of several Brassica plants (Rosa 1992) and in leaves and bulbs of swedes (B. napus Rapifera group) (Griffiths et al. 1991), with contents increasing toward the root tip of swedes (B. napus L. Napobrassica group) (Jurges and Robbelen 1980). The glucosinolate differences between plants and the large changes in the types of glucosinolates that occur in vegetative and floral tissues during ontogeny, underscore the necessity of making a comprehensive characterization of plant material when sampling for analysis. In 1979, VanEtten et al. studied the distribution of glucosinolates in the pith (relatively inactive tissue), cambial cortex (cambial and cortical tissues of actively dividing cells from which the leaves of the cabbage head originate) and leaves of the head in white, savoy and red cabbage. An analysis of top, center, and bottom portions of the cambial cortex indicated a similar pattern, with the basal section slightly, but not significantly, lower in total glucosinolate. There was a lower glucosinolate content in the older, outer leaves that were not a part of the cabbage head. The cambial cortex layer contained about twice the concentration of total glucosinolates compared to the pith or the leaves and was especially rich in 2-propenyl (white cabbage), 3-methylsulfinylpropyl (savoy cabbage), but-3-enyl (red cabbage), and 2-phenethyl glucosinolates. Leaves were generally high in indoles (white and savoy cabbage) and 4-methylsulfinylbutyl (red cabbage), and 3-methylsulfinylpropyl glucosinolate was also a major component. In pith, the highest components were 4methylthiobutyl (red cabbage), 3-methylthiopropyl (savoy and white cabbage), and 2-propenyl glucosinolates (white cabbage). The cambial cortex, despite its high content of glucosinolates, probably does not make a major contribution to human consumption of glucosinolates or derived products, since it represents only about 3.6% of the head weight. Leaves represent 94% with the remaining 2.4% being attributed to pith. Within the cabbage head (heart, inner central, outer central and outermost zones), there is a

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variation in glucosinolate levels, with a temporary tendency for lower concentrations in the two middle portions, explained as the result of rapid leaf expansion ("dilution effect"), while in the outermost zone the expansion was nearer completion (Pocock et al. 1987). This general pattern was followed loosely by 2-propenyl, 4-hydroxybut3-enyl, and indol-3-ylmethyl glucosinolates, whereas 3methylsulfinylpropyl glucosinolate showed an increase in concentration toward the outer leaves. However, in another cabbage variety, a different distribution was found, with a marked increase from inner to outer zones in 2-propenyl, 3-methylsulfinylpropyl, and 4hydroxybut-3-enyl glucosinolates although indol-3-ylmethyl glucosinolate decreased. In Brussels sprouts buttons (the immature small heads formed at each leaf) the glucosinolate levels increase toward the centre (R.K. Heaney, unpublished). Thus, the type and size of heads, the stage of their development and the proportion between young and old portions might influence the levels of glucosinolates. The seed pod position influences the glucosinolates content in rapeseed (Kondra and Downey 1970). The highest glucosinolate level was found in the lowest pods on lowest raceme, while the highest values were reported for the lowest pods on main flowering raceme. Maturity date (early or late maturing) influences the glucosinolate content of Brassica cultivars. Higher SCN- content has been found in later maturing cultivars of cauliflower (Ju et al. 1980a), radishes, turnips (Chong and Bible 1974b), and cabbages (Bible et al. 1980). C. Environment

Synthesis and degradation of glucosinolates can occur in a wide range of climate conditions, even at temperatures as low as O°C (Shattuck et al. 1990). Merrien (1989) reported pedoclimatic conditions affected levels of glucosinolates in oilseed rape. Bible and Chong (1975b) showed that climate can influence amounts of thiocyanate ion or its precursors and concluded that, despite variable effects of soil and climate, cold unit accumulation is a reliable index of root radish SCN- yield. Root radish SCN- content increased under cooler conditions and was associated with accumulated cold units (degree day accumulation below 18°C) on both organic and loam soils. Thiocyanate content was negatively correlated with mean daily air temperature on organic soil and positively correlated with rainfall accumulation only on loam soil. There are seasonal effects on glucosinolate content (Sang et al. 1986; Ishii and Saijo 1987; Rosa

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1992). Winter or autumn seasons seem to induce lower glucosinolate levels, due to short days, wetter conditions, cool temperatures accompanied by frosts, and less radiation. Synthesis of glucosinolates appears to be influenced by water availability, and a glucosinolate/ evapotranspiration relation was presented by Mailer and Pratley (1990). Periods of drought at pre- or postflowering stages greatly increased glucosinolate concentration in oilseed rape (Milford 1991) and in leafy material of Portuguese cabbage in spring and summer growing seasons (Rosa 1992). Large variation occurred in SCN- content of 14 cabbage cultivars between years (Bible et a1. 1980) and in total and individual glucosinolate content in several Brassica spp. (Rosa 1992). Total and individual glucosinolate concentrations can change significantly throughout the course of a single day (Rosa et a1. 1994) and under constant temperature conditions glucosinolate turnover was identified between the roots and the aerial part of the plant throughout the day (E. A. S. Rosa and R. K. Heaney, unpublished). Total glucosinolate content showed a site-to-site variation, although the relative proportions of the individual glucosinolates remained remarkably constant in Brussels sprouts (Heaney and Fenwick 1980a) and in rapeseed (Anony. 1989,1990). A study of the influence of photoperiod on glucosinolates, in which isothiocyanate content of radish was measured, was inconclusive (Neil and Bible 1973). Under a short photoperiod (8.5 h) radishes grown in loam soil had the highest isothiocyanate concentration, but under a longer photoperiod (15 h) radishes grown in organic soil showed the highest levels. It has been suggested that factors that modify root development may alter the ratio of secondary cortex cells (which have the highest glucosinolate content) to other cortical cells. D. Cultural Practices Density and spacing, spraying, and plant irrigation of Brassica all influence the glucosinolate content. To improve productivity, current trends are to use narrow spacings, which may affect flavor. Closer spacing (45 vs. 75 cm), which is likely to affect morphology by reduction of head size, increased concentrations of glucosinolates and other sulfur compounds to an undesirably high level (MacLeod and Nussbaum 1977), probably by reducing the "dilution effect" referred by Pocock et a1. (1987). Similar behavior was found in Brussels sprouts (MacLeod and Pikk 1978, 1979), and was attributable to stress conditions that induced increased biosynthesis of the low molecular weight precursors of flavor compounds. In rapeseed, there was no effect of plant population density in glucosinolate concentration

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despite morphological changes in the plant (the closely spaced plants have few branches and the widely spaced plants many) and in pod maturity as a result of light interception (Ishii and Saijo 1987). Increased plant density did not result in differences in seed glucosinolate concentrations in rapeseed (Evans et al. 1989) or isothiocyanate ion concentration in radish roots (Raphanus sativus) (Ishii and Saijo 1987). The influence of herbicides and pesticides on the glucosinolate content is not clear, but MacLeod and Nussbaum (1977) concluded that these inputs did not materially affect the flavor of cabbage. E. Pests and Diseases

Pest and disease infections can markedly alter glucosinolate levels. The turnip mosaic virus (TuMV), one of the most important viruses to which brassicacious plants are susceptible, increased total glucosinolate concentrations when infection occurred in the early stages of rutabaga root development, while the increase in 2hydroxybut-3-enyl glucosinolate was independent of time of infection and root growth (Stobbs et a1. 1991). An increase in total and individual glucosinolate concentration in mature seeds of TuMVinfected rapid cycling B. campestris was reported by Shattuck (1993), whereas an accumulation of indole glucosinolates in tissues of oilseed rape (B. napus) following infestation with flea beetle (Psylliodes chrysocephala 1.) was reported by Koritsas et al. (1989). Increased synthesis and degradation of indol-3-ylmethyl glucosinolate was also shown after clubroot infection (Rausch et a1. 1983; Butcher et al. 1984). Similarly, Lammerink et al. (1984) found levels of total glucosinolates to increase by up to 100% in seeds and seed meal of rape (B. napus) infested with Brevicoryne brassicae. However, cultivars vary in the synthesis of glucosinolates when submitted to fungal infection stress. An increase in aromatic and indole glucosinolates occurs in "single-low" and "double-low" oilseed rape cultivars after infection with Alternaria brassicae, while aliphatics increased only in "single-low" cultivars, mainly as a result of sulfur metabolism (Wallsgrove et a1. 1993). It is still unclear, whether stress-induced glucosinolate changes are regulated by the plant and have a biological purpose or are only by-products of abnormal plant metabolism.

F. Soils and Plant Nutrients 1. Soil Type. Soil type has been suggested to influence glucosinolate

content, with organic soils favoring increased levels of indole

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glucosinolates (Neil and Bible 1972), mean total glucosinolate (Ju et al. 1980b), and thiocyanate content (Chong et al. 1982). Increases of 15 and 38%, respectively, in mean glucosinolate content were found for rutabaga and turnip grown in organic soil (Ju et al. 1980b). Levels of indole glucosinolates, glucosinolates yielding volatile isothiocyanate hydrolysis products, and 2-hydroxybut-3-enyl glucosinolate in roots of both species increased throughout the growing season, probably due to higher S levels, increased soil moisture and aeration, and cooler prevailing temperature of the organic soil (Chong et al. 1982). 2. Organic Matter. The effect on glucosinolate content of applying organic matter to soil has been studied (Goodrich et al. 1988). Broccoli, Brussels sprouts, and cabbage were grown using either a sludge (pH 6.7; fertilizer N-P-K equivalent of 1.67-1.12-0.13; ash 68%) applied at a rate of 224 t/ha or cow manure at the same N-P-K rate as a control. In both amended soils, total glucosinolates in broccoli and Brussels sprouts were similar, but sludge-amended soil produced significant changes in specific glucosinolate levels and a significant reduction of glucosinolate in cabbage. 3. Sulfur. Brassicacious crops require considerably more S than most

other crops, due to its role in the synthesis of glucosinolates as well as sulfur aminoacids and proteins (Qinzheng et al. 1991a,b). Glucosinolates act as a vital storage for S (Schnug and Ceynowa 1990), although the remobilization of S via increased myrosinase activity is possible only from intact glucosinolates and not from the intermediary products (Schnug 1988, 1989). The influence of S on the glucosinolate content of several Brassicaceae species has been the subject of many studies which indicate that glucosinolate content tends to increase with the S content of the growing medium. In a recent study, S application was shown to increase the glucosinolate content in the vegetative parts and flowers of B. napus (Booth et al. 1991). An increase in thiocyanate ion with sulfate level has also been reported by Bible and Chong (1975a), suggesting that the effect of high nutrient concentration may alter the synthesis or accumulation of thiocyanate, and subsequently the correlation between Sand glucosinolates. This suggestion could explain the results obtained by Werteker (1991) who found that in soils with surplus sulfate concentrations between 100 and 160 ppm, there was no statistically significant correlation between glucosinolates and sulfate. Sulfur content in soils can be high enough to satisfy the crop requirements for sulfate, particularly in heavy soils (Josefsson 1970b).

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The use of fertilizers low in S did not significantly lower the glucosinolate content of oilseed rape, probably because S is not easily leached out from heavy soils and because S is also transported to the soil from the atmosphere (Josefsson 1970a). Similarly, when kale was grown in silt loam soils the glucosinolate content was unaffected by restriction of sulfate (McDonald et a1. 1981). On the other hand, an increase in myrosinase activity was shown in B. napllS and S. alba plants under S stress (Schnug 1990; Bones et a1. 1994), which may induce remobilization of S from glucosinolates and their subsequent decrease. Bodnaryk and Palaniswamy (1990) showed that a restriction in S induced a decline of 2-propenyl glucosinolate in B. jllncea. Thus, lower S supply can result in a decrease on glucosinolate content in the plant, either by the activation of myrosinase hydrolysis or by a reduction on the synthesis of glucosinolates. The form in which S is applied to the growing medium seems to influence the glucosinolate content of rapeseed, particularly when applied as sulfate rather than as elemental S (Anon. 1989). However, the growing medium itself influences the absorption and incorporation of S into glucosinolates (Qinzheng et a1. 1991b). 4. Nitrogen. Nitrogen is a widely used fertilizer for Brassica crops,

and is known to affect glucosinolate levels within and among plant tissues. Although N is a constituent of the glucosinolate molecule, early studies showed that increasing application of N tended to give lower glucosinolate levels and some more recent experiments seem to confirm this view. For example, such trends have been reported for kale by McDonald et a1. (1981) and for kohlrabi by Fischer (1992). Milford et a1. (1989a) found glucosinolate concentration in floral tissues of oilseed rape (B.naplls) grown with low N to be twice as much as those of well-fertilized crops however, Gustine and Jung (1985) reported a general increase in glucosinolate content in Brassica forage when N fertilization rates are elevated. Bilsborrow et a1. (1993a) demonstrated increasing levels of oilseed rape glucosinolates with N applications up to 150 kg / ha, where S supply was adequate. Recent work by Zhao et a1. (1993b) suggests that the balance between the Nand S supply plays an important role in the regulation of glucosinolate biosynthesis, whereby increasing N supply increases the seed glucosinolate concentration only if S is not at deficient levels. This balance was more important for alkenyl glucosinolates production due to the requirement of methionine in their biosynthesis (Zhao et a1. 1994). The physiological regulation of N-induced glucosinolate production in plants and the biological importance of stress-induced

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glucosinolate change remain to be established and several mechanisms have been presented to explain these changes. McDonald et al. (1981) suggested that at high rates ofN application, the synthesis of other substances in the plant that result in biomass increases will occur more readily than glucosinolate production. Since N fertilization presumably results in a more extensive use of the products of the tricarboxylic acid cycle for production of protein and relatively less carbohydrate, glucose might be a limiting factor for glucosinolate biosynthesis at high levels of N fertilization. The effect of N form on glucosinolate levels are not conclusive and no glucosinolate change was noted in rapeseed when N was applied as nitrate, ammonium, ammonium nitrate, and urea (Josefsson 1970a; Anon. 1989, 1990). Differences in plant material and development stage of the plants between experiments could account for the different responses to N fertilization. Sulfate utilization by higher plants may require the presence of nitrates in the nutrient medium. Another form of N, nitrogen dioxide (NO), which is an atmospheric pollutant, was shown to increase levels of indole glucosinolates in seedlings and older plants of B. campestris Chinese group (Shattuck and Wang 1993). In this case, the effective form of N may be nitrate or ammonium because gaseous NO z reacts with extracellular water or the cytoplasm to form nitrate or nitrite ions, which are rapidly converted to the ammonium ion by the activities of nitrate and nitrite reductase (Shattuck and Wang 1993). 5. Micronutrients. Boron nutrition is an important factor influencing glucosinolate content in Brassicaceae, according to Bible et al. (1981). These authors observed that induced boron deficiency significantly increased the yield of thiocyanate ion in both foliage and roots of radishes and altered the distribution of reducing sugars. They suggest that boron regulates the synthesis or accumulation of thiocyanate-yielding precursors and this was confirmed by Shelp et al. (1992) who showed a threefold increase in total glucosinolates and especially in indole glucosinolates in broccoli cultivars grown under conditions of boron deficiency. In roots of boron-deficient turnips the same tendency was observed, with those receiving 0.1 ppm boron showing brown heart but containing high levels of oxazolidinethione, accompanied by relatively high yields of thiocyanate ion Uu et al. 1982). In an in vivo study Jiracek et al. (1974a) demonstrated the positive effect of Zn in biosynthesis of indole glucosinolates, preceded by an increased biosynthesis of L-tryptophan. The effect of Cu 2 + ions

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on the glucosinolate content is rather complex. In the studies of Jinicek et a1. (l974b), the greatest accumulation of free Cu 2 + cations in the plant corresponded to a decrease in glucosinolate content. However, at stages before and after the greatest Cu 2 + accumulation (corresponding to 5 x 10-4 to 5 x 10-5 M concentration range), Cu 2 + appeared to stimulate the glucosinolate formation. G. Water Stress

Although other reviews (Fenwick et a1. 1983a) indicated that higher rainfall is linked to increased goitrogenicity (and, by implication, increased glucosinolate content) of cabbage, Freeman and Mossadeghi (1973) found an increase in glucosinolate production with increasing water stress conditions. Similar results were reported by Bible et a1. (1980) who found that while the marketable head weight of cabbage was lower, the thiocyanate content was more than twice that of irrigated plants. Later studies (Sang et a1. 1986) confirmed that years with rainfall below the average produce rapeseed with higher total glucosinolate contents. Increasing water stress increases inorganic and organic S in the plant (Freeman and Mossadeghi 1973). These results also suggested that Brassica crops submitted to relatively poor growing conditions accumulate large amounts of basic metabolites, such as sugars and amino acids, which could be differentially converted to secondary metabolites, Le., glucosinolates, rather than the expected conversion to cellulose and proteins during rapid growth (Freeman and Mossadeghi 1973; Bible et a1. 1980). This is in agreement with findings that plants grown under poor conditions, i.e., water shortage or nutrient deficiencies, tend to synthesize exceptionally high concentrations of secondary compounds (Janzen 1974). H. Growth Regulators

In brassicacious plants, indol-3-ylmethyl glucosinolate is often described as a precursor of the auxin IAA. It was suggested that this glucosinolate is translocated from the apex in a basipetal direction in savoy cabbage plants (Andersen and Muir 1969) and that GA treatment promotes the conversion of indol-3-ylmethyl glucosinolate to IAN, which is hydrolyzed by the enzyme nitrilase to IAA, promoting elongation of the stem. Most growth regulators act primarily on apical meristems, where enzymes are generally more active, influencing directly or indirectly auxin concentrations, which may in

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turn influence indole glucosinolates synthesis and/or related metabolic processes (Chong et al. 1982). Thiocyanate ion (corresponding to indole glucosinolates) increased with applications ofbutanedioic acid mono-(2,2-dimethylhydrazide) (darninozide), gibberellic acid (GA), 6-benzylamino purine (BA), and 2A-dichlorophenoxyacetic acid (2 A-D) to turnip (Brassica rapa L.) and radish (Raphanus sativus 1.) (Chong et al. 1982). I. Processing

The first step in preparing vegetables for human consumption, even when they are used raw, is cleaning/washing. These processes, which may cause the rupture of some plant cells, particularly in young and smooth leaves, brings myrosinase in contact with glucosinolates, increasing glucosinolate degradation. Cutting (slicing, dicing), cooking/blanching, and drying or freezing can influence the extent of hydrolysis of glucosinolates and the composition, flavor, and aroma of the final products. Other processes, sometimes following considerable storage, may include freezing, pickling, or fermentation, but literature on the effect of such procedures on glucosinolates is scarce. Cooking and blanching of Brassica vegetables may significantly decrease glucosinolate levels due to enzymic and thermal breakdown and due to leaching of the compounds into water. The common processing conditions and their effects on glucosinolate levels are considered below. 1. Cooking and Pulping. Glucosinolates and their hydrolysis products are water soluble and tend to be leached into the cooking water. Production of the volatiles 2-propenyl and butyl isothiocyanates and allyl cyanide was recognized by Macleod and Macleod (1968), and Shim et al. (1992b, 1993) noted that wet heating induces the release of high amounts ofthiocyanates. However, Michajlovskij et al. (1969a, 1970) found that on boiling, the volatile isothiocyanates disappeared completely and some other aglucones (5-vinyl-oxazolidine-2-thione and 3-methylsulfinylpropyl isothiocyanate) were partly or completely decomposed. All the liberated inorganic thiocyanate remained in the boiled material. However, about half to two-thirds of the original amount of glucosinolates remained unaffected in the boiled cabbage, cauliflower, kale, and kohlrabi. The glucosinolates were classified according to their decreasing heat stability in the following order: 2propenyl, 2-hydroxybut-3-enyl, indol-3-ylmethyl, 3-methylsulfinylpropyl, but-3-enyl, with the least thermo-stable being 3-methylthiopropyl glucosinolate.

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Sones et a1. (l984c), when cooking cabbage, Brussels sprouts, cauliflower, and swede-turnip, found the total glucosinolate content of the vegetables to decrease by an average of 36% with a considerable variation between different cultivars of the same species. Average losses of individual glucosinolates during cooking ranged from 34% (2-propenyl) to 59% (1-methoxyindol-3-ylmethyl). McMillan et a1. (l986) cooked Brussels sprouts for 9 min and reported average losses of 40, 30, and 63%, respectively for total glucosinolate content, 2hydroxybut-3-enyl, and indol-3-ylmethyl glucosinolates and similar results were described by Groot et a1. (1991) when cooking for 15 min, except that reduced losses were noted for indol-3-ylmethyl glucosinolate. In white and savoy cabbage cooked for 5 min, the latter showed the greater loss in glucosinolates (McDanell et a1. 1987). Indol-3-ylmethyl was reduced by 34 and 44% in white and savoy cabbage, respectively. A 50% reduction on total and individual glucosinolates was also reported for cabbages and kale cooked for 10 min, the loss being accounted for in the cooking water (Rosa and Heaney 1993). If such water is used for soup or gravy, nearly 100% of the intact glucosinolates can be ingested. Shredding has been shown to induce a more efficient release of glucosinolates into the cooking water. Glucosinolate content was shown to decrease with longer cooking times (Quinsac et a1. 1994). Myrosinase activity increases with temperature up to 60°C, and the enzyme is denatured at 100°C (Bjorkman and Lonnerdal 1973). Although some residual myrosinase was detected in Brussels sprouts that had been boiled for 6-7 min, cooking for 9 min strongly reduced myrosinase activity, thus limiting the production of 5-vinyloxazolidine-2-thione and thiocyanate ion (McMillan et a1. 1986). Bradfield and Bjeldanes (l987b) found a decrease of only around 20% in myrosinase activity on cooking cauliflower for 10 min, and autolytic products are thus likely to occur in cooked as well as raw vegetables. The effect of cooking on indole glucosinolate breakdown products in Brassica has been reported by Slominski and Campbell (1989). They found that heat treatment (lOO°C, 50 min) of cabbage, resulted in substantial decomposition of indole glucosinolates, with thiocyanate ion and indoleacetonitriles accounting for 50 and 30%, respectively, of the degraded indoles. Heat treatments of 10 min (steamed) or 40 min (cooked) of cabbage, broccoli, or cauliflower gave similar relative amounts of thiocyanate ion and indoleacetonitriles, despite a variation among samples. Cooking, as opposed to steaming, gives rise to increased leaching of the degradation products. Of the glucosinolates present in raw cabbage, 90% was recovered in cooked

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cabbage and in the cooking water as intact glucosinolates, thiocyanate ion, and indoleacetonitriles. Cooking water was found to contain over 50% of the glucosinolates or glucosinolate breakdown products. Overcooking of cabbage can generate undesirable odor and flavor. MacLeod and MacLeod (1970b) showed that allyl isothiocyanate reached a maximum at about 20 min and most is formed between 10 and 30 min. Steam blanching (3 min in a blancher) or blanching in boiling water of cabbage samples resulted in a 30% reduction of isothiocyanate-forming glucosinolates and a 50% reduction of indole glucosinolates (Srisangnam et al. 1980). Water blanching (99°C for 4 min) and steam blanching (99-102°C for 5.5 min) of Brussels sprouts, and broccoli did not significantly reduce glucosinolate levels in Brussels sprouts while water blanching of broccoli produced a significant glucosinolate loss (Goodrich et al. 1989). In broccoli, increased leaching of total glucosinolates was observed during water blanching (83%) when compared to steam blanching (40%), whereas for Brussels sprouts the proportions were 7 and 22 %, respectively. In another study, high amounts of volatiles were produced from blanching cauliflowers and Brussels sprouts (Langenhove et al. 1991). The fate of glucosinolates during blanching is determined by factors such as genotype, physical configuration of the product, and temperature and duration of the blanching process. Microwave heating resulted in enzyme inactivation and glucosinolate decomposition; however, these effects are dependent on the time of exposure to microwaves and the water content of the vegetable (Maheshwari et al. 1980). The higher the water content of the plant, the greater the absorption of microwave radiation by that plant and the reduction in cooking time (MacLeod and MacLeod 1970b). Some glucosinolate hydrolysis is to be expected when vegetables are chopped prior to cooking. However, fine chopping or homogenization of fresh plant tissues creates favorable conditions for myrosinase activity and a high degree of glucosinolate degradation can be expected. Daxenbichler et al. (1977) showed that while autolysis of fresh cabbage produced predominantly nitriles, isothiocyanates and oxazolidine-2-thione resulted when the same cabbage was air-dried at 50°C prior to autolysis. Indole-3-carbinol was the major glucosinolate metabolite generated when plant material was disrupted, although it is unstable in the autolytic milieu (84% conversion over 24 h to products such as 3,3-diindolylmethane, indole-3carboxaldehyde) (Bradfield and Bjeldanes 1987a). On the other hand,

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autolysis of raw cabbage, cauliflower, and Brussels sprouts indicated a very low production of indoleacetonitriles and high thiocyanate ion levels. 2. Freezing and Refrigerating. Freezing Brassica products at -18°C generally eliminates the production of off flavors, but marked differences have been noted between fresh and frozen Brussels sprouts. Fresh material showed higher thiocyanate and isothiocyanate contents, whereas frozen samples gave allyl cyanide, thiourea derivatives, and oxazolidinethiones. The difference is most likely due to inactivation of myrosinase during the blanching stage of the preservation process (Mac Leo d and MacLeod 1970a, Mullin and Sahasrabudhe 1978). Not all cultivars are well adapted to freezing. The differences are probably due to action of certain enzymes or coenzymes after blanching, which alter the hydrolytic breakdown route. Freezing without previous inactivation of myrosinase results in an almost complete glucosinolate decomposition after thawing, as shown in sea kale (Quinsac et a1. 1994). Low-temperature storage (below 10°C) alters the metabolism of glucosinolates (Shattuck et a1. 1991). Storage reduces glucosinolate contents in several Brassica (Shim et a1. 1992a), which may be due to the occurrence of uncontrolled hydrolyis, as noted in storage cabbage (1°C) for 215 days, which yielded, in descending order, isothiocyanates, thiocyanates, and goitrin (Chong and Berard 1983). 3. Dehydrating and Lyophilization. Dehydration of intact cabbage leaves in forced-draft ovens at temperatures between 50 and 65°C failed to destroy glucosinolates and myrosinase remained active (E.A.S. Rosa, unpublished). Similar findings were reported by Daxenbichler et a1. (1977). Lyophilization followed by grinding, homogenizing, and storing at -20°C has been described as a means of preserving glucosinolates, as myrosinase remains inactive until the addition of water to the dry material (Tiedink et a1. 1988). 4. Fermenting. Sauerkraut represents the most common fermented

product from cabbage. All the glucosinolates in fermented cabbage were hydrolyzed within 2 weeks (Daxenbichler et a1. 1980; de Vos and Blijleven 1988). In the finished canned sauerkraut, nitrile l-cyano-3-methylsulfinylpropane was the major component, representing 50% of the theoretical yield from the parent glucosinolate (3methylsulfinylpropyl). Thiocyanate ion was also generated, but isothiocyanate was undetected, probably due to the acidity of the

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medium. Losses have been attributed to heat applied during canning and to drainage of the juice. 5. Others. Coleslaw (shredded cabbage with salad dressing) is a common way to use raw cabbage. The generally high concentrations of allyl glucosinolate and vitamin C in the fresh cabbage, inassociation with the relatively low pH (about 4.5) of the coleslaw which mediated myrosinase hydrolysis, results in a final product with high levels of the corresponding isothiocyanate and a very low nitrile concentration, when compared to the fresh shredded cabbage (West et a1. 1977). Glucosinolate variation and production of degradation products can also result from extrusion and dry heating used in rapeseed processing, which have been partially effective in inactivating myrosinase, while autoclaving completely inactivated it, reducing isothiocyanate content (Ochetim et a1. 1980).

VI. BIOLOGICAL EFFECTS A. Humans and Other Animals 1. Cancer. Epidemiological studies of diet and cancer incidence

over the last decade reveal evidence of a protective effect of fruit and vegetables, especially those of the Brassicaceae and the genus Brassica. The role of fruit and vegetables in exerting a net protective effect against cancer has been the subject of several reviews (Hocman 1989; Steinmetz and Potter 1991a,b; Negri et a1. 1991; Block et a1. 1992; Kromhout et a1. 1993). A statistically significant protective effect of fruit and vegetables or the nutrients derived from them was found in 128 out of 156 studies of dietary intake (Block et a1. 1992). Most of the studies reported by Block et a1. (1992) for the protective effect of brassicacious vegetables are in stomach and colorectal cancers, the major cause of cancer death worldwide. Feeding brassicacious vegetables or glucosinolate derivatives can modify endogenous detoxication processes (McDanell et a1. 1989; Nugon-Baudon et a1. 1990b) and, thus, may interfere in a positive way with the metabolism of chemical carcinogens (Stoewsand et a1. 1988) or more generally of toxic chemical compounds. Wattenberg (1993) has reviewed the classes of compounds found in the diet that contribute to the protective effect against chemical carcinogens and it is clear that Brassica vegetables are important. The specific role of glucosinolates is discussed below. Much of the pioneering work on the anticarcinogenic properties of naturally occurring compounds has been carried out by Wattenberg

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and his coworkers. In an early study, Wattenberg (1971) showed benzo(a)pyrene hydroxylase activity in the intestine of rats fed a diet containing Brassica vegetables when compared to rats fed a purified diet and later proposed a system of classification of dietary anticarcinogens based on the stage of carcinogenesis at which they act (Wattenberg 1983, 1985). Anticarcinogens can then be divided in three major classes. The first class consists of compounds that prevent the formation of carcinogens from precursor substances. The second are called "blocking agents" and have been found to be effective when given immediately before or during treatment with chemical carcinogens, while the third class, called "suppressing agents," are thought to act by preventing the progression of initiated cells to fully transformed tumor cells, even when given after treatment with a complete carcinogen or a combination of incomplete carcinogen and promoting agent. According to Wattenberg (1985), aromatic isothiocyanates and indoles belong to the class of blocking agents, with benzyl isothiocyanate being considered also as a suppressing agent. Johnson et al. (1995) proposed a scheme (Fig. 3.7) for summarizing the various possible mechanisms of protective factors. The metabolic pathway of detoxification usually comprises three phases: phase I, which often involves carcinogen activation by oxidation reactions; phase II, which consists of conjugation reactions, whereby the molecule is made sufficiently polar to be easily excreted from the organism; and phase III, where the transport out of the cell occurs (Johnson et al. 1995) Diets containing Brussels sprouts and cabbage increased phase I oxidative metabolism of phenacetin and antipyrine in human volunteers (Pantuck et al. 1979) and glucuronide enhanced conjugation of paracetamol in human volunteers fed brassica-containing diets (Pantuck et al. 1984). Compounds that induce enzymes ofxenobiotic metabolism, particularly phase II enzymes, are considered to protect against cancer. Prochaska et al. (1992) developed a simple and rapid method for assaying the ability of plant extracts to induce enzymes that detoxify carcinogens (phase II enzymes), based on the direct assay of the activity of quinone reductase (NAD[P]H:[quinone acceptor] oxidoreductase, EC 1.6.99.2) in murine hepatoma 1c1c7 cells. The method, which uses microtiter plates, has facilitated the screening of large numbers of plants, especially those of the genus Brassica. Dietary cabbages and collards were shown to inhibit the yield of pulmonary metastases in mice injected with BALB/c tumor cells (Scholar et al. 1989). Miller and Stoewsand (1983) showed that Brussels sprouts enhanced hepatic aryl hydrocarbon hydroxylase (AHH)

"'CJ1"' o

NORMAL CELL

BLOCKING AGENTS

I PROCARClNOGEN

I

SUPPRESSING AGENTS

Fig. 3.7. Mechanisms and sites of interaction whereby protective factors may inhibit the carcinogenic process. (From Johnson et al. [1995], with permission.)

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activity, while a similar diet devoid of glucosinolates was inactive toward AHH but retained 0- and N-demethylase activities, thus indicating the presence of other protective compounds. Savoy cabbage has been found to be a stronger inducer of mixed-function oxidase activity than white cabbage and this was attributed to the higher levels of glucosinolate-derived indole compounds in the former (McDanell et al. 1987). Inclusion of mustard seed (Brassica nigra) in rat diets at levels as low as 1 % of total feed was shown to reduce the adverse biological effects of benzo(a)pyrene metabolites (Polasa et al. 1994).

Indole compounds. The involvement of hydrolysis products of indol3-ylmethyl glucosinolate in the activation of the drug metabolizing enzyme system was first reported by Loub et al. (1975) and reviewed by McDanell et al. (1988). Loub et al. (1975) identified from Brussels sprouts, cabbage, and cauliflower, indole-3-carbinol, indole-3-acetonitrile, diindolylmethane, and ascorbigen as inducers of benzo(a)pyrene activity in rat liver and intestine. The most potent was indole-3-carbinol (150 mg/kg body weight), which increased benzo(a)pyrene activity by 56-fold in the liver and 31-fold in the small intestine, followed by, in decreasing order of activity, indole3-acetonitrile, 3,3-diindolylmethane, and ascorbigen, which has also been recently reported as a modifier of P450 enzyme activity (Preobrazhenskaya et al. 1993). Several other studies have shown that of the hydrolysis products of indol-3-ylmethyl glucosinolate, indole-3-carbinol was the most active compound in inducing hepatic and intestinal enzyme activity. Testing the same compounds except ascorbigen, Pantuck et al. (1976) also found indole-3-carbinol (100 mg/kg body weight) the most potent inducer of intestinal 7ethoxycoumarin and benzo(a)pyrene hydroxylation in rats, increasing activity by 16- and 22-fold, respectively. A high induction effect for hepatic cytochrome P450 and benzo(a)pyrene activity was also obtained with indole-3-carbinol (167 mg/kg body weight) in rats and mice, when compared to indole-3-acetonitrile (83 mg / kg body weight) (Shertzer 1982). Indole-3-carbinollevels as low as 50 mg/kg diet were also reported to increase intestinal benzo(a)pyrene hydroxylase activity (Bradfield and Bjeldanes 1984). Indole-3-carbinol (167 mg /kg body weight) was also more effective than indole-3-acetonitrile (130 mg / kg body weight) in increasing ethoxyresorufin deethylation in liver and intestine of rats (McDanell et al. 1988). Loft et al. (1992) found myrosinase-catalyzed hydrolysis products of broccoli, derived mainly from indol-3-ylmethyl and 1-

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methoxyindol-3-ylmethyl glucosinolates, induced cytochrome P450 isoenzymes, with respect to the metabolism of metronidazole and antipyrine in rats, while no activity was found for the intact glucosinolates. Indole-3-carbinol, an hydrolysis product of indole glucosinolates, gives rise to a 7.5-fold increase in P450 1A1 in the liver, and a 14-fold increase in the small intestine, when fed to rats at a very high level (500 mg/kg body weight) for 14 days (Wortelboer et al. 1992b). The observation that indole-3-carbinol could induce activity of enzymes responsible for the 2-hydroxylation of estrogens in mouse and rat liver suggested an anticarcinogenic role for this compound (Baldwin and LeBlanc 1992; Jellinck et al. 1993). Michnovicz and Bradlow (1990, 1991) conducting an in vivo radiometric assay in humans found a 50% increase of the 2-hydroxylation of estradiol after daily exposure to 6-7 mg indole-3-carbinol/kg in feed for 7 days, with an increase of the urinary excretion of 2hydroxyoestrone when compared to that of oestriol. It was suggested that this induction was due to condensation products formed from indole-3-carbinol due to the acid conditions of the stomach, rather than indole-3-carbinol per se (Bradfield and Bjeldanes 1991). In a recent study Wortelboer et al. (1992a) showed that acid reaction condensation compounds ofindole-3-carbinol, such as 5,6,11,12,17,18,hexahydrocyclonona[ 1,2-b:4,5-b':7 ,8-b"]tri-indole and 2,3-bis[indol3 -y lmethy l]indole in addition to the already known 3,3'diindolylmethane, have a positive effect on cytochrome P450 and phase II enzymes in rat and monkey hepatocytes. The inhibitory effect of indole gl ucosinolates and their hydrolysis products, isothiocyanates, and Brassica on chemical carcinogenesis is summarized in Table 3.4. Despite the demonstrated anticarcinogenic effect of indole glucosinolate hydrolysis products, the timing of exposure to carcinogen and inhibitor can alter this effect. Studies by Bailey et al. (1987) showed that indole-3-carbinol (2000 ppm) fed to rainbow trout for a 12-week period after administration of 20 ppb AFB1 enhanced carcinogenesis when compared to diets given before and during exposure to carcinogen. It had already been suggested (Goeger et al. 1986) that indole-3-carbinol inhibition of hepatocarcinogens in the rainbow trout (Nixon et al. 1984) involves substantial changes in carcinogen distribution, metabolism, and elimination. Dashwood et al. (1988) found that, in in vivo studies with rainbow trout, AFB1-DNA binding was suppressed by almost 95% (indole-3-carbinol at 4000 ppm) and that even at low levels (1000 ppm) the inhibitor offered some protection against the chemical-induced neoplasia. A dose-

Table 3.4.

Inhibitory effects of Brassica, glucosinolates, and derived products, on chemical carcinogenesis.

Chemical Carcinogen

Diet

2 ppm AFB1 (rats) 1 ppm AFB1 (rats)

20% DW cauliflower 25% DW cabbage

Reduction of toxic effect (Stoewsand et al. 1978) Fewer tumors (Boyd et al. 1982)

9,12-Dimethylbenzanthracene

Indole-3-carbinol (15 mg/rat)

Inhibition of occurrence of mammary tumor (Wattenberg and Loub 1978) Inhibition of occurrence of mammary tumor (Wattenberg and Loub 1978) Ineffective (Wattenberg and Loub 1978)

Diindolylmethane (15 mg/rat) Indole-3-acetonitrile Benzo(a)pyrene (mice) (1 mg twice wk for 4 wk)

Indole-3 -carbinol Indole-3-acetonitrile Diindolylmethane (0.02-0.03 mmol/g diet)

~ w

Effect

Inhibition of occurrence of forestomach tumor (Wattenberg and Loub 1978) Inhibition of occurrence of forestomach tumor (Wattenberg and Loub 1978) Inhibition of occurrence of forestomach tumor (Wattenberg and Loub 1978)

Benzo(a)pyrene (mice)

Indole-3-carbinol (163 mg/kg body wt)

Anticarcinogenic in liver (Shertzer 1982)

N-Nitrosodimethylamine (mice)

Indole-3-carbinol (163 mg/kg body wt)

Anticarcinogenic in liver (Shertzer 1982)

Benzo(a)pyrene (3 mg)

Glucobrassicin (12 mg 3, 2, and 1 day prior B[a]P administration)

Inhibition of both forestomach and lung tumors (Wattenberg et al. 1986)

Benzo(a)pyrene

Glucobrassicin

Inhibition of both forestomach and

,... (Jl

;+:.

Table 3.4.

(Continued).

Chemical Carcinogen (3 mg)

Diet (4 h before B[a]P administration)

Effect lung tumors (Wattenberg et al. 1986)

Benzo(a)pyrene (rat)

Benzyl isothiocyanate (25 and 50 mg/diet) Phenethyl isothiocyanate (55 mg/diet) Phenyl isothiocyanate (23 mg/diet)

Inhibition of occurrence of mammary tumor (Wattenberg 1977) Inhibition of occurrence of mammary tumor (Wattenberg 1977) Inhibition of occurrence of mammary tumor (Wattenberg 1977)

7,12-Dimethylbenzanthracene (mice)

Benzyl isothiocyanate (5 mg/g diet) Phenethyl isothiocyanate (5.5 mg/g diet)

Inhibition of neoplasia of the forestomach and pulmonary adenomas (Wattenberg 1977) Inhibition of neoplasia of the forestomach and pulmonary adenomas (Wattenberg 1977)

7,12-Dimethylbenzanthracene (rats)

Benzyl isothiocyanate

Inhibition of breast tumor formation (Wattenberg 1977)

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dependent effect of the chemoprotection of indole-3-carbinol in mice was also reported by Shertzer and Sainsbury (1991).

Isothiocyanates. Pungent oils obtained from members of the Brassicaceae have been known for many centuries. Scientific investigations into the nature, origin, and composition of the oils (termed mustard oils) have been described for over 300 years and by the end of the last century it was known that the volatile oils were isothiocyanates that, although not themselves present in the plant, were derived from involatile precursors (McDanell et al. 1988). Specificity or high selectivity in the mode of action of therapeutic agents for tumor cells are desirable targets, and evidence for selective toxicity against transformed cells has been presented for several compounds (Johnson et al. 1995). Isothiocyanates have been postulated as monofunctional inducers (they induce phase II enzymes only) (Talalay and Prochaska 1987). Musk and Johnson (1993a) have shown that allyl isothiocyanate was more toxic toward transformed HT29 human colorectal adenocarcinoma cells than toward cells that have been experimentally detransformed in vitro. On the other hand, allyl isothiocyanate was not clastogenic toward SV40-transformed Indian muntjac cell lines, whereas benzyl, phenethyl, and phenyl isothiocyanates induced chromosome damage (Musk and Johnson 1993c). Mechanisms of action of several protective compounds remain uncertain and results are sometimes contradictory. Despite the blocking activity of phenethyl isothiocyanate (Morse et al. 1989a), this compound has been shown to inhibit P450 2El and thus decreases phase I metabolism (Yang et al. 1992). This is attributed both to chemical inactivation and to a competitive mechanism (Smith et al. 1993). Other isothiocyanates have been shown to decrease hepatic cytochrome P450 activity in rats, by covalent binding to this compound (Ozierenski et al. 1993) The suppressing activity of benzyl isothiocyanate was noted by Wattenberg (1981), who found that when this compound was administered 1 week following carcinogen challenge, it inhibited the appearance of 7, 12-dimethylbenz(a)anthracene-induced neoplasia of the breast in rats. Feeding cabbage or broccoli to rats for an 18 week period, starting 1 week after cessation of 7,12-dimethylbenz(a)anthracene treatment, decreased the number of animals with mammary tumors 40 to 50% for both vegetables compared to control groups, with an average number of tumors per rat of about 60% lower (Wattenberg et al. 1989a,b). Benzyl isothiocyanate (400 ppm) was also found to inhibit methylazoxymethanol acetate-induced (25 mg/

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kg body weight) intestinal carcinogenesis in rats, the effect being stronger than with benzyl thiocyanate (Sugie et al. 1991). Oral administration of benzyl isothiocyanate and benzyl thiocyanate to male "F344" rats resulted in the suppression of proliferative activity in hepatocyte primary cultures derived from them (Sugie et al. 1993). In vitro studies showed the anti-cancer activity of benzyl isothiocyanate against a variety of human and murine tumor cell lines (Pintao et al. 1995). However, different findings were reported by Musk and Johnson (1993b), who showed aromatic isothiocyanates to be selectively toxic toward transformed colorectal tumor cells. Phenethyl and benzyl isothiocyanates were also shown to induce aberrations and seE in mammalian cells (Musk and Johnson 1993b), with benzyl and allyl isothiocyanates giving a positive result in the Ames test (Yamaguchi 1980). Allyl isothiocyanate was also reported to be an inducer of aberrations in mammalian cells (Kasamaki et al. 1982), responsible for transformation of mammalian cells (Kasamaki et al. 1987), and an inducer of bladder tumors when fed to male F344 rats (Dunnick et al. 1982). Ioannou et al. (1984) suggested that this last effect was due to sex-related variations, with female rats excreting over twice the urine volume of male rats, resulting in males excreting a more concentrated solution of allyl isothiocyanate in urine, thus accounting for the toxic effects on the bladder of male rats. These results led to the suggestion by Johnson et al. (1995) that the same compound may exert quite different effects at different stages of carcinogenesis. A better understanding of tumor biology is necessary for a satisfactory classification of suppressing agents. The blocking activity of benzyl, phenethyl, and phenyl isothiocyanates was shown when these compounds, administered 4 h prior to 7,12-dimethylbenz(a)anthracene, effectively inhibited mammary tumor formation in rats (Wattenberg 1977). Similarly benzyl isothiocyanate inhibited benzo(a)pyrene-induced lung and forestomach tumors in "A/J" mice when administered 15 min prior to carcinogen (Wattenberg 1987). Benzyl glucosinolate, the parent compound from which benzyl isothiocyanate is formed by hydrolysis, had a comparable effect (Wattenberg et al. 1986). Inclusion of the recently isolated isothiocyanate, sulforaphane (1-isothiocyanate-(4R)(methylsulfinyl)butane), in diets (75 or 150 pmol per day for 5 days) fed to rats was shown to inhibit 9,10-dimethyl-l,2-benzanthraceneinduced mammary tumors, when administered 3 h before the carcinogen (Zhang et al. 1994). These results suggested that these naturally occurring compounds can inhibit carcinogenesis when consumed shortly before exposure to carcinogens; thus, they have a special role in cancer prevention.

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The assay of human exposure to these anticarcinogens can be facilitated by measurement of their urinary metabolites, as reported for phenethyl isothiocyanate (Eklind et a1. 1991; Chung et a1. 1992b). After volunteers ingested watercress, N-acetyl-S-(N-phenethylthiocarbamoyl)-L-cystein, the conjugate ofphenethyl isothiocyanate, could be detected in urine over several hours. An increase in benzo(a)pyrene metabolism was noted in hamster embryo cells treated either with m-methoxybenzyl isothiocyanate or the parent glucosinolate (m-methoxybenzyl) prior to the addition of the carcinogen (Baird et al. 1988). Other effects of benzyl isothiocyanate (doses of 100 and 150 mg/kg) were reported to be retarded fetal growth and ossification in rats (Bleyl and Lewerenz 1992).

Nitrosamines and Glucosinolates. N-Nitrosamines are compounds associated with human cancer and can be formed in vivo by endogenous nitrosation. Tiedink et a1. (1991) reported indol-3-ylmethyl and 4-hydroxyindol-3-ylmethyl glucosinolates to form N-nitroso compounds, but their contribution to Salmonella typhimurium mutagenicity was marginal. Isothiocyanates were shown to inhibit the metabolic a-hydroxylation of both N-nitrosopyrrolidine and N'nitrosonornicotine using in vitro studies (Chung et a1. 1984). Studies in vivo showed that in rat liver, phenethyl- and phenyl isothiocyanates and allyl glucosinolate exert a protective effect as blocking agents against two environmentally prevalent nitrosamines, N-nitrosodimethylamine and 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK), recognized as potent carcinogens (Chung et al. 1985). A pretreatment with phenethyl isothiocyanate in daily doses as low as 5 llmol for 4 days inhibited lung tumor induction by NNK (Morse et a1. 1989b), while post-treatment with benzyl and phenethyl isothiocyanates at a dose of 3 Jlmol/g of diet had little effect (Morse et a1. 1990). It was suggested that the basis of this inhibition was due to the reduction of DNA adduct formation caused by the inhibition of enzymes responsible for NNK activation. In studies with mice (Morse et a1. 1991) and with "F344" rats and "A/J" mice (Chung et al. 1992a) phenethyl isothiocyanate was identified, among other isothiocyanates, as the most effective inhibitor ofNNK-induced lung tumorigenesis. Inhibitory efficacy was found to increase as the alkyl chain elongates up to 6 carbon atoms, the 6-phenylhexyl isothiocyanate being approximately 50 to 100 times more effective than phenethyl isothiocyanate. Similar findings were reported by Guo et al. (1991, 1993). After administration of phenethyl isothiocyanate to "A/J" mice at 5 Jlmol/mouse, the maximal inhibi-

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tion of NNK-induced lung tumorigenesis occurred between 4 and 8 h after dosing (Eklind et al. 1990). Benzyl isothiocyanate administered 15 min before carcinogen treatment was found to inhibit Nnitrosodiethylamine-induced forestomach tumors in A/J mice (Wattenberg 1987). A reduction of hepatocarcinogenesis was noted in rats fed a diet containing 100 ppm of benzyl isothiocyanate for 1 week and then treated with diethylnitrosamine (200 mg/kg body weight) (Sugie et al. 1992). The inhibitory effect of benzyl isothiocyanate was found to be stronger than that of benzyl thiocyanate. Cabbage-containing diets also provide a protective effect against nitroso compounds. Inclusion of dried cabbage (5 and 10%) and collards (5%) in diets to rats that had been previously injected with Nmethyl-N-nitrosourea was shown to reduce the incidence of mammary cancer after initiation had occurred (Bresnick et al. 1990). Phase II Enzymes-GST, EH, and UDPGT Inducers. The glutathione transferases (GST) (EC 2.5.1.18) are a family of major detoxification enzymes that conjugate numerous reactive hydrophobic electrophilic compounds with glutathione and may also have a sacrificial function in covalently binding chemically reactive ligands (Benson and Barretto 1985). In rat liver cytosol at least 10 different isoenzymes of GST occur (Vos et al. 1988). Induction of GST activity by anticarcinogenic compounds is the major mechanism for carcinogen detoxification. Brassicacious vegetables that induce GST activity may thus inhibit chemical carcinogenesis in the same tissue (Wattenberg 1985). Induction of GST and reduced binding of aflatoxin Bl to DNA have been noted for both cabbage (Whitty and Bjeldanes 1987) and broccoli (Ramsdell and Eaton 1988). Brussels sprouts in the diet of rodents induced levels of liver and intestinal GST activity toward 1chloro-2,4-dinitrobenzene (Bogaards et al.1990) and increased rat liver glutathione levels (Nijhoff et al. 1993). In a recent study with humans, Bogaards et al. (1994) reported that consumption of 300 g of cooked Brussels sprouts by non-smoking volunteers increased glutathione S-transferase alpha in blood plasma, suggesting that this effect reflected the GST-a induction in tissues such as liver and small intestine. Powdered Brussels sprouts and cabbage were shown to have a similar GST effect in the liver and small intestine of mice (Sparnins et al. 1982a). However, Bradfield et al. (1985) found diets with 20% of Brussels sprouts did not induce GST-a in rat liver. Dupont et al. (1994) have reported brassicacious plants to induce

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GST in cultured human hepatoma cells. Increased activity of intestinal GST in rats was reported by Kore et al. (1993a) for diets containing 100 ~mol kg body weight of l-isothiocyanate-3(methylsulfinyl)propane. Benzyl isothiocyanate seems to be a good inducer of GST activity in several organs (Sparnins and Wattenberg 1981; Sparnins et al. 1982a,b; Benson and Barretto 1985). A three-fold increase in enzyme activity was shown in mice liver at benzyl isothiocyanate doses of 0.5% in the diet for 14 days and was more effective also in the small intestine and forestomach than in lung, kidney, urinary bladder, and colon (Benson and Barretto 1985). The inclusion ofindole-3-carbinol in mouse diets at about 4000 mg/kg diet (Cha et al. 1985) and 6000 mg/kg diet (Sparnins et al. 1982a) was reported to induce hepatic epoxide hydrolase (EH) (EC 3.3.2.3) and hepatic and intestinal GST, respectively. Both of these enzymes were also reported to be induced by oxazolidine-2-thione (Chang and Bjeldanes 1985), and the induction of GST by oxazolidine-2-thione was recently confirmed by Ozierenski et al. (1993). Sulforaphane was shown to induce GST activity in cytosols from a number of sites in mice (Zhang et al. 1992). The enzyme UDP-glucuronosyltransferase (UDPGT) (EC 2.4.1.17) conjugates xenobiotics with glucuronic acid, thus facilitating their excretion (Prestera et al. 1993). The UDPGT induction by drugs and other xenobiotics was reported by Sutherland et al. (1993), and Miners and Mackenzie (1991) inferred that diets high in cabbage and Brussels sprouts slightly increased this enzyme.

Quinone Reductase (QR) Inducers. Quinone reductase (QR) (EC 1.6.99.2), often considered as a phase II enzyme, promotes obligatory two-electron reduction of quinones, preventing their participation in oxidative cycling and depletion of intracellular glutathione transferases (GST) (Prestera et al. 1993). Brassica plants, particularly broccoli were found to be effective inducers of QR (Prochaska et al. 1992), and subsequently sulforaphane was identified as the main inducer of QR in cytosols from a number of sites in mice (Zhang et al. 1992). Sulforaphane is a monofunctional inducer, selectively activating phase II enzymes without induction of phase I enzymes. In this study, 4-methylthiobutyl glucosinolate (glucoerucin) was also identified as an inducer of QR. 3-Methylsulfinylpropyl glucosinolate is an abundant compound in Brassica oleracea and its derivative, 1isothiocyanate-3-(methylsulfinyl)propane, has been shown to increase intestinal QR activity in rats fed diets containing 100 Jlmol/ kg body weight (Kore et al. 1993a), but not in doses less than this.

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Ongoing studies confirm that many brassicacious plants induce QR in mouse cells (Tawfiq et al. 1994).

Other Glucosinolate and Derivative Products Activities. The effect of glucosinolates on animals that feed on brassicacious species has been known since 1928 when rabbits fed high levels of cabbage were found to develop goiter (Chesney et al. 1928). Forage Brassica, commonly fed to ruminants in temperate agricultural systems, contain glucosinolates that have been implicated as a cause of low feed intake by sheep, this effect presumably being due to either the taste or the smell (Armstrong et al. 1993), although no evidence was presented that these were factors. Early studies attributed the toxicity of glucosinolate-derived nitriles to their effects on the liver and kidney and have been recently confirmed by the observation of cellular damage in liver and kidney tissues of nonruminants after nitrile administration (Nishie and Daxenbichler 1980; Gould et al. 1985). Nitrile was also shown to suppress cellular respiration following release of free cyanide in the tissues and consequent inhibition of cytochrome oxidase activity (Ohkawa et al. 1972; Tanii and Hashimoto 1984; Willhite and Smith 1981). Intragastric doses above 0.3 mmol/kg body weight of 1-isothiocyanate-3-(methylsulfinyl)propane induced multifocal hemorrhages and erosions of the mucosal portion of the stomach (Kore and Wallig 1993). A decrease in food intake and growth depression together with an increase in kidney weights and impaired kidney function was observed in rats fed diets containing either cooked and uncooked Brussels sprouts (major glucosinolates: 2-propenyl, indol-3-ylmethyl, and 2-hydroxybut-3-enyl) at inclusion levels of 10% or more expressed on dry matter (Groot et al. 1991). In the same experiment an increase in prothrombin was noted if Brussels sprouts inclusion in the diet was 2.5% or more and 5% inclusion of the cooked vegetable caused an increase in liver weights. The antibiotic properties of benzyl isothiocyanate have been recognized for nearly 50 years. The compound has been marketed as a drug for the treatment of infections of the respiratory and urinary tracts. 2. Rapeseed. Rapeseed (Brassica napus and B. campestris) is a major oilseed crop grown widely in Europe and America, particularly in Canada, ranking fourth among the world's oilseeds. The name canola has been registered for rapeseed containing less than 2 % of the total fatty acids in the oil as erucic acid and less than 30 Jlmol of

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alkenyl glucosinolates per gram of oil-free dry matter of the seed (Bell 1993). Following extraction of the oil, which constitutes between 40 and 45% of the seed, the residual seed meal contains 3540% of high-quality protein together with less favorable compounds such as glucosinolates, phenolics, tannins, phytates, and lignin, and is used as a source of feedstuff for most species and classes of farm animals. During seed processing, glucosinolate breakdown products are formed that may impair feed intake, growth, and thyroid activity of the animal. Comprehensive reviews on the use of rapeseed meal containing glucosinolates as feedstuff and their antinutritional effects have been published (Hill 1991; Bell 1993; Mawson et al. 1993b; 1994a,b,c) and threshold levels for glucosinolates in diets for various animals have been suggested. There is some controversy about the quantity of glucosinolate that is tolerated by various animal species. Variables such as cultivar (which determines glucosinolate content), processing conditions, the age of the animals, the dietary supply of nutrients, and energy are often not sufficiently described to permit a comparison between the results of different studies. The degree of glucosinolate degradation depends on seed properties and seed processing conditions such as moisture level, pressure, or temperature (Bible et al. 1983; Mansour et al. 1993). For instance, autoclaving meal for 1.5 h reduced glucosinolate content from 35.4 !J.mol/g in the original raw meal to 2.3 !J.mol/g (Mansour et al. 1993). Treatment of rapeseed meal with Cu 2 + can reduce glucosinolates by more than 90% (Schone et al. 1990a). Ammonia used in conjugation with other processing has effectively reduced glucosinolate levels in canola seed (Keith and Bell 1983), canola meal (Keith and Bell 1982), frost-damaged canola seed (Bell and Keith 1986), and mustard meal (Bell et al. 1984). Ammonia has also been used to promote sinapine breakdown (Goh et al. 1982). A significant reduction in the glucosinolate content of extruded canola screenings was reported after treatment with a combination of mist heat and ammonia (Darroch et al. 1990). There have been many studies in which the use of high glucosinolate (HG-RSM) or low-glucosinolate rapeseed meal (LGRSM) in feeding trials with a variety of animals has allowed a direct comparison to be made of the effect of total glucosinolate content on various physiological parameters. The effects of feeding highglucosinolate (meal) to animals included reduced palatability and food intake, enlarged thyroids, reduced levels of circulating thyroid hormones, abnormalities in the liver, kidney, and suprarenal gland, and detrimental effects on growth and reproductive performance. A

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fishy taint in brown-shelled eggs from certain breeds of laying hen was traced to the inclusion of rapeseed meal in the diet. The problem was shown to be due to the ability of goitrin (from 2-hydroxybut3-enyl glucosinolate present in the meal) to inhibit the oxidation of trimethylamine, a compound derived from sinapine, also present in rapeseed meal, thus leading to its accumulation in the egg (Fenwick et al. 1984). Bjerg et al. (1987b) found that the glucosinolates present in rapeseed caused antinutritional or toxic effects when ingested at levels similar to those encountered when feeding diets containing "double-low" meal and recommended levels of glucosinolates in the diets to be below 2.5 Jlmollg.

Palatability. There is some evidence that palatability of feed can be adversely affected by inclusion of rapeseed meal, due to the presence of glucosinolates, the effect being dependent on the species of animal, age, and growth state. When fed to younger animals, particularly chicks, piglets, and calves, diets containing high levels of glucosinolates from high-glucosinolate rapeseed meal, appear to reduce palatability and food intake and consequently depress performance. Low-glucosinolate rapeseed meal containing 10-30 Jlmollg and very low glucosinolate rapeseed meal (VLG-RSM) containing 15 Jlmollg glucosinolates were reported to have less effect on palatability according to Mawson et al. (1993b), who suggested that LGRSM and VLG-RSM can be included in the diet at levels up to 20 and 30% for calves and dairy cows, respectively. Despite very low glucosinolate rapeseed meal (3.1 Jlmollg) fed to calves (300 g/kg) in a short period, can be less accepted when compared to soybean (Hill et al. 1990a), several studies reviewed by Hill (1991) suggest that LG-RSM, in a continuous feeding regime, can replace soybean meal in compound concentrates given ad libitum as "starter" feeds to calves. For grower and finisher pigs the recommended levels of LGRSM and VLG-RSM are 10 and 15%, respectively, while for piglets, rapeseed meal should be excluded from the starter diets (Mawson et al. 1993b). However, Schone et al. (1990b) reported growing pigs to discriminate between LG-RSM and HG-RSM at 2% inclusion level. Poultry appears to be less sensitive to palatability problems when fed rapeseed meal, but the low-energy and higher fiber contents of rapeseed requires the use of low levels in the diet. Food Intake and Growth Performance. Rapeseed meals have been shown to affect pig performance in the growing phase, an effect attributed to the presence of glucosinolates (McGregor 1978). Levels

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of 7.7 f..lmol/g reduced average daily gain (Spiegel et al. 1993), but, Latymer et al. (1991a) reported no significant effect on the performance of pigs when rapeseed meal was included in the diet to give a glucosinolate content between 9.3 and 10.5 f..lmol/g diet; canola meal with up to 15.62 f..lmol/g of glucosinolates seemed to reduce daily feed intakes, particularly when compared to soybean meal (Bell et al. 1991). Piglets appear to be more sensitive and diets containing rapeseed meal with total glucosinolate content as low as 4.2 f..lmol/g were reported to reduce food intake (Latymer et al. 1991b). In mice, decreased feed intakes and reduced growth rates were reported by Darroch et al. (1991) for diets containing indole glucosinolates (up to 8.2 f..lmol/g DM), primarily indol-3-ylmethyl glucosinolate, with no apparent effect on thyroid, hepatic or renal weights, or morphology. Intermediate levels of 4-6 f..lmol/g DM decreased palatability of the diet, attributed to the flavor effect of indole glucosinolates, while levels of 8 f..lmollg DM, in addition to producing off flavors in the diet, were reported to act as irritants in the gastrointestinal tract and so cause diarrhea. The reduction of feed intake was attributed to nitriles, the bitter garlic-like flavor compounds resulting from hydrolysis of indole glucosinolates in the acidic environmental conditions in the stomach. Indol-3-ylmethyl glucosinolate at levels below 3 f..lmol/g DM did not affect growth performance. Purified indol-3-ylmethyl glucosinolate fed to rats at a dietary concentration of 0.5 mg/g (ca. 1 f..lmollg) was shown to have no effect on feed intake (Vermorel et al. 1986). Allyl cyanide and allyl isothiocyanate (15 and 10 mmol/day, respectively) were tested in sheep and no anemic responses were found, the depressive effects on voluntary intake being associated with depressed reduced glutathione levels in the blood (Duncan and Milne 1990, 1993). Duncan and Milne (l992a) showed that allyl cyanide fed to lambs at levels up to 9.6 mmollday, depressed voluntary food intake, with only minor damage to liver and no effects on renal function, concluding that sheep showed considerable tolerance to allyl cyanide when compared to monogastrics, probably due to rumen microbial degradation of this compound (Duncan and Milne 1992b). Hill et al. (1990b) found that feed concentrates containing glucosinolates up to 17.5 f..lmol/g did not affect either food intake or weight gain in lambs but did influence the weight of the thyroid gland. With the production and use of LG-RSM as feedstuff in several animal species, food intake and growth seem to be less affected. For instance, the negative effects in rats start only when glucosinolate

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levels reach 4.4 /-lmol/g in the diet (Vermorel et al. 1988), but when alkenyl glucosinolates were over 20.3 /-lmol/g in rat diets a significantly decrease in weight gain could be observed (Rakowska et al. 1987). The 2-hydroxybut-3-enyl glucosinolate, in the presence of myrosinase, seemed to have a marked effect even at low levels (7.7 /-lmol/g diet) (Vermorel et al. 1988). In pigs, Mawson et al. (1994a) suggested that no deleterious effects are observed if total glucosinolate and oxazolidinethione levels are below 1.0 and 0.5 /-lmol/g diet, respectively. Clandinin and Robblee (1981) recommended the percentage of LG-RSM in the diet to be 8 and 12% for starter and growing pigs, respectively, and the use of only cereal protein in diets for finishing pigs. The deleterious effects of glucosinolates and RSM in poultry seem to be very dependent on the species of bird, with laying hens and turkeys being more sensitive than broiler birds (Mawson et al. 1994a). Mawson et al. reviewed several studies concerning detrimental effects of high glucosinolate intake on growth performance in chickens (broilers and laying hens), ducks, geese, quail, and turkeys. They suggested that glucosinolate levels in diets of young chickens (broilers) should definitely be below 10 /-lmol/g, because a trend in reduced growth was initiated between 2 and 4 /-lmol/g diet. A threshold of 100/0 for LG-RSM and 5% for HG-RSM in diets for laying hens avoids an increase in mortality rate and lowered egg production and egg weight, particularly during early lay. However, further studies are needed to show whether these effects are due to the low-energy value of rapeseed or to extreme sensitivity of laying hens to minor disruptions of liver/thyroid function caused by low levels of dietary glucosinolates. Ruminants appear to be less sensitive to high glucosinolate intake than monogastrics, and diets containing up to 10% of HG-RSM and 25% of LG-RSM are recommended (Mawson et al. 1994a). In fish, diets containing 20% of canola meal were shown to have no effect on rainbow trout growth (Hardy and Sullivan 1983), and it was suggested that dietary protein for chinook salmon could be provided by inclusion ofLG-RSM at levels up to 25% (Higgs et al. 1982).

Thyroid. After an enzymatic hydrolysis of glucosinolates present in several varieties of rapeseed meals, (-)5-vinyl-oxazolidine-2-thione (OZT) was found to be the predominant product from the heat-treated meal regardless of the source ofmyrosinase enzyme (Paik et al. 1980), which may explain the ability of rapeseed meal to induce thyroid hypertrophy (McKinnon and Bowland 1979). The extent to which

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thyroid function is impaired by glucosinolates is clearly related to species, intake, duration of feeding, and the nature of the compound. Glucosinolate enzymic breakdown products known to be goitrogenic to varying degrees, include OZT, isothiocyanates, and thiocyanate ion (derived from indole glucosinolates). The goitrogenicity of the latter is contingent on a low iodine status and the effect is ameliorated by dietary iodine supplementation. The goitrogenic effects of OZT and nitriles are irreversible and may be characterized by decreased iodine trapping by the thyroid gland, increased thyroid weight, and reduced levels of circulating thyroid hormones. Various studies with rats have demonstrated increased thyroid weight and inhibition of thyroxine synthesis (Rakowska et al. 19S7; Vermorel et al. 19S5). In the latter study, a comparison of two diets, one with rapeseed and myrosinase inclusion and the other with a similar amount of isolated 2-hydroxybut-3-enyl glucosinolate together with myrosinase, showed that the rapeseed diet resulted in much higher thyroid weight, pointing to the involvement of other factors as suggested by Fenwick et al. (19S3a). In pigs there is clear evidence that thyroid weight is strongly correlated with rapeseed intake (particularly HG-RSM) and 2-hydroxybut-3-enyl glucosinolate at levels as low as 2-3Jlmol/g diet showed a positive effect (Eggum et al. 19S5b). In poultry, a rapeseed diet containing 1.5 Jlmol/g of total glucosinolates was reported to induce thyroid hypertrophy (Mawson et al. 1994b). In general, rapeseed glucosinolates do not affect thyroid function in ruminants (Tyagi and Singhal 1993), due possibly to microbial breakdown of potential goitrogens in the gut. Christison and Laarveld (19S1) tested the antithyroid effect of including 15% canola and rapeseed meals in diets for pigs (starting at 13 kg weight) for about 50 days and found normal circulating levels of thyroxine but impaired thyroid reserve capacity. Rapeseed meal with low erucic acid and low glucosinolates, such as that derived from the cultivar Tower, appeared to have potential for use in the diets of early-weaned pigs (Ochetim et al. 19S0) but hyperplasia and hypertrophy of follicular epithelium were found in thyroids of pigs fed 10 and 20% autoclaved 'Tower' rapeseed meal. An increase in thiocyanate concentration in the urine was associated with increasing levels of rapeseed in the diets. Thyroid hormones play an important role in fetal development. In the chick embryo, normal development of several organs such as the liver, small intestine, and skeleton are dependent on thyroid hormones, which also playa role in the hatching process (Darroch and Bell 1991). No goitrogenicity was reported on developing chick em-

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bryos after injection with O.l-mL aliquots of indole glucosinolate extracts from Brussels sprouts into egg yolk and blood. In experiments with Large White gilts, dietary inclusion of up to 20% rapeseed meal containing 37.2 Jlmol of glucosinolates per gram induced larger thyroid, liver and kidney weights, with fetal weights at 111 days of pregnancy showing a linear decrease as rapeseed meal levels increased (Etienne et a1. 1990). Fetuses were sensitive to as little as 70/0 of rapeseed meal in the diet.

Effects on Other Organs. Liver enlargement has been reported in rats fed rapeseed meal, Brassica vegetables (Brussels sprouts), glucosinolates, or glucosinolate hydrolysis products, although the problem is less severe when LG-RSM is fed or after prior removal of glucosinolates. In certain breeds of chicken there is a tendency to liver hemorrhage as a consequence of feeding RSM. This problem, which may be genetically determined, is less severe when LG-RSM is fed, and rather than glucosinolates per se, it is probable that glucosinolate breakdown products such as epithionitriles are involved together with other factors. In cattle, rapeseed diets caused minor increases in liver size but these changes did not appear to be correlated with glucosinolate intake. Rapeseed silage containing high glucosinolates (21.7 mmol/g DM) produced no detrimental effects on digestive function or growth of beef steers (Lancaster et al. 1990). Animal Reproduction. Glucosinolates may affect animal reproduction but species and age of the animal and the dietary levels can induce differences in the results. According to Rakowska et a1. (1987) diets with a glucosinolate content higher than 0.5 Jlmol/g resulted in reduction of female fertility in rats. Etienne et a1. (1991) reported no negative effects on sow fertility at levels up to 17 Jlmol/g of diet, but a decrease was noted in the number of foetuses and in the size of uterus. After reviewing this subject, Mawson et a1. (1994c) suggested that no detrimental effects are likely if the glucosinolate intake in pigs is under 4.0 l-lmol/g of diet, while in poultry the effects of glucosinolates on reproduction have been variable, although diets with less than 30% of RSM are not detrimental. In ruminants, inclusion of HG-RSM in the diet up to 30% had no effect on behavioral or hormonal aspects (Vincent et al. 1988). B. Effects of Glucosinolates on Insects and Mites

Despite the variability of data for different groups of adapted insects, results of field and laboratory studies indicate that most spe-

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cies of crucifer feeders respond to glucosinolates or to their hydrolysis products. Glucosinolates and the products of their degradation are considered to function as part of the plant's defence against insect attack (reviews by Fenwick et al. 1983a and Chew 1988a), to act as phagostimulants (Chew 1988a; Bartlett and Williams 1989; Traynier and Truscott 1991; Renwick et al. 1992), and to have a major role in host plant location and colonization by many phytophagous insects specifically adapted to brassicacious plants and also in the location of these insects by their parasites. The biological activity of glucosinolates and isothiocyanates seems to be determined by the nature of the side chain R (Table 3.1), by the compound concentration and by the type of pest. However, the idea that glucosinolates are mainly responsible for host recognition and specifity by all crucifer-feeding insects has been challenged (Nielsen 1978, Chew 1988b). Melchior Larsen et al. (1985) reported that a plant lacking glucosinolates would not be recognized as a host plant by brassicacious specialist insects, and some insects, like the potentially devastating Phyllotreta spp., are able even to distinguish between cultivars on the basis of their glucosinolate content. Grasshoppers (Melanoplus sanguinipes) can also des criminate cultivars based on isothiocyanate levels-the lower the isothiocyanate content, the greater the feeding preference (Pawlowski et al. 1968). The relation between isothiocyanates and insects may be concentration dependent. Finch (1978) suggested that during evolution, isothiocyanates were used by plants for defensive purposes and that with time some insects have adapted to these hydrolysis products, presently using them to locate host plants. In addition to being toxic to many phytophagous insects, the hydrolysis products of the secondary plant substances are often toxic to the plant themselves. In general, plants do not contain high concentrations of these chemicals free within their tissues. Normally, they are found combined as glycosides, a form in which they are less toxic, and are spatially separated from their specific hydrolysing enzymes. Support for this theory was given by Dawson et al. (1993a). Several studies have shown stimulation of the flea beetle Phyllotreta cruciferae (Goeze) and the striped flea beetle P. striolata (F.) in the presence of glucosinolates and their hydrolysis products, the degree of stimulation being dependent of the type of glucosinolate (Hicks 1974; Nielsen 1978; Nielsen et al. 1979). Of several compounds examined, allyl glucosinolate had a strong stimulating effect on Phyllotreta armoraciae (Koch), but this glucosinolate was not solely responsible for this effect (Nielsen et al. 1979); other glucosinolates, such as 2-phenethyl, benzyl, p-hydroxybenzyL and 2-butyl, were also

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recognized as stimulants. Thus, the feeding habit of this insect within brassicacious species cannot be attributed to any single glucosinolate or any particular combination of glucosinolates. Antixenosis (nonpreference) and tolerance have been identified as two mechanisms in seedlings of 5. alba that probably account for flea beetle resistance in this species (Bodnaryk and Lamb 1991). The effect of glucosinolates on insect feeding depends on the developmental stage of the host plant, their concentration, and the insect species being considered. High concentrations of phydroxybenzyl glucosinolate in young cotyledons (up to 20 mM) and leaves (up to 10 mM) of mustard seedlings (5. alba) have been identified as responsible for deterring feeding by the flea beetle P. cruciferae, while lower concentrations (2-3 mM) offered little protection or may even act as stimulant of feeding (Bodnaryk 1991). On the other hand, in cotyledons of mustard (E. juncea 1.) and rape (E. napus 1.), Bodnaryk and Palaniswamy (1990) showed that allyl and indol-3-ylmethyl glucosinolates do not determine the feeding rate of the flea beetle P. cruciferae. On the contrary, Pivnick et a1. (1992), testing several isothiocyanates at the same concentration, showed allyl isothiocyanate to be more attractive to P. cruciferae and P. striolata than benzyl, ethyl, phenyl, and 2-phenethyl isothiocyanates, nitriles being the least attractive compounds. Melchior Larsen et a1. (1985) reported indol-3-ylmethyl glucosinolate to be one of the most stimulatory glucosinolates for flea beetles (Phyllotreta spp.). However, damage by insects can also alter glucosinolate concentration, as evidenced by increased levels of indol-3-ylmethyl and 4hydroxyindol-3-ylmethyl glucosinolates in cotyledons of young oilseed rape seedlings due to feeding by the flea beetle P. cruciferae or after mechanical wounding (Bodnaryk 1992). The importance of glucosinolates in the feeding and oviposition of cabbage small white (Pieris rapae L.) and large white (Pieris brassicae 1.) butterflies has been known since the beginning of the century. However, Traynier (1986) and Renwick and Radke (1988) suggested that oviposition of P. rapae was not stimulated by the presence of allyl glucosinolate, but was due to a learning effect associated with visual cues and contact chemoreception. Renwick and Radke (1983) found allyl glucosinolate to confuse the attraction of P. rapae, which thus did not show a typical response to cabbage extracts. Testing plant species, Huang and Renwick (1993) showed that plant chemistry is a key factor in differential selection of potential hosts by P. rapae and Pieris napi oleracea Harris and found allyl and indol-3-ylmethyl glucosinolates to be the stimulants in oviposition.

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No differences were noted in the stimulatory effect in P. rapae between phenethyl and 2-hydroxy-2-phenylethyl glucosinolates when compared to indol-3-ylmethyl glucosinolate, and the insect was less sensitive to aliphatic glucosinolates, while P. napi oleracea was strongly stimulated by the aliphatic group (Huang and Renwick 1994). In other studies 3-methylsulfinylpropyl and 3-methylsulfonylpropyl glucosinolates were identified as the most active stimulants to P. napi oleracea (Huang et a1. 1993a), while 3-methylsulfinylpropyl was recognized to be as strong as 2-propenyl glucosinolate in stimulating oviposition by this species (Huang et a1. 1993b). In this latter study, 2-0-~-D-glucosyl cucurbitacin I and 2-0-~-D-glucosyl cucurbitacin E were identified as the most active deterrents of P. rapae, while P. napi oleracea was insensitive to these compounds. Despite these differences it was concluded that the behavioral responses of P. rapae and P. napi oleracea depend to a large extent on the concentration of the stimulant (Huang et a1. 1994). Among glucosinolates, the indoles have been demonstrated to be the most powerful oviposition kairomones for P. brassicae and P. rapae (Traynier and Truscott 1991; Renwick et a1. 1992; Loon et a1. 1992). In these studies, using leaf surface glucosinolates, 2-propenyl was shown to be only slightly active, while 3-methylsulfinylpropylglucosinolate was completely inactive as stimulants of oviposition. The strong effect of indole glucosinolates is attributed to their precursor, the amino acid tryptophan, which is nutritionally essential to insects (Koritsas et a1. 1991; Loon et a1. 1992). Moreover, an amino acid receptor cell sensitive to tryptophan was identified in P. brassicae and P. rapae. Another advantage of indol-3-ylmethyl glucosinolate in host recognition could be that indole glucosinolates do not yield volatile aglycones following enzymatic hydrolysis by myrosinase and thus will not attract natural enemies such as parasitoids or generalist predators that use such signals to locate their host or prey (Loon et a1. 1992). In the same way, the identification of the bitter allyl glucosinolate and its related isothiocyanate in the pupae, larvae, and adults of P. brassicae and P. rapae might playa role in the butterfly defense mechanism against predators, by virtue of distasteful qualities. The cabbage root fly, Delia radicum (L.)/Delia brassicae (Wiedemann), attacks a wide range of brassicacious plants, suggesting that several volatile chemicals are probably involved in attracting the flies and in stimulating them to lay (Finch 1978). Oviposition stimulation was found in species yielding different types of isothiocyanate, while in plants lacking volatile glucosinolate break-

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down products no stimulation of oviposition was found. Finch suggested that during cell transformations involved in plant growth, volatile chemicals can be formed and this process is more marked in fast-growing species, which will thus be the most attractive. Similar findings were reported for early maturing cauliflowers, Brussels sprouts, and swedes. However, it was observed that the concentrations of glucosinolates changed during the life of the plant and that at high concentrations, the isothiocyanates repelled larvae (Finch 1978). Later it was observed that the cabbage root fly can locate sources of allyl isothiocyanate solely by olfactory cues, providing the basis for very effective yellow water traps using mixtures of volatile isothiocyanates (Finch and Skinner 1982). Recently, Roessingh et al. (1992) found that the D sensilla on segments 3 and 4 of the tarsus of D. radicum females contain a receptor cell sensitive to glucosinolates, especially to indol-3-ylmethyl, phenethyl, and pent4-enyl glucosinolates. The turnip root fly Delia floralis (Fall) was also shown to differentiate between glucosinolates, the type D tarsal sensilla being identified as the most responsive to pent-4-enyl, but3-enyl, and indol-3-ylmethyl (Simmonds et al. 1994). However, differentiation by both Delia species has been attributed not only to glucosinolate composition of the leaf surface on which the flies walk, but also to an unidentified nonglucosinolate compound (Birch et al. 1993). The feeding of turnip root fly larvae, Delia floralis on the roots of a range of Brassica species, including forage and oilseed rape, was shown to significantly increase the proportion of indole glucosinolates (particularly 1-methoxyindol-3-ylmethyl glucosinolate) in the host roots (Birch et al. 1990, 1992). Similar findings were reported by Griffiths et al. (1994) in roots of B. napus when damage by the turnip root fly larvae occurred, but not if damage was artificially induced. Further studies are needed to explain this difference. Reed et al. (1989) reported indol-3-ylmethyl glucosinolate to be as stimulatory as aromatic and aliphatic glucosinolates in oviposition of Plutella xylostella. Several other studies have shown these compounds to be at least partially responsible for host-plant selection by Plutella maculipennis (Curtis) (Thorsteinson 1953) and the cabbage aphid Brevicoryne brassicae (1.) (Wensler 1962). Recently, Cole (1994) failed to correlate glucosinolates with resistance to the cabbage aphid. Comparative studies have indicated but-3-enyl, pent-4-enyl, benzyl, and p-hydroxybenzyl glucosinolates to be more stimulatory than 2-propenyl glucosinolate to feeding by the cabbage seed weevil

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Ceutorhynchus assimilis Payk., while indol-3-ylmethyl glucosinolate has no effect (Melchior Larsen et al. 1985). These results are in agreement with the finding that antennae of Ceutorhynchus assimilis and Psylliodes chrysocephala L. possess a high proportion of olfactory receptors which respond strongly to but-3-enyl and pent-4-enyl isothiocyanates but not to allyl isothiocyanate (Blight et al. 1989; Garraway et al. 1992). In other studies (Larsen et al. 1992), the lesscommon glucosinolates, 6-methylsulfinylhexyl (glucohesperalin), 3,4-dihydroxybenzyl (glucomatronalin), and 3-0-apiosylglucomatronalin, were identified as the most powerful feeding stimulants for Ceutorhynchus inaffectatus Gyllenhal, when compared to allyl, 3-methylsulfinylpropyl, benzyl, and p-hydroxybenzyl glucosinolates. The number of live cabbage stem flea beetles Psylliodes chrysocephala L. in oilseed rape was reduced by application of 2phenethyl isothiocyanate precursors, while application ofbut-3-enyl isothiocyanate precursors reduced feeding by both flea beetles and damage by the adult seed weevils (Griffiths et al. 1989). The pent-4enyl isothiocyanate was shown to be repellent (the effect being concentration dependent), while no significant response was elicited by 2-phenethyl isothiocyanate (Garraway et al. 1992). Traynier (1965) reported oviposition preferences of Hylemia brassicae (Bouche) to be correlated with the presence of glucosinolates in hosts. He suggested that chemostimuli provided by plants might be more potent than the physical characteristics of hosts in eliciting oviposition and found allyl glucosinolate to be less effective than juice squeezed from the root of swede (B. napus Napobrassica group), a favored host of H. brassicae, pointing to the involvement of other compounds. Nair and McEwen (1976) studied the ovipositional behavior of this insect and confirmed that allyl, phydroxybenzyl, and benzyl glucosinolates induce oviposition equally, while 3-methylsulfinylpropyl and 3-methylsulfonylpropyl were significantly less effective. When host plant material was not available, oviposition by these insects was severely restricted. Allyl isothiocyanate was shown to stimulate flies into greater activity and attracted them to its source. Because oviposition may be dependent on the presence of glucosinolates, development of plant varieties containing a very low concentration of the most active glucosinolates might confer some degree of resistance, but the "glucosinolate pattern" of a given species is more important than the total glucosinolate content in determining the acceptability of a plant. Another recent pest of oilseed rape is Lygus spp. (Hemiptera: Heteroptera, Miridae), but L. borealis (Kelton), L. elisus Van Duzee, and L. lineolaris (Palisot de Beauvois) showed no preferences when

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feeding on high- or low-glucosinolate cultivars (Butts and Lamb 1990). In "double-low" rapeseed cultivars, severe attacks by Meligethes spp. have been reported (Milford et a1. 1989b). Selective grazing on rapeseed plants by the field slug Deroceras reticulatum (Muller) has been demonstrated (Glen et a1. 1989), with high levels of glucosinolates having a negative effect on attack (Moens et a1. 1992). High concentrations of glucosinolates can protect plants against insects. For example, 2-propenyl glucosinolate deters the mustard aphid Lipaphis erysimi (Rohilla et a1. 1993). The desert locust (Schistocerca gregaria Forsk.) is repelled by high levels of glucosinolates (214 Jlmol/g dry matter), while low concentrations (21 Jlmol/g dry matter) acted as phagostimulants (Ghaout et a1. 1991). Thus, new cultivars with reduced glucosinolate levels may be more susceptible to particular insects. Relatively few reports of the insecticidal activity of glucosinolates and hydrolysis products have been produced, but methyl isothiocyanate has been offered commercially as an insecticide and fumigant. Lichtenstein et a1. (1962) reported that 2-phenethyl isothiocyanate in the edible parts of turnips was insecticidal toward vinegar flies (Drosophila melanogastor Meig), and house flies (Musca domestica L.) and confused flour beetles (Tribolium confusum Duvae), spider mites (Tetranychus atlantic), and pea aphids (Macrosiphum pisi). Crambe (Crambe abyssinica) seed meal in diets of house flies resulted in high mortalities and was repellant and/or deterred feeding of the beetles Tribolium castaneum and Oryzeaphilus surinamensis (Tsao et a1. 1993). Soil amended with allyl isothiocyanate showed both lethal and sublethal effects on Limonius californicus Mann. wireworms, depending on concentration (Williams eta1. 1993). Isothiocyanates can also provide some protection against the Bertha armyworm Mamestra configurata Walker (McCloskey and Isman 1993). A similar effect was reported for sinalbin (Bodnaryk 1991). Phenethyl isothiocyanate, the myrosinase hydrolysis product of watercress, was shown to be a feeding deterrent to amphipods (Gammarus pseudolimnaeus) , caddisflies (Hesperophylax designatus and Limnephilus spp.), and snails (Physella spp.) (Newman et a1. 1992). The occurrence of insecticidal substances in Brussels sprouts, red cabbage, kohlrabi, kale, broccoli, white cabbage, radish and cauliflower has been evidenced by mortality in vinegar flies and houseflies, root extracts being most effective (Lichtenstein et a1. 1964). Allyl isothiocyanate was shown to be acutely toxic to Papilio polyxenes L. larvae, which normally attack only plants of the

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Umbeliferae. Larval growth of Spodoptera eridania, a generalist feeder, was inhibited by high but not low concentrations of the compound, while larval growth of Pieris rapae was not affected (Blau et al. 1978). Insecticidal activity of phenethyl isothiocyanate and phenyl isothiocyanate to larvae of the soldier fly, Inopus rubiceps (Macq.), at high dosage levels was reported by Lowe et al. (1971). The insecticidal activity of glucosinolates is a result of changes in the metabolism of the insect, specifically the inhibition of the the glycolysis-Krebs cycle, by decreasing the total 02 uptake and CO 2 expired, as demonstrated using yellow mealworm larvae (Tenebrio molitor 1.) by Pracros et al. (1992a,b). C. Effects of Glucosinolates and Breakdown Products on Microorganisms and Viruses In the search for natural antimicrobial substances, the effects of glucosinolates and their hydrolytic reaction compounds, isothiocyanates and thiocyanates, on bacteria, yeasts, and fungi have been reported in several studies. Isothiocyanates seem to have stronger antifungal and antibacterial properties than the counterpart glucosinolates or the thiocyanates. Bacteriological tests have indicated a very pronounced inhibitory effect of 3-methylsulfonylpropyl, 4-methylsulfonylbutyl, and 4-methylsulfinylbutyl isothiocyanates on a large selection of pathogenic bacteria and fungi (Gilliver 1946; Kjaer and Conti 1954). A strong antimicrobial activity against bacteria, yeasts, and fungi was also shown for 4-methylthio-3-butenyl isothiocyanate (Uda et al. 1993). McKay et al. (1959) reported benzyl isothiocyanate to be a much more effective bacteriostat than cyanoalkyl isothiocyanates and phenyl derivatives. However, Tang et al. (1972) found that the bacteria Enterobacter cloacae is able to degrade benzyl isothiocyanate. The 2-propenyl glucosinolate is also susceptible to microbial degradation (Brabban and Edwards 1994). The antibiotic activity of benzyl and p-methoxybenzyl isothiocyanates was shown against Candida albicans, Escherichia coli, Pseudomonas fluorescens, and Staphylococcus albus (Johns et al. 1982). Inhibition of Staphylococus aureus and Penicillium glaucum was particularly effective with 4methylsulfonylbutyl isothiocyanate and 2-phenylethyl thiocyanate (Virtanen 1962), while 2-phenethyl and 5-methylthiopentyl isothiocyanates were active against Aspergillus niger 11/13, Penicillium cyc10pium 11/17, and Rhizopus oryzae 5/1 (Drobnica et al. 1967). Allyl isothiocyanate was reported to be mutagenic to Salmo-

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nella typhimurium TA100 (Neudecker and Henschler 1985). Recently, Mari et al. (1993) showed the isothiocyanates from 4-methylsulfinylbut-3-enyl, p-hydroxybenzyl, and 2-propenyl glucosinolates to be particularly effective in inhibiting germination of the major postharvest fungal pathogens Botrytis cynerea Pers., Fr., Rhizopus stolonifer Vuill., Monilinia laxa Honey, Mucor piriformis E. Fisch, and Penicillium expansum Link. It has been suggested that the ringspot of young leaves of Brassica is inhibited by allyl isothiocyanate (Hartill and Sutton 1980). Downy mildew, Peronospora parasitica (Pers. ex Pers.) Fr. is a worldwide problem in Brassica crops. In 1975, Greenhalgh and Dickinson, noted some commercial cultivars of Brassica oleracea, Capitata group, to be resistant to downy mildew, which did not appear to be so race-specific, and linked the resistance to the level of 2-propenyl glucosinolate derived volatiles. One year later, Greenhalgh and Mitchell (1976) found tissue macerates of the resistant cultivar to yield the greatest concentration of volatiles. Allyl isothiocyanate proved to be highly toxic to the pathogen and very inhibitory to sporangium germination, and was produced in much greater concentrations in macerates of cotyledon than first leaf tissue. The longest established wild populations possessed the highest average concentrations of flavor volatiles, and it was suggested that selective breeding has resulted in a reduction in the amounts of these compounds in commercial cultivars. This change may be a contributory factor toward the lack of resistance shown by modern cultivars, but minor differences were detected between lines of high and low glucosinolate contents in turnip and winter rape (Grantoft 1993) and slightly lower susceptibility was found in oilseed rape with high glucosinolate and erucic acid content when compared to "double low" accessions (Nashaat and Rawlinson 1994). Further studies are needed to clarify these observations. Greenhouse experiments (Chan and Close 1987) and field experiments (Muehlchen et al. 1990) showed amendments with several Brassica spp. of soils infested with Aphanomyces euteiches reduced pea root rot. Allyl isothiocyanate was shown to have a markedly inhibitory effect on mycelial growth and zoospore formation and germination. Cabbage tissue used as an amendment also significantly reduced root rot of beans (Papavizas et al. 1970) and root rot of sesame, both caused by Thielaviopsis basicola (Berk & Br.). Other antifungal studies suggest that high levels of alkenyl glucosinolates such as 2-propenyl and but-3-enyl and their hydrolysis products are associated with resistance to stem canker disease Leptosphaeria maculans (Desm.) Ces and de Not., with growth of

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the pathogen being completely inhibited by 2-propenyl glucosinolate at concentrations greater than 40 /lg/mL in the presence of myrosinase (Mithen et al. 1986; 1987a,b). The derived isothiocyanate was the most toxic and the antifungal activity of indole hydrolysis products, especially indole-3-carbinol was described for the first time (Mithen et al. 1986). In a recent study, it was suggested that glucosinolates are intermediates in the defence reactions to 1. maculans and are not directly involved in the resistance despite the high levels of alkenyl glucosinolates in the resistant lines of Brassica (Mithen and Magrath 1992). Buchwaldt et al. (1985) reported a preliminary study of the effect of 2-propenyl glucosinolate on Alternaria brassicae (Berk.) Sacc., Sc1erotinia sc1erotiorum (Lib.) de Bary, and Phoma lingam (Tode ex Fr.) Desm. and found a weak stimulation in the growth of Alternaria. Doughty et al. (1991) reported oilseed rape leaves infected by the fungal pathogen Alternaria brassicae to accumulate aliphatic, indole, and aromatic glucosinolates at different rates. The symbiosis between vesicular-arbuscular mycorrhizal (VAM) fungi (Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe and Gigaspora gigantea) and glucosinolate concentration in Brassica has been studied by Glenn et al. (1988). Although they failed to correlate glucosinolate in the roots with VAM, El-Atrach et al. (1989) showed extracts and volatile compounds from cabbage plants to reduce spore germination in Glomus mosseae. Isothiocyanates, which can be produced at a low level even in intact plants (Tang and Takenaka 1983), and not the intact glucosinolates, have been identified as the inhibitory agents of spore germination (Vierheilig and Ocampo 1990). These results have been confirmed in recent findings (Schreiner and Koide 1993a,b). Isothiocyanates have been shown to inhibit virus multiplication (Rada et al. 1971). The action of glucosinolates and hydrolysis products on plant viruses has been recently reported (Spak et al. 1987), although no correlation was found between glucosinolate content and the resistance of rape cultivars to turnip mosaic virus (TuMV). Later studies showed that 2-propenyl glucosinolate and allyl isothiocyanate caused a significant decrease in turnip mosaic virus infectivity in Nicotiana tabacum 1. Similar effects were obtained when p-hydroxybenzyl glucosinolate and phenyl isothiocyanate were tested (Spak 1988). The different results obtained in these two reports have been attributed to the different types and content of glucosinolates in plant material. The highest antiviral (TuMV) effect in vitro was reported for the hydrolysis products of p-hydroxybenzyl and indol-3-ylmethyl glucosinolates (Spak et al. 1993).

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Rapeseed meal (13 mmol of glucosinolates/kg) soil treatments were shown to reduce the number of soil-borne microsclerotia of Cylindrocladium crotalariae (Adamsen et a1. 1992). Brassica spp. were suggested for use as green manure crops after allyl isothiocyanate was shown to be as fungitoxic as methyl isothiocyanate (a commercialized fungicide) against strains of the potato silver scurf Helminthosporium solani, while but-3-enyl isothiocyanate had no effect (Vaughn et a1. 1993). D. Glucosinolates and Plasmodiophora brassicae

Clubroot, caused by Plasmodiophora brassicae Wor., is one of the most destructive deseases ofbrassicacious crops in Europe and North America. This desease is restricted to the Brassicaceae, a family known to accumulate indol-3-ylmethyl glucosinolate and certain other glucosinolates, which may act as auxin precursors (Butcher et a1. 1984). Early studies suggesting a link between glucosinolate content and clubroot resistance have been confirmed by Butcher et a1. (1974, 1976), who showed that susceptibility to clubroot appeared to be related to presence of indol-3-ylmethyl glucosinolate. It was found that the concentration of indol-3-ylmethyl glucosinolate in infected tissues during the early stages of infection was much higher than in comparable healthy tissues, while no significant difference was found in 1-methoxyindol-3-ylmethyl glucosinolate levels, suggesting that the clubroot pathogen brought about the exposure of this and/or related compounds to myrosinase. Searle et a1. (1982) reported the conversion of indol-3-ylmethyl glucosinolate to 3-indoleacetonitrile in swede tissues infected with P. brassicae, while Rausch et a1. (1983) and Butcher et a1. (1984) observed that the development of the infection symptoms is associated with large increase in both the synthesis and degradation of indol-3-ylmethyl glucosinolate yielding higher concentrations of 3-indolylacetonitrile (IAN), an immediate precursor of 3-indolylacetic acid (IAA). In addition there is an increased capacity to hydrolyze IAN to IAA. The IAN and/or its metabolite IAA so formed induced the tissue proliferation (Ockendon and Buczacki 1979), thus indol-3-ylmethyl glucosinolate appears to be a special, inactive storage and transport form of the active auxin. There is some controversy on the levels of IAN and IAA after infection, which might depend on the infection phase at which those levels were measured and the extent to which IAN had been converted to IAA. Ludwig-Muller et a1. (1993) found IAA content to increase in infected plants of both susceptible and tolerant Chinese

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cabbage, but a decrease was noted after 14 days of incubation in susceptible accessions, while levels in the tolerant plants increased. According to the review by Fenwick et al. (1983a) the cell divisioninducing hormones, cytokinins, reached high concentrations in infected tissues. Buczacki and Ockendon (1979) found differences between collections of some species of wild Brassicaceae in susceptibility to P. brassicae populations and reported that indol-3 -y lmethy I glucosinolate cannot be correlated with susceptibility to all populations of P. brassicae. Similarly, total glucosinolates in oilseed rape were not correlated with susceptibility to this pathogen (Rod 1992). This then led to the suggestion that other glucosinolates are involved in this process. After the analysis of 22 cultivars of rutabaga and 21 cultivars of turnip, Mullin et al. (1980) found no correlation between combined indole glucosinolate content (thiocyanate ion) and clubroot resistance. However, a linear increase in SCN- content with increasing clubroot severity was found in the heads, but not in roots, of susceptible cabbage (Chong et al. 1981; 1985). Similar findings have been reported by Chiang et al. (1989) using broccoli cultivars for which no significant difference in distribution or average concentration of oxazolidine-2-thione and volatile isothiocyanates between susceptible and resistant cultivars was identified. Using garden cress (Lepidium sativum 1.), Butcher et al. (1976) found two glucosinolates other than indol-3-ylmethyl and 1methoxyindol-3-ylmethyl that give rise to compounds with auxin activity: benzyl glucosinolate, which can break down to phenylacetonitrile and possibly phenylacetic acid, and p-hydroxybenzyl glucosinolate, a precursor of p-hydroxyphenylacetonitrile and phydroxyphenylacetic acid. However, the four hydrolysis products are less active than IAN and IAA. Studying the susceptibility of brassicacious weeds to clubroot, Ockendon and Buczacki (1979) have suggested that benzyl glucosinolate might have an influence on this disease. In another study, Wheeler (1980) has showed that 3phenylpropionitrile isolated from water-cress, exhibited auxin-like plant growth activity. The studies by Butcher et al. (1976) suggest that Brassica that are low in glucosinolates may be relatively resistant to clubroot, but not all the resistance should be attributed to this mechanism. E. Allelopathic Effects There is an increasing interest in reducing or replacing pesticide application by use of natural phytotoxic plant residues and in this

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respect brassicacious plants may have a role to play. The inhibitory influence of aqueous extracts from parts of B. oleracea plants on the germination and growth of clover (Trifolium repens 1.) and on growth of rye-grass roots (Lollium perenne L. and Lollium multiflorum Lam.) was first described by Campbell (1959). Kutacek (1964) has shown indol-3-ylmethyl glucosinolate and 3-indoleacetonitrile, at concentrations of 10-3 and 10-4 M respectively, to inhibit germination and growth in wheat and clover, the inhibitory effect being attributed to the effect of 3-indoleacetonitrile. At lower concentrations, both substances gradually enhance growth. Allyl isothiocyanate was ineffective on Medicago sativa 1. growth (Choesin and Boerner 1991), probably due also to low concentrations of this compound. Different allelopathic potential of some species and cultivars of Brassica on wheat in laboratory and field trials has been reported by Mason-Sedun et al. (1986) and Mason-Sedun and Jessop (1988). Oleszek (1987) has shown exposure of germinating seeds of lettuce, barnyard grass (Echinochloa crusgalli L.B.P.), and wheat to volatile substances released from pulverized leaves of B. juncea and B. nigra resulted in delayed germination and reduction of overall growth, this effect being attributed to degradation products of glucosinolates. Wolf et al. (1984) showed benzyl isothiocyanate to inhibit germination and growth of velvet leaf (Abutilon thephrasti). Yamane et al. (1992) recently isolated 8-methylsulfonylocty 1, 9-methylsulfonylnonyl, and 10-methylsulfonyldecyl isothiocyanates from Rorippa indica Hiern. and showed inhibition of lettuce hypocotyl and root growth at 0.1 M or above. In a comprehensive study, Bialy et al. (1990) tested several glucosinolates and isothiocyanates and sinapine thiocyanate for effects on germination and growth of wheat seedlings and found no effect of glucosinolates on seed germination, while allyl isothiocyanate was highly active against and 2phenethyl isothiocyanate completely inhibitory of germination, the other isothiocyanates being only moderately active. Although slight growth depression was noted for indol-3-ylmethyl glucosinolate and sinapine thiocyanate, isothiocyanates had a marked effect. The glucosinolate 4-methylsulfinylbut-3-enyl (glucoraphenin) and the counterpart isothiocyanate sulforaphane isolated from stock (Mathiola incana (1.) R. Br.) were shown to inhibit velvet leaf seedling root growth, the glucosinolate being less phytotoxic than the isothiocyanate (Brinker and Spencer 1993). All these results suggest that Brassica species might be used as intercrops or forecrops for weed control (Bialy et al. 1990). Little is known concerning the fate of glucosinolates in the soil environment,

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although Borek et al. (1994), testing 2-propenyl glucosinolate decomposition in different soil types, found allyl isothiocyanate to be the predominant compound regardless of soil characteristics. However, the effect of isothiocyanates can fade away in a relatively short time. In soil amended with 30 g defatted winter rapeseed meal/kg, soil isothiocyanate production reached a maximum of 301 nmol/g soil at 2 h, decreasing by 90% within 24 h, while thiocyanate reached a maximum of 180 nmol/g of soil but persisted longer (Brown et al. 1991). F. Nematodes Nematodes are controlled primarily by soil fumigation compounds, such as methyl isothiocyanate, which are expensive and detrimental toward the environment. Sustainable alternatives to fumigation for control of soil-borne pests are being sought. The effect of extracts of several brassicacious plants and allyl isothiocyanate in reducing soil population densities of the potato cyst nematode in vitro was first reported by Ellenby (1945). In vitro studies also showed leaf extracts of some Brassica species to immobilize the nematode Pratylenchus penetrans, but differences were noted between species and cultivars (McFadden et a1. 1992). Johnson et al. (1992) reported an absence of feeding, development, and egg production of Meloidogyne incognita (Kofoid & White) and M. javanica on rapeseed roots, but the incorporation of 30 to 61 t ha green rapeseed biomass did not affect population densities, this being attributed to the low glucosinolate concentrations in the green material. Mojtahedi et al. (1991, 1993) demonstrated that soil incorporation of rapeseed (B. napus cv. Jupiter) reduces Meloidogyne chitwoodi Golden population densities, particularly in the zone of incorporation where protection from colonization lasted for 6 weeks. These studies revealed the second-stage juveniles to be more sensitive than egg masses. A good protective effect against the proliferation of the M. chitwoodi population was achieved when rapeseed was grown in the fall and incorporated in the spring as green manure (Mojtahedi et al. 1993). This effect, which was probably a result of production of isothiocyanates and/or thiocyanate ion after amendment (Brown et al. 1991), was later confirmed by Lazzeri et al. (1993). They showed that intact glucosinolates have no nematocidal effect on Heterodera schachtii Schmidt, while isothiocyanates, especially from 2-propenyl, but-3-enyl, benzyl, and 4-methylthiobutyl, demonstrated a mortality rate that varied with both the concentration and exposure time.

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They suggest that isothiocyanates act by degradation of the syncytium after a few days and, therefore, interrupt the life cycle of the phytophage. No nematocidal activity was shown for the hydrolysis products of 4-methylsulfinylbut-3-enyl and p-hydroxybenzyl glucosinolates. The 2-propenyl glucosinolate also suppresses progeny production on the nematode Steinernema carpocapsae (Weiser) (Epsky and Capinera 1994). Developing seedlings of Raphanus sativus and Sinapis alba were shown to hatch and attract larvae and then disrupt normal reproduction of the sugarbeet cyst nematode Heterodera schachtii (Martin and Hoefert 1991). Thus, the cultivation of certain Brassica species was recognized as particularly promising to maintain the degree of nematode infestation below the tolerance threshold, even though plants of this family are generally regarded as classic hosts of certain nematode species (Lazzeri et al. 1993).

G. Milk and Meat Panter and James (1990) reviewed the natural toxicants in milk and reported glucosinolate derivatives transferred to milk to cause thyroid enlargement in young animals or in humans ingesting such milk. Epidemiological surveys show a correlation between endemic goiter and the consumption of brassicacious vegetables or milk from cows fed on Brassica crops (Clements 1955; Michajlovskij et al. 1969b; Mitjavila 1986). Thiocyanate production can be a result of the hydrolysis of glucosinolates by the ruminal microbes. Concentration in milk depends on the inclusion level of Brassica oilseed cake/meal in the animal feed (Panter and James 1990). Diets with less than 4% of glucosinolates on dry matter basis, fed ad libitum to ruminants, showed a twofold increase in thiocyanate in the milk (around 15 mg/L) (Saha and Singhal 1993). When compared to allyl isothiocyanate, allyl thiocyanate is more likely to be absorbed from the forestomachs and possibly transferred to milk (Majak 1992). Meat quality seems not to be affected by glucosinolate-containing diets. In young bulls, inclusion of whole rapeseed at 30% of the diet gave meat of a high standard with high protein and low fat content (Krelowska-Kulas et al. 1991). VII. FLAVOR A review of glucosinolates would not be complete without a brief reference to the nature of flavor and to some of the volatile flavor

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components of some Brassicaceae species. The identification of glucosinolate-derived flavor is quite complex because of the number of glucosinolates involved and the additional volatile compounds that do not arise from glucosinolate catabolism. The perception of flavor is a complex phenomenon involving olfactory, gustatory, and tactual sensations. Gustatory and tactual components are involved in the taste and the texture of the food, while the olfactory component deals with the smell of the food. The volatile flavor compounds in foods frequently occur in trace concentrations (ppm or ppb) surrounded by other organic materials and water. Sulfur is particularly associated with odor, and sulfur compounds contribute to the agreeable as well as the disagreeable flavors of many foods. Included among such compounds are the odoriferous breakdown products (isothiocyanates, thiocyanates, and nitriles) of glucosinolates (flavorless sulfur precursors). Isothiocyanates are of considerable flavor importance (MacLeod 1976) and descriptions such as pungent, lachrymatory, acrid, garlic-like, horseradish-like, and bitter have been associated with these compounds (Fenwick et al. 1983a). Pungency has been associated with the volatiles 2-propenyl, but-3-enyl, and 4-methylthiobut-3-enyl isothiocyanates (Fenwick et al. 1983a; Ishii 1991). In cabbage, 2-propenyl isothiocyanate formed from 2-propenyl glucosinolate is a pungent, lachrymatory, very bitter compound (Fenwick et al. 1983b) and has a low flavor threshold value (MacLeod 1976). A low level of 2-propenyl isothiocyanate results in abnormally flat and dull product. It is generally agreed that the heart of the cabbage has a much stronger flavor than the outer leaves. This is in agreement with the high glucosinolate content (particularly 2-propenyl) found in the cabbage heads, both raw (Rosa 1992) and cooked (MacLeod and MacLeod 1970b). The bitterness associated with 2-hydroxybut-3-enyl glucosinolate is due to its decomposition product, the goitrogen 5-vinyloxazolidine-2-thione (Fenwick et al. 1983b). These authors found a good correlation between bitterness score of Brussels sprouts and goitrin content. Other flavor compounds have been characterized in other Brassica vegetables in relatively high concentrations, such as 2-phenethyl isothiocyanate in cabbage, 4-methylthiobutyl isothiocyanate in broccoli, and 3-methylthiopropyl isothiocyanate in cauliflower. All corresponding cyanides have also been reported. A good source of volatiles is horseradish root, in which the following isothiocyanates have been identified: methyl, ethyl, isopropyl, 2-butyl, allyl, pent-

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4-enyl, and 2-phenethyl, the latter being the major component despite the lack of pungency and lachrimatory character (Gilbert and Nursten 1972). Flavor compounds are formed from the parent glucosinolates and factors affecting the glucosinolate content must be taken into account in any evaluation of Brassica flavor. VIII. CONCLUDING REMARKSIPROSPECTS

The importance of glucosinolates as flavor precursors in brassicacious vegetables and the need to produce rapeseed meal of acceptable quality for feeding to animals have led to considerable advances in glucosinolate research over the last 20-30 years. More recently, the role of glucosinolates as potential anticarcinogens has generated renewed interest and the study of this important class of compounds has become truly interdisciplinary. By selecting for reduced glucosinolate levels in rapeseed, plant breeders have solved many of the problems associated with feeding rapeseed meal to animals. However, the further reduction of glucosinolates in animal feed by processing techniques continues to attract considerable at'tention. Genetic manipulation of glucosinolates may be used to produce a glucosinolate profile more suited to a particular purpose, such as enhanced flavor or improved resistance to pests or diseases. The role of glucosinolates during plant ontogeny and the relationship of these compounds or their metabolites to insect or fungal attack are still not clearly understood. In addition, there is also a need for more information about the fate of glucosinolates and their metabolites in the human digestive tract. The development of new procedures for the preparative isolation of a range of glucosinolates together with new and highly sensitive analytical methods has facilitated further study in these important areas. The possible protective effects of glucosinolates and their derivatives against the initiation and progression of cancer has prompted the suggestion that Brassica breeding programs should be developed with the objective of enhancing such effects by increasing the levels of particular glucosinolates in the plant. Although results from in vitro cell bioassays and in vivo studies with experimental animals have been encouraging, the importance of the timing of dietary administration of such compounds in animal studies, the possible synergistic or antagonistic effects of other dietary components, and the genotoxic nature of some glucosinolate breakdown products in cell

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bioassays merit further study. The relevance of such findings to humans suggests that further work is necessary before advocating the production of such "designer" Brassicaceae. With exciting possibilities ahead, it is certain that glucosinolate research will benefit from renewed interest and will continue to provide challenging problems for scientists for many years to come. LITERATURE CITED Adamsen, F. J., D. M. Porter, and D. L. Auld. 1992. Effects of rapeseed meal soil amendments on microsclerotia of Cylindroc1adium crotalariae in naturally-infested soil. Peanut Sci. 19:92-94. Amarowicz, R., M. Shimoyamada, and K. Okubo. 1991. The content of crude glucosides and glucosinolates in Japanese rapeseed varieties. Die Nahrung 35:671673. Andersen, A. S., and R. M. Muir. 1969. Gibberellin induced changes in diffusible auxins from savoy cabbage. Physiol. Plant. 22:354-363. Anon. 1989. Joint report for the period November 1987 to March 1989 on the effects of agronomy and husbandry on glucosinolates and nutrition value of oilseed rapeagronomy. Rothamsted Exp. Stat. and Univ. of Newcastle-upon-Tyne, UK. Anon. 1990. Joint report for the period April 1989 to March 1990 on the effects of agronomy and husbandry on glucosinolates and nutritional value of oilseed rapeagronomy. Rothamsted Exp. Stat. and Univ. of Newcastle-upon-Tyne, UK. Appelqvist, L. A., and E. Josefsson. 1967. Method for quantitative determination of isothiocyanates and oxazolidinethiones in digests of seed meals of rape and turnip rape. J. Sci. Food Agr. 18:510-519. Armstrong, R. H., M. M. Beattie, and E. Robertson. 1993. Intake and digestibility of components of forage rape (Brassica napus) by sheep. Grass Forage Sci. 48:410415. Bailey, G. S., J. D. Hendricks, D. W. Shelton, J. E. Nixon, and N. E. Pawlowski. 1987. Enhancement of carcinogenesis by the natural anticarcinogen indole-3-carbinol. J. Natl. Cancer Inst. 78:931-934. Baird, W. M., T. M. Zennie, M. Ferin, Y.-H. Chae, J. Hatchell, and J. M. Cassady. 1988. Glucolimnanthin, a plant glucosinolate, increase the metabolism and DNA binding ofbenzo(a)pyrene in hamster embryo cell cultures. Carcinogenesis 9:657660. Bajaj, K. L., P. P. Kaur, and D. S. Cheema. 1991. Quality evaluation of some varieties of chinese cabbage (Brassica campestris L.). J. Res. Punjab Agr. Univ. 28:37-40. Baldwin, W. S., and G. A. LeBlanc. 1992. The anticarcinogenic plant compound indole-3-carbinol differentially modulates P450-mediated steroid hydroxylase activities in mice. Chem.-Biol. Interact. 83:155-169. Bartlett, E, and 1. H. Williams. 1989. Host plant selection by the cabbage stem flea beetle (Psylliodes crysocephala). Asp. Appl. BioI. 23:335-338. Bell, J. M. 1993. Factors affecting the nutritional value of canola meal:a review. Can. J. Anim. Sci. 73:679-697. Bell, J. M., and M. O. Keith. 1986. Growth, feed utilization and carcass quality responses of pigs fed frost-damage canola, see (low glucosinolate rapeseed) as affected by grinding pelleting and ammoniation. Can. J. Anim. Sci. 66:181-190.

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4 Fruit Skin Splitting and Cracking Linus U. Opara, Clifford J. Studman, and Nigel H. Banks* Massey University Palmerston North, New Zealand I.

II.

III.

IV.

Introduction A. Overview, Scope, and Importance B. Terminology C. Historical Review D. Assessment and Induction of Cracking Types of Cracking A. Skin Cracking B. Star Cracking C. Splitting Causes of Cracking and Splitting A. General B. Biotic C. Skin Abnormalities D. Genetic Factors E. Environmental Factors 1. Soil Moisture 2. Rainfall and Irrigation 3. Relative Humidity and Evaporation Rates 4. Temperature Fluctuations and Light Exposure F. Cultural Factors 1. Rootstock and Tree Vigor 2. Mineral Nutrition 3. Pesticides G. Fruit Internal Factors Reducing Fruit Cracking A. Cultural Methods

* The authors acknowledge the assistance of Steven Northover with the on-line literature search. This review was part of a research project on "Fruit Splitting in 'Gala' Strains," with financial assistance from the New Zealand Apple and Pear Marketing Board. Horticultural Reviews, Volume 19, Edited by Jules Janick ISBN 0-471-16529-8 © 1997 John Wiley & Sons, Inc.

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V.

1. Nutrient Sprays 2. Fertilization, Pruning, and Scoring 3. Moisture Management B. Application of Plant Growth Regulators 1. Daminozide 2. Gibberellin 3. Paclobutrazol 4. Promalin Summary and Conclusions A. General Review B. Prospects for Future Research 1. Assessment and Induction 2. Developmental Studies 3. Control Literature Cited

I. INTRODUCTION

A. Overview, Scope, and Importance Bruises, cuts, and abrasions are quality defects in fruit induced during harvest and postharvest handling. However, one of the most widespread physical defects limiting the production and delivery of sound, blemish-free fruit is the cracking of the skin and splitting of the underlying flesh while the fruit is still attached to the plant. This occurs extensively in both pome and stone fruit crops (Beattie et al. 1989), grain crops (Lague and Jenkins 1991a,b; Srinivas et al. 1977, 1978), and vegetable crops (Lutz et al. 1949; McGarry 1993). The cracking and splitting of detached fruit during postharvest handling (Mohsenin 1972; Khan and Vincent 1990) and in cold storage (Mezzeti 1959; DeEll and Prange 1993) is also a problem. Skin splitting and cracking is a serious economic problem in many crops and the severity of damage varies with the cultivar. In apple, 'Stayman Winesap', 'Cox's Orange Pippin', 'James Grieve' (Flore and Dennis 1990), and 'Gala', 'Royal Gala', and 'Fuji' (Opara 1993) are especially susceptible. In 'Stayman Winesap', fruit cracking can exceed 90% during the season (Byers et al. 1990) and this cultivar is declining in importance in some fruit growing regions due to extensive cracking problems (Marini 1991). Field investigations in the United States (Walsh et al. 1991) and New Zealand (Hodson 1991) found that up to 40 and 24%, respectively, of 'Gala' were affected by splitting. A survey conducted in 1990 by the New Zealand Apple and Pear Marketing Board (NZAPMB) on the problem of apple splitting in the Hawke's Bay district found that the packed tray carton

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equivalent affected by splitting at fruit packers ranged from 27% for 'Royal Gala', 62% for 'Fuji', and 78% for 'Gala' (S. McLeod, personal communication). The total export value of packed fruit affected and at risk from splitting was estimated to be over NZ$10 million for the three cultivars during the season. This was a particularly disturbing problem since the NZAPMB allowed a maximum of 2% of apples in box to contain splits and any box checked exceeding 2% of split fruit required the packer to repack the entire batch. In certain sweet cherry cultivars, serious crop losses due to cracking are common in areas where rains occur near harvest. Crop losses of crack-susceptible cultivars have reached 50% in many seasons, exceeding 75% in many some seasons, and may lead to loss of entire crop (Davenport et al. 1972; Harrington et al. 1978; King and Norton 1987; Perez et al. 1992). Due partly to high risk of fruit cracking, only 5% of the sweet cherries in the Eastern areas of the United States were grown for fresh market in the 1970s (USDA 1973). Analysis of the fruit-splitting problem in cherry orchards in Marlborough, New Zealand, over the period 1942-1993 showed that significant fruit damage occurred in 3 out of 4 years (Kearney and Neal 1993). Fruit cracking and splitting is also a major commercial problem in other crops, including tomato (Reynard 1960; Peet 1992) citrus (Garcia-Luis et al. 1994), pear (Mendoza and Ortiz 1984), and pomegranate (Malhotra et al. 1983). Losses due to cracking and splitting in these crops can affect the market value of 90% of yield. The presence of fruit cracking alters structural integrity and lowers mechanical strength (Lague and Jenkins 1991a). Cracks produce lines of weakness along which the otherwise intact food material is more likely to undergo further damage when subjected to mechanical stresses. The presence of cracks accounts for excessive crushing of soft, fleshy fruits in harvesting containers and loss of fruit juices (Reynard 1960). Cracks or splits provide open wounds that facilitate rapid moisture loss and excessive shriveling, which lowers fruit quality and storage life (Meyer 1944; Mezzeti 1959; Goode et al. 1975). Prior to harvest, insects and chemical sprays may contaminate the cracked or split fruit (Shear 1971). Fruit with cracks are also prone to chemical injury during washing to remove spray materials as in apple (Fisher 1937a,b), or during postharvest fumigation, as in grape (Laszlo and Saayman 1991). Crack resistance in tomato is an important attribute in processing because fruit cracking leads to high mold counts in the processed product (Iverson 1938; Reynard 1960; Berry et al. 1993). The presence of crack on fruit also permits infection by

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opportunistic fungi such as Penicillium spp. and Gnomonia erythrostoma in cherry (Ceponis et a1. 1987; Perez et a1. 1992), and Aspergillus aculeatus, which causes bunch rot in grape (Jarvis and Traquair 1984). When fruit is transported in water channel as part of the postharvest chain (Schilstra and Janse 1986), cracked fruit absorb more water than fruit without cracks. In addition to increase in a water uptake, the shelf life of cracked fruit is also reduced. In one study in which fruit were in a water channel for 3 h, fruit with no skin cracking had a shelf life of 12.7 days compared to 6.4 days for those with severe skin cracking (Schilstra and Janse 1986). Since the severity of cracking or splitting usually increases as fruit approaches its peak of ripeness, growers of susceptible cultivars tend to pick early to avoid or reduce the proportion of cracked fruit. This often results in the delivery of fruit of nonuniform quality and undercolored fruit to the market and processing plants. Scientific interest in apple fruit cracking and splitting has increased markedly since the beginning of this century, but this development has not guaranteed an understanding of the exact nature of the problem and possible control measures. A review devoted mainly to the causes of fruit cracking was presented over 20 years ago by Teaotia and Singh (1970); while Walter (1967) reviewed the literature on russeting and cracking in apple. Researchers have also provided valuable summaries of previous research together with original work on apple fruit cracking and splitting (Cunningham 1925; Verner 1935, 1938; Shutak and Schrader 1948; Byers et a1. 1990). There are more general reviews on disorders and diseases of fruits include cracking and splitting of apple (Posnette 1963; Salter and Goode 1967) and on mineral-related disorders (Shear 1971, 1975; Bangerth 1973,1979). Fruit cracking is a major problem in the softer fruits and there are a number of research reports on this problem in tomato (Frazier 1947; Reynard 1960), cherry (Bullock 1952; Christensen 1976), prune (Uriu et a1. 1962; Mrozek and Burkhardt 1973), grape (Meynhardt 1964b; Considine 1979; Considine and Brown 1981), and citrus fruit (Randhawa et a1. 1958). The literature on fruit cracking in tomato has recently been reviewed by Peet (1992), and work on several aspects of cracking in cherry (Verner 1937,1938, 1939, 1957; Verner and Blodgett 1931; Christensen 1968, 1970a,b, 1972a-d 1973, 1975, 1976; Webster and Cline 1994a,b; Cline and Webster 1994) and grape berry (Meynhardt 1957 1964a,b; Considine 1979, 1982; Considine and Kriedman 1972; Considine and Brown 1981) are well documented by researchers. Some of these are cited as we discuss the individual sections with which they are concerned.

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Although fruit cracking is important problem in apple, the causes are still less well understood and there are currently no guaranteed management strategies to prevent the incidence of the disorder in susceptible cultivars. This review summarizes research over the past 80 years on the occurrence of physical defects resembling fissures on the skin of apple fruit. Although the problem of fruit cracking and splitting in apple is emphasized, we refer to other fruits as needed to establish a particular point. A distinction is made between cracking (sometimes described as pitting, false sting, star cracking, or even russetting), where the defect is contained within the skin layer, and splitting, where the damage extends into the interior of the flesh. Causes of fruit cracking and splitting are discussed, and the effects of cultural practices and growth regulator sprays to reduce the incidence of damage are considered. We conclude this review by identifying several prospects for future research, including the development of techniques for inducing the various types of cracking, and the development of strategies for effective control of fruit cracking and splitting. B. Terminology Cracking is a general term that has been applied to certain physical disorders of fruits that are expressed as fractures in the cuticle or skin. These fractures may be microscopic or easily seen, sometimes extending deep into the inner flesh as well-defined cavities. Cracking has been defined as the physical failure of the fruit skin (Milad and ShackeI1992), and is generally believed to result from stresses acting on the skin. It could be due to normal processes of growth or damaged-induced (Walter 1967). Stiles et al. (1959) classified 'Stayman Winesap' apples having visible cracks in the skin 6 mm or longer as cracked. Splitting is an extreme form of cracking in which the cracks penetrate deep into the flesh. They range in size from thin splits, a few millimeters long, to wide splits of about 60 mm in apples (Verner 1935). Thus, a practical difference between splitting and other forms of fruit cracking is that a split causes gross exposure of the internal tissue to the atmosphere, whereas in a crack the interior is not completely exposed (that is, it is contained in the cuticular layers). Cracking and splitting in apples have been described in many terms which usually reflect either the perceived cause or symptom of the problem, or both. During the early part of this century, apple cracking was synonymous with the terms blister, blister disease, and

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Coniothecium blister (Cunningham 1925; Goodwin 1929). These terms were derived from the symptoms of the fungus Coniothecium chomastospoTum Cordia, which was widely reported in South Africa ( Evans 1907; BijI1914), New Zealand and Australia (Kirk 1907; Cunningham 1925; Campbell 1928; Goodwin 1929) and Britain (Moore 1931) as the causative agent. In Canada, Hockey (1941) used the term "false sting" to describe a virus disease of apples in which the affected fruit exhibited a degree of deformity with well-defined cracks, but he did not make any reference to fruit cracking. Jenkins and Storey (1955), Schmid (1960, 1961), and Cropley (1963,1968) used the term star cracking to refer to a viral disease of apples. The term "boron deficiency pitting" is used to describe various mineral disorders of pear, including cracking (Kienholz 1942; Pierson et a1. 1971; Raese 1989), and has been applied to the cracking of 'Rymer' apples in India (Dube et a1. 1969). Other terms used have been derived from the position of the crack on the fruit surface. Skin or lenticel cracking has been used to describe fruit cracking in many apple cultivars (Fisher 1937a,b; Schrader and Haut 1938; Jackson et a1. 1977). Stem-end fruit splitting in apple refers to splitting that originates from the base of the stem and radiates toward the crown (shoulder) of the fruit. A stem-end split is a breach of both the skin and the underlying tissue. It occurs extensively in three important commercial cultivars, 'Gala', 'Royal Gala', and 'Fuji'. Stem-end splitting is preceded by the formation of internal ring crack, which occurs near the joint between the stem and the fruit flesh (Opara 1993; Opara et a1. 1993). Crack defects at the stem end of fruit also occur. Verner (1935) observed that late in the growing season of 'Stayman Winesap' apples, cracks originating near the fruit stem and extending outward in straight meridional lines toward the cheek were common. Severe stem-end cracking has been reported in 'Stayman Winesap' apples in the United States (Masden and Bailey 1959) and in 'Cox's Orange Pippin' apples in Britain (Montgomery 1959). Although the term cracking is popular, it may be inappropriate for all physical failures that breach the skin of the fruit. Sometimes the term cracking is incorrectly applied to clearly different symptoms such as russet (Walter 1967; Taylor and Knight 1986) and this makes it difficult to compare results from different researchers. For consistency, the terminology of the original researcher is used in the following sections of this review unless indicated otherwise where

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the original terms are altered in order to categorize the failure more clearly. C. Historical Review

Cracking or splitting occurs in practically all important apple-growing regions. It has been reported in Australia (Carne 1925), Britain (Tetley 1930; Moore 1931; Jenkins and Storey 1955; Skene 1965, 1980), Canada (Mezzetti 1959; Proctor and Lougheed 1980), Denmark (Pilgaard 1957), Italy (Costa et a1. 1983; Visai and Marro 1986; Visai et a1. 1989), India (Dube et a1. 1969; Teaotia and Singh 1970), Japan (Tomana 1961; Watanabe 1987), Korea (Kim et a1. 1991), New Zealand (Kirk 1907; Cunningham 1925; Campbell 1928; Goodwin 1929; Irving and Drost 1987; Opara, 1993), Russia (Fischer 1955; Schmid 1960, 1961), South Africa (Evans 1907; Bijl 1914), Sweden (Nilsson and Fernqvist 1956; Nilsson and Bjurman 1958; Rootsi 1962; Goldschmidt 1962), and the United States (Verner 1935, 1938; Fisher 1937; Schrader and Baut 1938; Shutak and Schrader 1948; Stiles et a1. 1959; Byers et a1. 1990; Unrath 1991). Reports from New Zealand (Kirk 1907; Cunningham 1925) and South Africa (Evans 1907; Bijl 1914) are probably the first records devoted to apple cracking and the Coniothecium disease, which was then believed to be the main cause of the problem. Cunningham (1925) confirmed the prevalence of the disorder in New Zealand with limited distribution elsewhere. In Australia, Carne (1925) concluded that the cracking and russeting disorders of 'Dunn's Favorite' and other apples are connected with climatic and growth conditions. Other early reports from New Zealand extended the focus to include the causes of the problem and possible remedial measures (Campbell 1928; Goodwin 1929). In a British study of the anatomical development of the apple, Tetley (1930) observed extensive cracking on the sun-exposed side of 'James Grieve' and 'Beauty of Bath'. Moore (1931) conducted detailed investigations on the fungus Coniothecium chomastosporum in association with cracking and russeting of apple fruit and blistering of the twigs and concluded that the existence of other similar fungi complicated the investigation. Recognition of cracking as a major commercial problem coincided with expanding apple production in the United States and particularly in New Zealand and Australia, which exported apples to Europe and elsewhere with increasing quality requirements. A disorder that developed prior to harvest (Verner 1935) and during storage (Mezzetti 1959; Goode et a1. 1975) was particularly devastating to

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growers who relied heavily on markets around the world (Goodwin 1929). Verner (1935) documented the first detailed field and laboratory studies on the problem of fruit cracking in apples. His pioneering work and those of other researchers on cherry (Hartman and Bullis 1929; Verner and Blodgett 1931) and tomato (Frazier 1934; Brown and Price 1934) laid the foundation for further in-depth studies on cracking and splitting in these and other fruits. Cracking and splitting are erratic in occurrence, causing heavy losses in some years, seasons, and locations, and almost none in others (Cunningham 1925; Campbell 1928; Goodwin 1929; Tetley 1930; Moore 1931; Verner 1935, 1938; Hockey 1941; Jenkins and Storey 1955; Teaotia and Singh 1970). In New Zealand, the stem-end splitting of 'Gala' and 'Fuji' apples has been observed in Hawke's Bay and Canterbury, but has not been reported in Central Hawke's Bay, Nelson, or Blenheim (Hodson 1991). During the 1920s, the cracking of 'Dunn's Favorite' and 'Cox's Orange Pippin' was reported throughout New Zealand irrespective of climatic conditions or quality of soil (Campbell 1928; Goodwin 1929). With few exceptions (Verner 1935), most observers seem to agree that cracking and splitting occur only later in the season in mature apples. Susceptibility to cracking and splitting varies distinctively from fruit to fruit, and is partly cultivar dependent. Cunningham (1925) noted that although the Coniothecium disease causing apple cracking was prevalent in New Zealand, it was common only on certain cultivars. Within susceptible fruit cultivars, the amount of cracking and splitting varies considerably among individual trees in the same orchard, branches of the same tree, and even spurs on the same branch (Verner 1935, 1938; Posnette and Cropley 1963). During a 3-year study of cracking in 'Stayman Winesap', Stiles et al. (1959) found that during one year, cracking varied widely from tree to tree, ranging from 3.6 to 24.9%. D. Assessment and Induction of Cracking White and Whatley (1955) suggested the use of a planimeter to measure the length of cracks in apple and tomato. This method is objective but it is slow, and limits the number of fruit that can be evaluated. Furthermore, it gives only a measure of the amount of cracking on fruit but does not provide a measure of the tendency of the fruit to crack under certain conditions. Ordinarily, investigators classify cracked fruit arbitrarily as slight, moderate, or severe. Several numerical rating systems have also been used to evaluate crack susceptibility of fruit (Iverson 1938; Reynard

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1951; Prashar and Lambeth 1960). These systems are rapid and suitable for distinguishing between relatively large differences in crack resistance (Armstrong and Thompson 1969). Proctor and Lougheed (1980) assessed the cracking of 'Golden Russet' apples by using a severity rating on a scale of 1 (no cracking) to 5 (severe cracking), while Reynard (1951) used "crack-resistant scores" of 10 to 100 (no visible cracks). A crack-resistant score near 75 was considered the dividing line between resistant and susceptible tomato. In a later study, Reynard (1960) applied a weighted average using the number of plants falling within each class times the value of each class. Byers et al. (1990) induced water uptake and cracking of 'Stayman Winesap' apples in the laboratory by submerging fruit in nonionic and anionic surfactant-water solutions. Within 24 h, both water uptake and fruit cracking increased linearly with increasing concentrations of X-77 surfactant solution, and the authors suggested that submerging apples in X-77 solution could be used to predict the potential for fruit to crack under field conditions. Using this immersion technique, Opara (1993) was able to induce skin cracking at the stem end, calyx end, and equatorial regions of 'Gala' apples but not stem-end splitting.

II. TYPES OF CRACKING Cracking can occur in a number of ways to produce different types of the disorder. Skene (1965) summarized three mechanisms for fruit cracking in apple: (1) the formation of cuticle cracks associated with the initiation of russet; (2) the cracking of the outer layers of skin when these are sloughed off during the final stages of russet development; and cracks that penetrate deeply into the flesh and are responsible for serious downgrading of fruit quality. This third mechanism would account for the splitting (Section I.B) that occurs mainly as stem-end splitting. According to Walter (1967), the various types of cracking which occur in apples appear to be partly cultivar characteristics. In broad terms, these kinds of fruit cracks can be classified into skin cracks, star cracks, and splits.

A. Skin Cracking This defect, also referred to as "checking," lenticel cracking, or cuticle cracking, is characterized by the presence of numerous minute superficial cracks on the fruit surface, followed by the gradual peel-

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ing off of the skin in patches, giving the affected apple a russeted appearance (Kirk 1907; Reed and Crabill 1915; Goodwin 1929; Moore 1931; Fisher 1937a,b; Schrader and Haut 1938; Gourley and Howlett 1941; Meyer 1944; Shutak and Schrader 1948; Fischer 1955; Tomana 1961; Jackson et al. 1977; Taylor and Knight 1986). Some of these cracks heal over during the growth of the apple (Schrader and Haut 1938) by cork formation with a light deposit of suberin on the cell walls. Meyer (1944) noted that the unhealed shallow cracks accounted for the excessive shriveling of apples during storage. Fisher (1937a) described the skin cracking of 'York' apples as varying from barely noticeable to as much as 1.5 mm wide. According to Schrader and Haut (1938), skin cracks on 'York' apples may show as slight "checking" of the skin, resulting in a rough feel or so-called poor finish of the fruit, but in severe cases, many small open cracks, usually 3 mm or less in length may occur. Several authors noted that skin cracking was limited almost entirely to the green (shaded) side of the fruit (Reed and Crabill 1915; Fisher 1937a,b; Schrader and Haut 1938; Shutak and Schrader 1948). These workers and Pilgaard (1957) have also reported that skin cracking is prevalent in the calyx region of the fruit. Skin cracks usually developed perpendicular to the axis of the apple but, if insect or some similar injury was present, cracks generally ran concentrically around the injured spot (Fisher 1937a,b; Schrader and Haut 1938; Shutak and Schrader 1948). The presence of extensive cuticular cracks has been associated with the development of extensive russeting, although some russeted cultivars have virtually no cuticle cracks during their early stages of development (Skene 1965; Costa et al. 1983). B. Star Cracking

Affected fruit are marked with star-shaped cracks in the skin, sometimes on the side of the fruit, but more frequently near the calyx end (Ramsfjell 1952; Jenkins and Storey 1955; Gilmer and Einset 1959; Schmid 1960, 1961; Cropley 1963, 1968). Fruit are usually undersized and become heavily russeted when about half grown (Posnette and Cropley 1963). In severely affected fruit, the star cracks develop deep cracks that usually heal, resulting in severely scarred fruit (Jenkins and Storey 1955; Cropley 1963; Posnette and Cropley 1963), and the affected fruit also tend to have irregular shape (Jenkins and Storey 1955). Star cracking is caused by virus diseases.

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C. Splitting This defect, also referred to as flesh cracking, occurs in the form of breaks in both the skin and flesh of the affected fruit (Verner 1935). Individual splits vary from almost invisible short slits to splits several millimetres deep that extend around the fruit. Proctor and Lougheed (1980) observed extensive early season fruit cracking of 'Golden Russet' apple, which consisted of deep (up to 40.3 mm) and wide (up to 20 mID) equatorial furrows containing easily detached cork tissue, and occurred mainly on the stem half of the fruit. On the basis of evidence provided by Verner (1935), splits on apples can be classified into those originating in regions with structural deformities (such as russet and scar lesions), and those originating in or near the stem depression. Unlike the other types of fruit cracking, which occur on parts of the fruit with certain injuries and virus diseases that alter the fruit surface, stem-end splitting often occurs on apples that appear from the outside to be in excellent condition except for the presence of the split (Verner 1936; Montgomery 1959; Opara et al. 1993). Evaluation of five strains of 'Gala' apples showed that all developed stem-end splitting on the third harvest (Greene and Autio 1993) and extensive stem-end splitting was observed in several sports of 'Royal Gala' apples (L. U. Opara, unpublished), suggesting that all strains of 'Gala' are particularly susceptible. III. CAUSES OF CRACKING AND SPLITTING

A. General It is commonly believed that cracking and splitting in a wide range

of fruits, such as cherry and tomato (Verner and Blodgett 1931; Frazier and Bowers 1947; Reynard 1960; Niiuchi et al. 1960; Westwood and Bjornstad 1970; Christensen 1972d), occur as a result of a sudden increase in the water content of the soil, atmospheric humidity, or temperature. Rixford (1918) reported the splitting of figs under conditions of high atmospheric humidity without rain or irrigation. Cracking in many kinds of fruit such as apple, peach, and cherry is caused by the osmotic absorption of water through the skin of the fruit (Bohlmann 1962) as well as by increased water absorption by roots. The rupture of fleshy parts was discussed by Sorauer (1922), who considered the causal relations to be similar for fruit cracking in cherry, plum, and grape, bursting of carrots and beets, and split-

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ting of stems in kohlrabi, rape, bean, and potato. The author was of the opinion that "all these phenomena have one characteristic in common-that they are initiated only when, after a considerable period of normal development, or still more after a dry period, an unusual supply of water is given suddenly." Discussing the causes of splitting in orange, Coit (1917) stated that "the most common theory in regard to the cause of splits is that an irregular water supply causing wide variations in the moisture content of the soil, produces a greater fluctuation in the growth of the interior than in the skin of the orange." He maintained, however, that such a cause should be regarded as only a contributing factor, because only a proportion of the fruit on any given tree would split. Chandler (1925) discussed the water relations of deciduous fruits in general and stated that "certain injuries, such as cracking of fruit, may result from a heavy irrigation late in its development, if growth has been checked by lack of water earlier." Gardner et al. (1927) concluded that splitting in apples and in some stone fruits was most likely to occur shortly before maturity when rain or late irrigation occurred following a long period of drought. The problem of fruit cracking and splitting in cherry has been extensively studied during the last 70 years (Hartman and Bullis 1929; Kertesz and Nebel 1935; Verner 1937; Bullock 1952; Christensen 1976; Anderson and Richardson 1982; Callan 1986; Webster and Cline 1994a,b; Cline and Webster 1994) and many researchers agree that cracking occurrs as a result of excessive water absorption by the fruit, either directly through the skin in wet weather or by way of the root system and vessels. However, Verner and Blodgett (1931) were unable to observe any relationship between soil moisture and cracking in three cultivars of sweet cherry. Similarly, Sawada (1931) concluded that extremes of soil moisture played no direct part on the splitting of sweet cherries. Peet (1992) has recently summarized factors contributing to fruit cracking in tomato and concluded that cracking occurs when there is a rapid net influx of water and solutes into the fruit at the same time that ripening and/or other factors are reducing the strength and elasticity of the fruit skin. After a 10-year study on tomato fruit cracking, Frazier (1947) concluded that fruit cracked most severely after heavy irrigation at the end of a prolonged dry period. Cracking was less severe in plots with frequent irrigation, which prevented excessive drying of the soil, and it was least severe in plots where the soil-moisture content remained low throughout the growing season. Shaded fruits cracked much less than those exposed to the sun.

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The causes of cracking and splitting in grape have been studied extensively in Australia (Considine and Kriedman 1972; Considine et a1. 1974; Considine 1979,1982; Considine and Brown 1981; Swift et a1. 1974), Germany and New Zealand (Lang and Thorpe 1988; Lang and During 1990), and South Africa (Meynhardt 1957, 1964a,b). Studies by Meynhardt (1957, 1964a) show that spray irrigation can increase the incidence of berry cracking in certain cultivars of grapes when the atmospheric moisture content is high. The splitting of grapes and other fruit by unseasonal rainfall has been attributed to the development, under conditions of high availability of water and low evaporative demand, of a high hydrostatic pressure in the fruit (turgor pressure) in excess of the tensile strength of the cell walls (Considine and Kriedman 1972; Considine et a1. 1974). According to Gourley and Howlett (1941) the cracking of apples and sweet cherries occurs due to excessive cell enlargement of the fruits following a marked increase in the soil moisture. Mrozek and Burkhardt (1973) identified 23 factors believed to be associated with the cracking of apple, tomato, avocado, cherry, and prune. Water, high humidity, rain, and fruit maturity were factors applicable to all types of fruit. Fruit cracking in apple is varied in extent, and the various types of cracking appear to be partly cultivar characteristics. Therefore, this review emphasizing apple, is based on the underlying causes rather than on symptoms. Nearly 100 years of observation, speculation, and research have implicated no less than 20 factors correlated with apple fruit cracking. With the exception of the "viral disease" theory (Powers and Bollen 1947; Posnette 1963; Cropley 1968), none of these may be summarily discarded, and it is likely that the cause is due to the interaction of several factors. In the following review, the causes of fruit cracking and splitting in apples is summarized under biotic, genetic, environmental, cultural, and fruit internal factors. B. Biotic

Most early researchers on fruit cracking in apple concluded that the disorder is caused by the fungus Coniothecium chomatosporum (Evans 1907; Kirk 1907; Bijl1914; Cunningham 1925; Moore 1931). However, factors other than fungal infection were later implicated. Bijl (1914) assumed that cracking was also brought about by uneven growth of the fruit, due to climatic conditions. In a survey of 21 apple growers in New Zealand, Campbell (1928) found that 19 were

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of the opinion that the trouble was physiological, and 2 believed that it was due to disease. Similarly, Goodwin (1929) concluded that the coniothecium disease was an aftereffect, and that the problem could be regarded as due more to general debility of the tree. Fruit cracking in apple has also been ascribed to virus diseases (Jenkins and Storey 1955; Fischer 1955; Schmid 1960,1961; Posnette 1963; Cropley 1968), and the disorder has been claimed to be transmissible (Schmid 1960, 1961; Cropley 1963). Virus diseases that cause apple cracking have been reported in many countries (Cropley 1963, 1968). However, the relationship between the viruses and the susceptibility to fruit cracking remains obscure. In Switzerland, viruses causing fruit cracking and russeting have been transmitted within and between apple cultivars (Schmid 1960,1961,1963), but according to Cropley (1963), it is not yet possible to assess with certainty the relationship of these diseases to the disorder. In Britain, 'Granny Smith' apples from Australia and New Zealand were unaffected when inoculated with four strains of star-crack virus and 'Boskoop' and 'Glockenapfel' scions grafted on star crack-diseased 'Cox's Orange' were similarly unaffected (Cropley 1963). These results indicate that some fruit cultivars are resistant to viruse-induced cracking while others are particularly susceptible. Powers and Bollen (1947) found no correlation between cracking and the number and kinds of microorganisms in cherry. No recent literature associates cracking and splitting in apple and other fruits with viral or fungal diseases. For example, Montgomery (1959) ignored forms of fruit cracking in 'Cox's Orange Pippin' apple due to virus diseases to investigate the "more serious" cracking due to unusual climatic conditions. According to Fawcett and Lee (1926), splitting in citrus fruit is commonly associated with diseased tissues, such as lesions. These diseased tissues absorb water exceptionally when the water supply is plentiful and cause rupture through abnormal swelling. Gardner et al. (1927) and Goodwin (1929) found fruit cracking in apple and pear fruit to be associated with severe scab, blotch, and russeting. C. Skin Abnormalities

Physical defects on fruit constitute points of weakness where rupture generally occurs first. These defects may arise from physiological disorders, diseases, insects, or mechanical injury (Fisher 1937a,b; Schrader and Haut 1938; Shutak and Schrader 1948; Montgomery 1959; Walter 1967; Teaotia and Singh 1970). Fruit cracking and split-

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ting are both associated with a disintegration of the cuticular layer, either localized or general (Walter 1967). In 'Stayman Winesap' apple, Gardner and Christ (1953) noted an eightfold increase in the number of incipient cracks and splits that subsequently developed on severely russeted specimens compared with smooth-skinned specimens. The authors also noted increased skin permeability to water vapor in russeted areas. Simons and Aubertin (1959) studied the effect of wounding on the development of fruit tissues by inducing damage by cutting, abrasing, or scraping fruits of 'Golden Delicious' at various stages of growth. Cutting the skin 2 days after fruit set stimulated periderm formation, accompanied by sloughing off of cells in the exposed tissues, and this persisted throughout the fruit development. On the normal areas adjacent to the injury, macroscopic effects were not pronounced, but cuticular cracks developed as a result of irregular growth. There was no malformation of the fruit, and consequently no secondary cracks when fruits were injured during later stages of development, but periderm activity was insufficient to form a protective covering over the wound. The effects of abnormalities of peripheral tissues in relation to apple cracking was studied extensively by Verner (1935), who concluded that cracking is less likely to occur on sound apples than on those with some abnormality such as sunburn or russet. During one season 880/0 of the cracks formed on the fruits of one tree were directly associated with russeted skin, sunburn, or scab spots. The remaining 12% were most often on the sound cheek of fruit surfaces that was most exposed to sunlight. Cracking appeared to be independent of the abnormalities themselves, but abnormalities rendered affected portions of the fruit more susceptible to cracking than normal portions when environmental influences promoted cracking. Stiles et al. (1959) found that cracking of 'Stayman Winesap' apple increased with an increase in russet or other injury to the fruit. According to Montgomery (1959), the mechanism involved water uptake through the skin, which ruptured cells. In addition, moisture fluctuations may cause the cracking of skin already finely russeted, or in combination with temperature changes lead to uneven cell division or enlargement, with consequent stresses that might lead to cracking. Other authors found a histological analogy between cracking and russeting in apple (Walter 1967; Skene 1982; Proctor and Lougheed 1980). According to Visai et al. (1989), cracking was considered the last and the more serious stage of skin russet. However, on the basis of observations on 'Stayman Red' apple, Costa et al.

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(1983) found it difficult to relate fruit cracking to russeting, although they pointed out that fruit cracking originated from small russeting plates and/or hypertrophic lenticels. The authors suggested that these anatomical features led to a reduction of cell elasticity, which, associated with high fruit growth rate, could be a basic factor in determining the onset of cracking. D. Genetic Factors

Susceptibility to fruit cracking is considered to be under genetic control. Posnette and Cropley (1963) attributed fruit cracking in apples to a genetic disorder. Visai et al. (1989) quoted unpublished data that showed that cracked 'Neipling Stayman' fruit had less gibberellic acid-like (GA-like) substances than intact ones and concluded that this factor was associated with cracking in 'Stayman Winesap'. In Canada, Proctor and Lougheed (1980) found extensive early season fruit cracking of 'Golden Russet' apple but not on 'Pomograte Russet'. 'Cox's Orange Pippin' is more frequently affected by star crack than other cultivars in England (Jenkins and Storey 1955). Researchers in the United States have found that 'York Imperial' has severe skin cracking (Fisher 1937a,b; Schrader and Haut 1938; Shutak and Schrader 1948). Genes controlling fruit cracking have been identified in tomato. Reynard (1951) and Young (1957, 1959) concluded that radial crack resistance in tomato was controlled by two recessive genes, while radial cracking was determined by two genes designated as cr cr and ri ri. Young (1959; 1960) found crack-resistance genes to be associated with pink fruit color, high number of fruits per plant, low average number of locules per fruit, and small fruit diameter and determinate plant growth habit. Resistance to fruit cracking in tomato has also been associated with wide calyx base and lobes (Frazier 1951,1958). Prashar and Lambeth (1960) studied the inheritance of radial cracking in tomato and concluded that resistance was not controlled by the same gene in all cultivars. Reynard (1960) found that radial and concentric cracks in tomato are governed by separate gene systems, and through cross-breeding, crack-resistant cultivars have been produced (Frazier 1959; Reynard 1960). Zielinski (1964) studied the genetic resistance to fruit cracking in 38 sweet cherry cultivars by immersing fruit in distilled water at 20 ± 2°C and found that some cultivars were 5- to 7-fold more resistant than others. Zielinski observed large genetic variability for resistance

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to fruit cracking among sweet cherry cultivars and suggested that breeding had potential to solve the problem. E. Environmental Factors The incidence of fruit cracking and splitting varies greatly among climatic regions, and by years, seasons, and orchard (Verner 1935, 1938; Walter 1967; Teaotia and Singh 1970). Environmental factors associated with fruit cracking include soil moisture, rainfall, relative humidity, temperature, and exposure to sunlight. 1. Soil Moisture. It has often been suggested that the major factor responsible for the splitting of various fruits is a sudden marked increase in soil moisture content late in their development, especially if growth has been checked earlier by lack of water (Gardner et al. 1922; Chandler 1925; Bohlmann 1962; Walter 1967). In Japan, the fluctuation of soil moisture from low to high induced more cracks on tomato fruit (Niiuchi et al. 1960). In Sweden, Nilsson and Bjurman (1958) observed that cracking of 'Ingrid Marie' apples was promoted by rapidly changing weather conditions. Proctor and Lougheed (1980) suggested that cracking of 'Golden Russet' apple was related to fluctuating water supply in the early growing season. The cracking disorder of stone fruits (mainly cherry and apricot) and grape has been attributed to excess uptake of water by fruit shortly before harvest, leading to cell rupture (Beattie et al.

1989).

Verner (1935) observed no increase in the incidence of splitting when he caused sudden and pronounced soil moisture fluctuations by inducing drought in 'Stayman Winesap' apple followed by flood irrigation. He attempted to induce splitting by forcing water into the cut ends of detached fruit-bearing branches when these were exposed to air but was unable to induce splitting even though the treatment continued up to 3 h. In a study of fruit splitting in 'Mutsu' apple, Watanabe et al. (1987) reported that soil types, moisture content, and bagging had no clear effects on the incidence of the disorder. Irving and Drost (1987) found that water deficit imposed during phase one of fruit growth increased the proportion of cracked 'Cox's Orange Pippin' apples by 2- to 3fold. The incidence of bitter pit was marginally reduced, but mean fruit size and titratable acidity were not altered. Trought and Lang (1991) investigated the role of water in cherry splitting and observed that significant fruit splitting occurred on blocks where fruit were protected from rain with plastic covers. The

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authors suggested that water uptake through the root systems, may be of greater significance in causing fruit splitting than had been realized in the past. 2. Rainfall and Irrigation. Although the experiments of Verner

(1935) showed no relation betw~en apple cracking and soil moisture, he succeeded in inducing severe splitting when branches bearing attached fruit were submerged in water for several days. In further experiments in which rain was artificially diverted from large branches, some of the fruit on those branches cracked, indicating that the presence of a film of water on foliage or fruit was unnecessary to promote cracking. Verner concluded that wetting of fruit and leaf surfaces for a long period might aggravate the tendency to crack, but it was not the primary factor. Reed and Crabill (1915) found that skin cracking of apple occurred very rarely in dry seasons but usually after late rains followed drought. Gardner and Christ (1953) kept detached half-grown fruit of several apple cultivars continually covered with a film of water for 4 or 11 days and found that no cracking was induced in either 'Rome Beauty' or 'Delicious'. In 'Stayman Winesap' some splitting occurred after 4 day's soaking, and after 11 days, half the fruit showed splits. Montgomery (1959) associated the widespread cracking and russeting of 'Cox's Orange Pippin' in England in 1958 with the exceptionally heavy rainfall during June and August. In addition to excessive water absorption by the roots, Bohlmann (1962) found that peach, apple, and cherry fruit tend to crack more easily when they corne into contact with rain or mist or when immersed in water. He concl uded that fruit protected from rain will not crack. Goode et al. (1975) were able to induce skin cracking of 'Cox's Orange Pippin' apples by maintaining water stress. In experiments with'Stayman Winesap', Byers et al. (1990) found that over-tree or under-tree sprinkling for one 12-h night period did not cause fruit cracking. After 6 nights of sprinkling, over-tree sprinkling caused 9% and under-tree sprinkling caused 7.6% of the fruit to crack. Fruit covered with bags or petroleum jelly on over-tree sprinkled trees did not crack, while 7.6% of the wetted fruit cracked. These results agree with the conclusion of Bohlmann (1962) on apple, peach, and cherry but disagree with those of Verner (1935) also on 'Stayman Winesap' apples, and Trought and Lang (1991) on sweet cherry, where fruit under a tarpaulin cracked. 3. Relative Humidity and Evaporation Rates. Tukey (1959) found

that prolonged periods of high relative humidity, especially while

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the apples are small, may inhibit the potential formation or modify the composition of the cuticle sufficiently to cause it to lose its protective capacity. Increase in water supply and decrease in water loss from leaves due to saturated relative humidity promoted fruit cracking and splitting in apples (Verner 1935, 1938), and several other fruits (Teaotia and Singh 1970). Verner (1935) obtained a fairly close relationship between fruit cracking and relative humidity when a heavier rain accompanied by humidity well below 90% caused no cracking while relative humidity between 99 and 100% caused severe cracking. Under natural orchard conditions, Verner (1935) found a definite association between low rates of evaporation and the incidence of fruit splitting in apple. There was extensive splitting during several periods of prolonged slow evaporation rates, even when there had been no rain for up to 6 days. Outbreaks of splitting were generally preceded by markedly depressed transpiration, maintained for 6 h or more. Verner concluded that splitting in 'Stayman Winesap' is promoted by increased water supply to the fruit tissues as a result of reduced transpiration under conditions of high humidity. Low humidity during fruit development has been associated with apple cracking (Mrozek and Burkhardt 1973; Walter 1967). Under conditions of water stress, low relative humidity would accentuate the effects of drought, and thus tend to promote cracking associated with the outer tissues of the fruit. The combination of these factors accounted for the greater incidence of fruit cracking of 'Ohenimuri' apple in drier inland regions in South Africa compared with humid regions (Louw 1948). 4. Temperature Fluctuations and Light Exposure. Koske et al. (1980) found that increasing growing soil temperature of tomato up to 32°C had no effect on cracking. Peet and Willits (1991) tested the effects

of solar energy and temperature on tomato fruit cracking. When nighttime temperatures were maintained below 21°C by air conditioners, the percentage of fruit cracking decreased significantly because the total number and weight of fruit increased more than the number and weight of cracked fruit. Verner (1935) found that the occurrence and severity of cracking of 'Stayman Winesap' apples was unrelated to air-temperature fluctuations; however, cracks on otherwise sound fruits occurred most often on the cheek exposed to sunlight. Bohlmann (1962) noted that in apple, peach, and cherry, there is increased tendency to crack as the temperature of water on fruit skin rises.

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The degree of sun exposure of fruit has been associated with the cracking of apple, tomato, and cherry (Fisher 1937a,b; Verner 1938; Mrozek and Burkhdart 1973). In 'Rome Beauty' apples, Magness and Diehl (1924) found that the exposed side of the fruit developed a thicker skin than the shaded side. Reed and Crabill (1915) and Fisher (1937a,b) noted that the skin cracking of 'York' apples is limited almost entirely to the green (shaded) side of the fruit. Reed and Crabill suggested that perhaps "the skin on the shaded side of the fruit may be actually stretched to bursting by the unusual rapid multiplication and growth of pulp cells due to a sudden increase in water supply." The prevalence of skin cracking of 'York' apple on the shaded side of fruit was also reported by Schrader and Haut (1938) and Shutak and Schrader (1948). However, Tetley (1930) found that in 'James Grieve' and 'Beauty of Bath' cultivars, most of the cracks were formed on the sunny side of the apple. Seasons that had extensive fruit cracking also had a long dry, cold period when the fruit was setting, followed by a warm rainy period when the apple was ready to swell. Tetley concluded that the cold period had produced a comparatively thick, inelastic cuticle, especially on the exposed side of the apple, with the result that the epidermis was unable to resist the rapid swelling of the cells within and consequently cracked. During 3 years of study on 'Stayman Winesap' apples, Verner (1938) found sound, densely shaded fruit growing in the innermost parts of the tree with virtually no cracking. When apples in different parts of the tree were enclosed in brown paper bags for 3 to 4 weeks before harvest, the incidence of splitting was 5% compared to 41 % in the control. Surveys in Sweden by Rootsi (1962) showed that direct exposure to sunlight may increase the incidence of apple fruit cracking. Rootsi also found that the resistance to pressure of the skin of several cultivars was lower on the shaded side of the fruit, and concluded that the lower incidence of cracking was related to the greater elasticity of the shaded tissues. It is apparent that there are cultivar differences on the effect of exposure of fruit to sunlight on the occurrence of cracking in apple. Although cracking occurs predominantly on the shaded side of 'York' cultivars (Shutak and Schrader 1948) and on the exposed side of many other cultivars, such as 'James Grieve' and 'Beauty of Bath' (Tetley 1930) and 'Stayman Winesap' (Verner 1935, 1938), cracking defects are on the side of the fruit with a thicker, inelastic cuticle. This means that thicker cuticle is on shaded side of 'York Imperial' and the exposed side of 'James Grieve', 'Beauty of Bath,' and 'Stayman

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Winesap' apples. More studies are required to elucidate these cultivar differences on cuticle thickness in relation to light exposure and cracking. In prunes, Mrozek and Burkhardt (1973) found that the exposed side of the fruit located on the south side of the tree experienced the highest temperatures and highest incidence of side cracking. F. Cultural Factors Factors such as choice of rootstock, supplemental water supply, mineral nutrition, chemical sprays, pruning, thinning, and other practices that influence the nature of fruit growth exert much influence on fruit cracking and splitting. Measures that increase fruit size are apt to accentuate cracking in apples (Nilsson and Bjurman 1958). In stone fruits, cracking is negatively related to fruit load (Beattie et al. 1989). 1. Rootstock and Tree Vigor. The association between rootstock, tree vigor, and incidence of fruit cracking has received continued attention from several researchers. Goodwin (1929) attributed the cracking or blister disease of apples to a general debility of the tree, rather than to other causes. He found that practically all sound fruit on affected trees were located near the top where the growth was stronger. Goodwin concluded that the lower buds in debilitated trees had become so weakened and immature that it was impossible for them to maintain sufficient vigor to produce fruit without cracks. Verner (1935) observed that fruit splitting was more pronounced and extensive when the foliage was sparse than when it was dense. The greater incidence of skin abnormalities, such as sunburn and russet, on the sparsely foliated branches was also believed to be a contributory factor to higher incidence of fruit splitting. Fisher (1937a,b) found that the tendency of apples to crack increased as the fruit approached maturity, with greater severity on trees low in vigor and bearing a light crop. Investigations by Schrader and Haut (1938) and Shutak and Schrader (1948) on the cracking of 'York Imperial' confirmed that low vigor and light crop were conducive to cracking. In addition, small, highly finished fruit with deep green ground color was less susceptible to skin cracking. Louw (1948) provided further evidence that vigorous growth was conducive to reduced incidence of cracking in apples. When trees of 'Ohenimuri' apple in a neglected orchard in South Africa that had not been pruned for a number of years were severely pruned,

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fruit cracking was almost entirely eliminated as a result of vigorous growth that developed. On well-tended trees in which growth was not a limiting factor, severe pruning did not affect the incidence of cracking. Cracking and russeting of 'Cox's Orange' apples in East MaIling, Kent, in England were also more frequent on poorly grown trees having inadequate leaf cover (Anon, 1961). Proctor and Lougheed (1980) found that rootstock and crop load influenced the cracking of 'Golden Russet' apples in Canada, with fruit cracking more severe in trees on the more dwarfing rootstocks, which were also younger and bore fewer fruit per centimeter of trunk circumference. In contrast to the above results, Nilsson and Fernqvist (1956) in Sweden observed that vigorous rootstocks, such as M.16, and seedlings were more conducive to the development of fruit cracking in 'Ingrid Marie' apples. However, cracking was more marked in large and red-colored fruits than in small and green fruits, respectively. This agreed with the findings of Shutak and Schrader (1948) in the United States. Watanabe et al. (1987) associated fruit splitting in 10 apple cultivars with very early flowering and suggested that conditions conducive to rapid fruit growth were related to splitting. In addition to rootstock effects, large fruits were also more susceptible to splitting. Cobianchi et al. (1984) also reported rootstock effects on fruit cracking of 'Stayman Winesap' apples, but studies by Comai and Widman (1981) did not find significant rootstock effects on fruit cracking in 13 'Stayman' clones. In sweet cherry cultivars, Granger and Frensham (1991) reported significant differences in fruit splitting up to 40% due to rootstock effects. 2. Mineral Nutrition. Nutritional conditions of the tree and fruit have been suggested to account for the differences in the cracking susceptibility of fruit on different trees, or even on the same tree (Schrader and Haut 1938). Shallow soil conditions and inadequate soil moisture have been indicated as factors influencing tree nutrition, leading to susceptibility to fruit cracking. Calcium, nitrogen, and boron appear to be the mineral nutrients that affect fruit cracking (Tomana 1961; Dube et al. 1969; Shear 1971,1975; Bangerth 1973, 1979). Deficiencies in Ca and B may lead to the development of cracks, while high N would aggravate the disorder (Shear 1971). Fischer (1955) found no evidence to attribute apple fruit cracking to nutrient deficiency or spray damage. Stiles et al. (1959) found no influence of urea sprays on cracking of 'Stayman Winesap' apple. Experiments on 'Cox's Orange Pippin'

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showed that cracking was worse on clean cultivated plots, especially where Nand K were applied (Montgomery 1959). Cracking was very much less where the trees were in grass or were receiving potash only. Results from long-term manurial trials obtained similar results (Greenham 1965). Tomana (1961) found that in 'Jonathan' apples, when seed development ceased and the fruit began to enlarge, the N content of the flesh increased rapidly, causing cracking of the skin around the lenticels. A positive relationship between N manuring and the cracking of 'Holstein Cox' apple fruit was reported by Weissenborn and Gottwald (1965). Fruit cracking in 'Rymer' apples has been attributed to B deficiency (Dube et al. 1969). 3. Pesticides. Fruit cracking has been reported to be aggravated by pesticide sprays (Schrader and Haut 1938; Asquith 1957; Anon. 1962a). However, in other cases, both sprayed and unsprayed fruit have been affected similarly (Reed and Crabill 1915; Moore 1931; Fischer 1955; Byers et al. 1990). Applications of Bordeaux mixture caused cracking and general distortion of apple fruits (Moore 1931). Similar injury was also observed on fruit from unsprayed "control" trees or those sprayed with lead arsenate only, although the injury was greatest where Bordeaux mixture was used. Schrader and Haut (1938) obtained similar results on the cracking of 'York Imperial' aggravated by late arsenate sprays. Fungicide sprays have been observed to affect the cracking of apples (Asquith 1957). In trials to control mites in 'Stayman Winesap' apple orchards, phosdrin caused severe cracking round the stem end, while captan caused the least cracking. In trials to control fruit pests (Anon. 1962b), high-volume sprays of 2,2,2-trichloro-1,1-bis(4chlorophenyl)ethanol at petal fall caused the cracking and russeting of 'Cox's Orange Pippin' fruits. Surfactants, often applied with herbicides, fungicides, or insecticides as emulsifying, dispensing, and spreading agents, may cause distinctive stress symptoms that affect fruit quality. They are known to increase the penetration of water, spray chemicals, and nutrients through fruit cuticles (Marios et al. 1987; Westwood and Batjer 1960; Byers et al. 1990). Many workers have found that the use of surfactant enhances fruit cracking in apple (Noga and Bukovac 1986; Noga and Wolter 1990; Byers et al. 1990). Submerging'Stayman Winesap' apples in several nonionic and anionic surfactant-water solutions caused increased water uptake and fruit cracking (Byers et al. 1990). Submerging apples in pesti-

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cide combinations or nutrient solutions generally did not affect fruit splitting, while a nutrient-surfactant combination increased fruit cracking. It was concluded that the surfactant was the constituent primarily responsible for the cracking. Similar results were found by Opara (1993) using 'Gala' apples. G. Fruit Internal Factors

The anatomical and physiological conditions of roots, branches, and fruit have major effects on fruit splitting in apple (Tetley 1930; Verner 1935, 1938; Rootsi 1962; Goldschmidt 1962; Skene 1965), cherry (Verner and Blodgett 1931; Christensen 1972d), grape (Meynhardt 1964b; Considine 1979), and tomato (Cortner et al. 1969; Hankinson and Rao 1979). Verner (1935) and Teaotia and Singh (1970) reported that some incipient cracks originated at hypertrophied lenticels, which may be caused or promoted by greatly retarded transpiration from the plant, accompanied by a plentiful water supply to the regions of hypertrophy. Schilberszky (1918) concluded that hypertrophy of lenticels in apple fruits is related to an excessive water supply in the soil. According to the author as reviewed by Verner (1935) and Teaotia and Singh (1970), the proliferation that constitutes lenticel hypertrophy may decrease the extensibility of the neighboring peripheral cell layers and lower their mechanical resistance to being torn apart; and if that be true, lenticels might be expected to make the weakest point, at which rupture should begin, whenever peripheral tissue strain became sufficiently excessive. Periods of drought result in the development of strengthening tissues, which usually appear first in the xylem and phloem (Graebner 1920). Graebner suggested that, as a general rule, strengthened cells have lost their ability to divide and most of their capacity to enlarge. In this condition, if water supply is greatly increased after a dry period, the meristematic group quickly resume growth but not the strengthened cells. Resultant differences in growth rates between contiguous mechanical and meristematic tissues may thus lead to excessive tensions and failure of the mechanical tissue. In some fruits, the structure of the cutin may have a definite correlation with cracking. Tetley (1930) found that apple cultivars having cutin deposited on the tangential wall, which touches only the apex of mature epidermal cells on the radial wall, are less susceptible to cracking than cultivars having their cutin deposit extended throughout the length of radial wall or even completely surrounding the cell.

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Shutak and Schrader (1948) obtained a significant positive correlation between thickness of cutin and the percentage of cracked apple fruits on a given tree. The red side of the fruit, which is less subject to skin cracking, possessed thin, regular cutin and showed little distortion of the epidermal and subepidermal layers of cells. On the shaded side with the greater incidence of cracking, the cuticle was thicker and more regular than on the exposed side. The thickened cuticle was usually sharply indented, and thick wedges of cutin were often found between the epidermal cells. Such irregularities in the structure of the cuticle and the underlying epidermis were considered to be the main factors involved in the increased susceptibility to cracking of these tissues. The greater thickness of the fruit cuticle on the shaded side of 'York' apples reported by these authors disagrees with Magness and Diehl (1924), who found that the sun-exposed side of 'Rome Beauty' apples developed a thicker skin than the shaded side. By enclosing young fruit in polythene bags, Tukey (1960) found that fruit of 'Rome Beauty', which has a moderately thick cuticle, cracked less than 'Golden Delicious', which has a thin cuticle. Nikitina (1959) did not find any consistent correlation between skin thickness and apple keeping quality. Physiological studies on fruit cracking in 'Stayman Winesap' apples (Verner 1935) showed that cracks generally appeared first in restricted areas, which indicated that peripheral tissues became exceptionally weak in such regions. On two separate branches of a single tree, Verner found 31 and 70% fruit cracking in each branch and concluded that physiological conditions within the tree or fruit not directly related to current weather conditions were also influential. Histological studies by Verner (1938) suggested that the susceptibility of 'Stayman Winesap' apple to fruit cracking was due chiefly to premature cessation or restriction of growth in the hypodermal layer. Verner maintained that cracking is due to the failure of the peripheral fruit tissues to keep pace in growth with that of the cortex, rather than their inability to repress and contain excessively rapid growth of this region. Skene (1965) proposed that the variations in fruit growth rate may account for the time at which cracking occurs. Microscopic examinations of cool, stored apples showed that dissolution of the intercellular pectic membranes allowed excessive swelling and separation of the pulp cells and the resulting pressure caused cracking of the fruit skin (Mezzetti 1959). A possible explanation for loss of cell cohesion derives from the increase in air space, which in turn implies a decrease in average area of contact between cells (Hatfield and Knee 1988).

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Anatomical studies by Costa et al. (1983) showed that fruits of 'Stayman Winesap' apple (highly susceptible to cracking) were characterized by a lack of transition cells between the hypodermis and fruit parenchyma. The hypodermic cells were small, thick-walled, tangentially oriented, and depressed. The fruit parenchyma had large isodiametric cells with thin walls. Cell division in the hypodermic tissue ceased earlier than in the fruit parenchyma and, as a consequence, the outer part of the fruit could not follow the growth of its inner part. During the high-growth periods, cracking of the hypodermic cells could occur. This agreed with the results of Verner (1938). Weiser (1990) obtained similar results and hypothesized that the inability of the hypodermis to keep pace with the expansion of the fruit was due to a difference in cell wall composition and the consequent effect on wall extensibility. Taylor and Knight (1986) studied the cuticular morphology of apple fruits and found a greater occurrence of deep flanges protruding between epidermal cells. This suggested that there were areas of weakness where cracking could arise and that would also cause russet development. It has been suggested that fruit splitting of 'Gala' apple is induced by high internal turgor of the fruit, and that the additional stress caused by the wrenching of the stalk may cause the cortex cells about the peduncle entry area to pull apart (M. C.Trought, personal communication). In cherry, Kertesz and Nebel (1935) found that cultivars that crack most readily had smaller cells and thus, presumably, more cell-wall material than those resistant to cracking. Greater retention of liquid by pulp of the cultivars that cracked badly was attributed to the imbibitional properties of the greater amount of colloidal substance in these fruit. Hankinson and Rao (1979) found that tomato cultivars particularly resistant to concentric cracking possessed flattened epidermal and hypodermal cells for their first few rows while for the cultivars resistant to radial cracking, the cutin penetrated into the third layer of cells. IV. REDUCING FRUIT CRACKING

Many orchard management practices have been recommended to control or reduce fruit cracking, but their effectiveness varied with the degree of susceptibility, and this in turn varied greatly among fruits, cultivars, growing areas and conditions, and seasons. To date, success achieved in reducing fruit splitting experimentally has not

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been translated into the commercial fruit industry due partly to the difficulties of reproducing controlled conditions in the field. In some instances, the cost of implementation would not be justified by the economic value of the crop (Bohlmann 1962; Kearney and Neal 1993; Cline and Webster 1994). Despite increasing contributions to our knowledge and awareness of the phenomenon of fruit cracking in apples and other fruits, there is still no agreement in the literature on the exact origin or cause of the problem, and, to date, the search for reliable methods to adequately control the problem remains elusive. Fruit growers have adopted a number of strategies to minimize losses, such as harvesting fruit early, reducing irrigation, protecting fruit from rain by installing temporary covers or shades, and applying spray materials to minimize water uptake by fruit. In severe cases, growers topwork susceptible cultivars, plant new cultivars resistant to cracking, or choose orchard sites where the probability of rainfall at the critical stage of the season is low (Schmid 1960; Trought and Lang 1991). A. Cultural Methods It is generally agreed that cultivation measures resulting in the pro-

motion of tree vigor reduce fruit cracking. Earliest control measures advocated spraying with certain chemicals, manuring, and severe pruning (Kirk 1907; Evans 1907; Carne 1925; Cunningham 1925; Campbell 1928; Goodwin 1929). 1. Nutrient Sprays. Spraying trees at various stages of fruit devel-

opment with bordeaux mixture, copper sulfate, or slaked lime had been recommended to reduce cracking (Kirk 1907; Evans 1907; Bijl 1914; Cunningham 1925; Powers and Bollen 1947). Calcium in the mixture is believed to prevent fruit cracking (Verner 1939; Bohlmann 1962); however, Powers and Bollen (1947) concluded that the benefit reported from the use ofbourdeaux spray appears to be due more to Cu than Ca. A major limitation of these nutrient materials is that they cause spray damage on certain cUltivars and leave harmful residue on the fruit. It has been suggested that such spraying should be carried out early in the season so that the residue may decrease as a result of weathering and an increase in fruit size (Moore 1931; Bohlmann 1962). In boron-deficient soil, the percentage of cracking has been reduced by boron applications (Bohlmann 1962); however, in a trial with 'Rymer' apples, Dube et al. (1969) found that soil application of

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boron was ineffective. Foliar sprays of 0.3% boric acid reduced fruit cracking considerably. Foliar or injection applications of CaCl z solution reduced fruit cracking of 'Sekaiichi' apples (Kim et al. 1991). 2. Fertilization, Pruning, and Scoring. Campbell (1928) and Goodwin (1929) recommended that in order to produce fruit free from crack-

ing, tree vigor should be increased by such practices as heavy pruning, combined with manuring and cultivation. Following the system of pruning advocated, Goodwin obtained an increase of 285 % export apples from the same trees in the subsequent year. According to Schmid (1960), growers may be able to overcome the russeting and cracking of apples by top-working affected trees. Byers et al. (1990) found that two scores around the trunk of 'Stayman Winesap' apple trees with a carpet knife reduced fruit cracking by 22 % and they noted that neither fruit size, fruit color, nor return bloom were affected by the treatment. Although no explanation was given for the effectiveness of the treatment, the authors claimed that a greater effect on fruit cracking might have been realized if scoring had been done every 2 to 3 weeks. 3. Moisture Management. Maintaining an adequate moisture supply has been found to reduce fruit cracking in apples (Rootsi 1962; Goode et al. 1975). Mezzetti (1959) suggested that cracking in stored apples could be prevented by reducing the intensity and duration of the skin-yellowing process and by keeping the humidity in cold storage relatively low. Protecting apples from rain by shading during critical growth periods produced fruit with less russet and cracking (J ackson et al. 1977). The cracking of sweet cherry on the tree has been prevented by enclosing fruit in paraffined paper (Sawada 1931) or by excluding rain by means of waterproof tarpaulins (Verner and Blodgett 1931) when severe cracking occurred on the exposed parts of the tree. However, Trought and Lang (1991) observed a significant splitting of cherry fruit on blocks that were protected from rain by plastic covers. They concluded that water uptake through the root system may be of greater significance in causing fruit splitting because the small vapor pressure deficits that occur under covers can reduce transpirational rate, causing fruit growth to increase toward the sum of the transpiration and growth rates. Other field practices recommended for reducing fruit cracking include removing the water drops from the tree after rain using strong wind machines or space heaters (Levin et al. 1959; Bohlmann 1962),

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but there is no evidence that these practices reduce fruit cracking. In a study on the effect of insolation levels on stem-end fruit splitting in 'Gala' apple, Opara (1993) reported that the proportion of fruit with stem-end splitting was significantly higher in well-exposed fruit compared with naturally shaded fruit. Fruit quality attributes were also superior in well-exposed fruit. Therefore, successful application of fruit shading to reduce fruit cracking and splitting will depend largely on the compromise between the level of fruit splitting and changes in other quality attributes of fruit. B. Application of Plant Growth Regulators It has been proposed that cracking occurs due to high tensions in

the fruit skin and the outermost flesh (Verner 1938), as, for example, when the fruit is growing most rapidly in surface area (Skene 1965). This suggests that growth-regulating substances may be applied to fruit to manipulate the growth process and thereby control susceptibility to growth-induced cracking. Due to their ability to modify cuticular and epidermal morphology such as to increase the plasticity of frui tlet skin, researchers have investigated the use of growth hormones such as daminozide (succinic acid 2,2-dimethylhydrazide), gibberellin, promalin (GA 4 +7 + benzyladenine), paclobutrazol, and ethephon ([2-chloroethyl]phosphonic acid) to reduce the incidence of fruit cracking and splitting. 1. Daminozide. Several workers have reported the effectiveness of spray application of daminozide to reduce fruit cracking and splitting in apples (Gardner and Christ 1953; Sullivan and Widmayer 1970; Cobianchi and Rivalta 1974; Kried11974; Comai and Widman 1979; Joosse 1982; Costa et al. 1983). Kriedl (1974) reported a 93% reduction in fruit cracking in 'Stayman Winesap' apples following spray application of daminozide with best results in applications 2 months before harvesting. Daminozide reduced fruit cracking in 'Stayman Winesap' only in some years (Byers 1990; Byers et al. 1990). Costa et al. (1983) reported a 66% reduction in fruit cracking of 'Stayman Red' apples following spray application of daminozide (2000 ppm x 4 times) but there were adverse effects on fruit size, color and yield in the year of application, and in fruit size and shape the following year. Similarly, Joosse (1982) reported that the application of daminozide once at full bloom or in combination with gibberellic acid applied 4 times from petal fall reduced fruit splitting in 'Discovery' apple, but in contrast to the adverse effects on fruit

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quality in 'Stayman Red' apple, daminozide also increased the yields by 1 kg, and by 2 kg in combination with gibberellic acid. Gorini et al. (1982) obtained similar results on plums, including increased flesh softness and total soluble solids. Despite considerable evidence that daminozide is effective in reducing fruit cracking, its application in industry is limited due to the adverse effects on fruit quality, especially when applied at high concentrations. It appears from the results discussed that the extent to which daminozide can reduce fruit cracking and its effects on fruit quality are affected by a combination of factors, including cultivar and the time, number, and rate of applications. 2. Gibberellin. The incidence of fruit cracking and splitting has been markedly reduced in 'Cox's Orange Pippin', 'Discovery', and 'Golden Delicious' apples following gibberellic acid sprays (Joosse 1982; Taylor and Knight 1986). The primary effect of gibberellic acid treatment was considered to be the alleviation of stress within the fruitlet, which reduced the susceptibility to splitting (Taylor and Knight 1986). Byers et al. (1990) found that 4 airblast spray applications of gibberellic acid in July, August, and September 1988 reduced the incidence of cracking from 56 to 21 %, and 5 applications during the same period reduced fruit cracking from 93 to 75%. Combination treatments of gibberellic acid, daminozide, naphthaleneacetic acid (NAA), and Vapor Gard (anti-transpirant) reduced fruit cracking from 93 to 22%. The reduction of fruit cracking by gibberellic acid alone and in various treatment combinations was not consistent in other years (Byers 1990). This inconsistency has also been reported in citrus (Garcia-Luis et al. 1994). Fruit splitting in 'Nova' hybrid mandarin was increased when gibberellin was applied at flowering, but the treatment reduced splitting when applied shortly after the end of fruit drop. Unrath (1991) studied the influence of concentration, spray interval, and number of applications of gibberellic acid on suppression of 'Stayman' fruit cracking and found that 50 ppm concentration at 3-week intervals and the use of 5 applications were superior. Fruit cracking was reduced by more than 80% compared to the no-gibberellin treatment. Cracking and splitting in fruits such as cherry (Looney 1985; Barsy et al. 1988; Perez et al. 1992), pear (Maotani et al. 1990), and pomegranate (Sharifi and Sepahi 1984; Sepahi 1986) have been reduced by spray application of gibberellic acid or attachment of gibberellin tapes on fruit. However, the effectiveness of giberrellin on cherry splitting appears to be cultivar dependent (Webster and Cline 1994b),

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and in pomegranates fruit quality was adversely affected in the following year due to undesirable delay in leaf yellowing (Sepahi and Sharifi 1986). Reduction of fruit cracking in Japanese pear (nashi) following the attachment of gibberellin tapes to the calyx and/or peduncle ends of fruit was attributed to the maintenance of balanced growth by advancing the enlargement of the calyx end (Maotani et al. 1990). In grape berries, gibberellic acid treatment for increasing fruit size also increased the incidence of berry splitting (Hiratsuka et al. 1989; Laszlo and Saayman 1991). 3. Paclobutrazol. Soil application of paclobutrazol (8 mg/tree) significantly reduced fruit cracking in 'Seb' apples (Sankhla et al. 1989) by minimizing excessive moisture and thermal stress. However, spray application at 250 ppm increased the incidence of cracking in 'Niepling Stayman' apples. In sweet cherry, soil application of paclobutrazol at 750-1500 mg/tree reduced fruit cracking and also increased fruit size in the next 2 years (Belmans 1989). The positive influence on fruit quality in the following years was related to high persistence and immobility of paclobutrazol in the soil.

4. Promalin. Trials with promalin failed to reduce fruit cracking in 'Stayman Red' (Costa et al. 1983) and 'Niepling Stayman' apples (Visai et al. 1989), despite very high incidence of fruit cracking dur-

ing the season. Promalin affected fruit shape of 'Niepling Stayman' but did not have significant effects on fruit shape, size, and color of 'Stayman Red' both in the year of application and on returning bloom and fruiting. Visai et al. (1989) attributed the ineffectiveness of promalin to unsuitable timing of treatments, which were applied too early before the period of maximum fruit susceptibility to cracking. In trials to reduce fruit cracking in 'Stayman Red' apple with ethephon, spray application at 50 ppm (4 times) was found ineffective (Costa et al. 1983). Fruit attributes such as shape, color, and size were also not significantly affected. Although some growth regulators have been shown to reduce fruit cracking while others were ineffective, these results must be interpreted with caution because of considerable differences in region, cultivar, method and rate of application, and time of application relative to developmental stage of fruit. For individual growth regulators, the effects of these factors and their interaction need to be established with a consideration of fruit quality attributes so that a more reliable spray strategy can be developed for controlling the occurrence of fruit cracking and splitting.

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V. SUMMARY AND CONCLUSIONS A. General Review The problem of preharvest cracking and splitting occurs widely in many cultivars of apples and other fruits. Published recognition of the problem in a commercial sense dates back to the early part of this century (Kirk 1907; Cunningham 1925; Goodwin 1929). Fruit cracking has been reported from all major fruit growing areas in the world. Various terms have been used to describe the disorder, and most terms reflect the perceived cause or symptom. The term "cracking" has been generally used to refer to many forms of breakage on the fruit surface. In apples, three types of cracking are clearly identifiable: skin cracking, star cracking, and splitting. A practical difference is that a split causes gross exposure of the internal tissue to the atmosphere, whereas in other forms of cracking the defect is contained in the outer cell layers. Each type of crack is most prevalent in particular cultivars, with peculiar mechanisms of occurrence (Skene 1965). Skin cracks occur mainly on the green (shaded) side of the fruit and are most common in 'York Imperial' and 'Cox's Orange Pippin' apple (Fisher 1937a,b; Shutak and Schrader 1948; Goode et al. 1975). Star cracks occur on fruit infected with certain virus diseases (Montgomery 1959; Posnette 1963; Cropley 1968), and 'Cox's Orange Pippin' is more frequently affected than other cultivars (Jenkins and Storey 1955). Fruit splitting occurs mainly on the red (exposed or sunny) side of fruit (Tetley 1930; Verner 1935; Rootsi 1962), and is very common in 'Stayman Winesap', 'Gala', and 'Fuji'. Recently, Opara (1993) reported the occurrence of a type of fruit splitting called stem-end splitti;ng, which originates from the presence of an internal ring crack at the fruitstem interface of 'Gala', 'Royal Gala', and 'Fuji' apples. A review of the literature showed a dearth of information focused toward understanding the phenomenon of stem-end splitting, whereas considerable amount of literature was found on the causes of the other forms of fruit cracking in apples. Frequently, the information in the literature did not clearly differentiate the types of fruit cracking and the word "cracking" was often used as a generic term to refer to several disorders, possibly including stem-end splitting. Fruit cracking occurs sporadically across orchards, seasons, cultivars, trees of the same cultivar, branches of the same tree, and spurs on the same branch. In all types of fruit, the problem has been attributed to a multitude of cultivation, environmental, and fruit internal factors. Viral and fungal diseases have also been associated with fruit

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cracking. There is a general belief that fruits crack when there is a sudden, marked increase in soil moisture content, and atmospheric water content or excess free water on the fruit skin following a period of dry weather. However, experimental results on apple by some researchers failed to confirm this belief (Verner 1935; Watanabe et al. 1987). It is evident from the literature that the causes of cracking cannot be considered sufficiently in terms of environmental conditions alone or in terms of fruit internal conditions alone. Both external and internal influences need to be taken into account. Factors associated with cracking need to be classified into genetic or internal factors (which account for cultivar differences) and external or environmental factors (which influence the degree of splitting within susceptible cultivars). Despite the apparent cUltivar differences on the effect of fruit exposure to sunlight on cracking, researchers agree that both skin cracking (Shutak and Schrader 1948) and splitting (Tetley 1930; Verner 1935) occur on the side that has a thicker inelastic cuticle (shaded or exposed). Efforts to control or reduce fruit cracking in apples and other fruits include cultural measures, and the use of plant regulators and other chemicals that modify the fruit growth process. The degree of success with growth hormones differed considerably between types of chemical applied and was possibly due to time of application. For instance, while daminozide reduced fruit splitting in many studies, trials with promalin failed to reduce it, and paclobutrazol significantly increased it. Most trials that have successfully reduced fruit cracking have not been translated into commercial use due to problems of controlling field conditions. Spray chemicals that reduce cracking also have adverse effects on fruit quality, and may reduce crop yield (Powers and Bollen 1947; Costa et al. 1983). Although differences in cultivar susceptibilities are well known, the possibilities of genetic control of fruit splitting in apples have not been exploited or documented in the literature. To date, there is no guaranteed strategy recommended or widely accepted for commercial growers to control fruit cracking and splitting in apples and other fruits. Harvesting fruit before the onset of cracking and the selection of crack-resistant cultivars offer the best protection against crop damage. Crack-resistant cultivars of many crops, including tomato (Frazier 1947; Reynard 1960; Metcalf et al. 1985; King and Norton 1987; Gardner 1993; Berry et al. 1993), cherry (Christensen 1983), peach (Battistini 1985), apricot (Layne 1991a), and nectarine (Layne 1991b), have been developed by breeding. Most commercial cultivars are still susceptible to cracking damage.

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To date, only the water immersion technique alone (Byers et al. 1990; Opara 1993) or in conjunction with vacuum infiltration (Studman 1994) have been successfully applied to induce fruit cracking in apples. However, the results reported by Opara (1993) showed

that some types of field cracking such as stem-end splits or internal ring cracks were not formed following fruit immersion. These results suggested that water immersion induces only certain types of cracking. Continued advances in our understanding of splitting and cracking in fruits, and apples in particular, have been hindered by many factors. In addition to the well-known difficulties of apples or any biomaterial as an research material, especially in fluctuating weather conditions, there appears to be a general lack of sustained and controlled research studies in this area in comparison to other forms of defect in fruit such as bitter pit or bruising. Although current efforts by apple growers to reduce fruit splitting and cracking may be useful, the development of new cultivars with good consumer acceptance that are susceptible coupled with the continued popularity of susceptible cultivars, especially in the export market, assures a future concern with the problem. Despite the earliest efforts to control fruit splitting and cracking in apples through a series of cultural measures and using growth regulators, there is no guaranteed strategy to control fruit splitting and cracking except selection of crack-resistant cultivars. There is a need for sustained research to gain a better understanding of the relationships between fruit biophysical properties and susceptibility to splitting and cracking in order to facilitate the development of suitable strategies to produce fruit resistant to splitting without compromising the overall quality. B. Prospects for Future Research 1. Assessment and Induction. There is a need for an objective method to assess susceptibility of fruit to cracking and splitting. Such methods would be valuable in assessing new cultivars during breeding or for evaluating existing ones during growth and maturation and under different management conditions. Although previous researchers have induced cracking in detached fruit by submerging fruit in various osmotica alone (Byers et al. 1990) or in combination with vacuum infiltration (Studman 1994), results of laboratory immersion tests using 'Gala' apples showed that fruit developed skin cracks that were distinct from the stem-end splitting that occurs while fruit is on the tree (Opara 1993). Perhaps, more attention needs to be

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given to identifying tests that measure some mechanical or biophysical properties of fruit. 2. Developmental Studies. There is a need to determine the onset and chronological development of fruit cracking or splitting in susceptible cultivars using tagged random fruit samples. This will help us to understand the critical growth period when fruit splitting occurs and the development of suitable management strategies for reducing or controlling fruit splitting. Management practices such as fertilization, thinning, nutrient sprays, and irrigation can be timely applied to provide optimum benefits in reducing fruit cracking and splitting. Studies on 'Gala' apple have shown that the development of fruit splitting is related to the presence of imbalances in fruit growth rates (Opara 1993). Although this provides useful information on the mechanism of fruit splitting in 'Gala' apple, it does not explain why this cultivar is particularly susceptible to stem-end splitting. Further studies are required to understand the relationships between splitting and fruit internal structures, such as cell size, cell shape and orientation, and intercellular space, which could provide an understanding of differences in resistance to splitting among cultivars. A more complete understanding of the processes that determine size and shape in fruits requires elucidation of the sites and orientation of cells (Green 1976). For instance, studies by Vincent (1989,1990) and Vincent et al. (1991) have shown that in parenchymatous tissues, the mode of failure can be dependent on the anisotropic arrangement of the cells, the presence of air spaces, and the ratio of thickness of the cell wall to the diameter of the cell. Simi1arly' histological studies in grapes by Meynhardt (1964a) indicated that in cultivars susceptible to berry splitting, the ratio between the longitudinal to radial subepidermal cell dimensions of the berry was comparatively small and such berries usually had an epidermal cell layer consisting of relatively few cells. Further work is needed to understand how stresses develop at parts of fruit where splitting occurs. This may help explain, in part, how any differences in shape and other physical attributes of cultivars may be related to susceptibility to splitting. In grapes, theoretical analysis of surface growth forces suggested that fruit shape and structural attributes can cause stresses that affect the occurrence of rain-induced splitting (Considine 1979; Considine and Brown 1981). 3. Control. There is a need for better understanding of the relationships between overall fruit quality, growth characteristics, and

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orchard management practices that affect the incidence of fruit splitting and cracking. In view of the many external and internal factors that are associated with high incidence of fruit cracking and splitting, experimental studies should determine how these factors interact with fruit growth rates to induce stem-end splitting. Of particular interest are the effects of frequent water supply, low crop load, and degree of fruit exposure on growth rates, and the effects of irrigation treatments applied during the critical growth period when ring cracking and stem-end splitting are initiated. This information could be used to develop recommendations to reduce the incidence of stem-end splitting in apples. To eliminate the problem of fruit cracking in apples, it important to understand why certain cultivars resist cracking while others are susceptible. A study is needed to identify fruit properties and biophysical attributes that govern resistance and susceptibility to cracking and splitting. Finally, the inheritance of fruit cracking needs to be determined. LITERATURE CITED Anderson, P. C. and D. G. Richardson. 1982. A rapid method to estimate fruit water status with special reference to rain cracking of sweet cherries. J. Amer. Soc. Hart. Sci. Vol. 107:441-444. Anon. 1961. p. 9. In: Report of East MaIling Research Station for 1960. Anon. 1962a. p. 9-10. In: Report of East MaIling Research Station for 1961. Anon. 1962b. Horticulture. Results of research in 1961. p. 52-61. In: 74th Annual Report, Kentucky Agr. Exp. Sta. Armstrong, R. J. and A. E. Thompson. 1967. A diallel analysis of tomato fruit cracking. Proc. Am. Soc. Hort. Sci. 91:505-513. Armstrong, R. J. and A. E. Thompson. 1969. A rapid and accurate system for scoring tomato fruit cracking. HortScience 4:288-290. Asquith, D. 1957. Some results on the control of the red-banded leaf roller and the two-spotted mite. Pennsylvania State Hort. Assoc. News 36: 39, 41-45. Bangerth, F. 1973. Investigations upon Ca-related physiological disorders. Phytopath. Z. 77:20-37. Bangerth, F. 1979. Calcium-related physiological disorders of plants. Annu. Rev. Phytopathol. 17:97-122. Barsy, T. de., R. Bronchat, K. Belmans, and J. Keulemans. 1988. GA a on the level of splitting in cherries cv Brabanders and on the morphology of their epidermis. Arch. Int. Physiol. Biochim. 96: 6. Batal, K. M., J. L. Weigle, and D. C. Foley. 1970. Relation of strain-stress properties of tomato skin to cracking of tomato fruit. HortScience 5:223-224. Battistini, M. 1985. Springbelle: a new peach cultivar with yellow flesh. Riv. Frutticoltura Ortofloricoltura. 47:16-19. Beattie, B. B., W. B. McGlasson, and N. L. Wade. 1989. Postharvest diseases of horticultural produce, vol. 1: temperate fruit. NSW Agr. and Fisheries, CSIRO, Australia.

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Belmans, K. 1989. Study of growth, yield and fruit quality of sweet cherry, cv. Hedelfinger R. after soil application of paclobutrazol. Acta Hort. 239:443-446. Bernstein, Z., and 1. Lustig. 1985. Hydrostatic methods and measurement of firmness and turgor pressure of grape berries (Vilis vinifera L.). Sci. Hort. 25: 129136. Berry, S. Z., K. L. Wiese, and T. S. Aidrich. 1993. 'Ohio 8556' processing tomato. HortScience 28: 751. Bijl, van der P. A. 1914. "Apple cracking" and "apple branch blister": caused by the fungus Coniothecium chomatosporum, Corda. Agr. J. Union S. Africa. 8: 64-69. Bohlmann, T. E. 1962. Why does fruit crack? Farming S. Africa. 38:12-13. Brown, H. D., and C. V. Price. 1934. Effect of irrigation, degree of maturity, and shading upon the yield and degree of cracking of tomatoes. Proc. Am. Soc. Hort. Sci. 32:524-528. Bullock, R. M. 1952. A study of some inorganic compounds and growth promoting chemicals in relation to fruit cracking of Bing cherries at maturity. Proc. Am. Soc. Hort. Sci. 59:243-253. Byers, R. E. 1990. 'Stayman' fruit cracking as affected by surfactants, plant growth regulators, and other chemicals. Pennsylvania Fruit News. 70:4, 28-30. Byers, R. K, D. H. Carbaugh, and C. N. Presley. 1990. 'Stayman' fruit cracking as affected by surfactants, plant growth regulators, and other chemicals. J. Am. Soc. Hort. Sci. Vol. 115:405-411. Callan, N. W. 1986. Calcium hydroxide reduces splitting of 'Lambert' sweet cherry. J. Am. Soc. Hort. Sci. 111:173-175. Campbell, J. A. 1928. Cracking of Dunn's and Cox's orange apples: investigations in Nelson District. New Zealand J. Agr. 85-86. Carne, W. M. 1925. Cracking and rusetting of Dunn's and other apples. J. Dept. W. Australia. 2d. Ser. 2, 214. Ceponis, M. J., R. A. Cappellini, and G. W. Lightner. 1987. Disorders of sweet cherry and strawberry shipments to the New York market, 1972-1984. Plant Dis. 71:472475. Chandler, W. H. 1925. Fruit growing. Houghton Mifflin, Boston. Christensen, J. V. 1968. A study of some factors responsible for the cracking of cherries. 1.S.H.S. Symposium on cherries. Bonn. Christensen, J. V. 1970a. Numerisk undersogelse af morfologiske kendetegn hos sodkirsebaer. Bestemmelsesnogle til 34 sorter. (Numerical studies of morphological distinction marks in sweet cherry cultivars: identification key for 34 cultivars.) Tidsskr. Planteavl 74:44-74. Christensen, J. V. 1970b. Sortsforsog med sodkirsebaer. (Cultivar trial with sweet cherries.) Tidsskr. Planteavl 74:301-312. Christensen, J. V. 1972a. Revner i kirsebaer 1. Rytme og hastighed af frugternes vandoptagelse i relation til revnetilbojelighed. (Cracking in cherries, I: fluctuation and rate of water absorption in relation to cracking susceptibility.) Tidsskr. Planteavl 76:1-5. Christensen, J. V. 1972b. Revner i kirsebaer II. Klimaets indflydelse fa revnetilbojelighed. (Cracking in cherries,II: the influence of climatic conditions on cracking susceptibility). Tidsskr. Planteavl 76:191-195. Christensen, J. V. 1972c. Cracking in cherries, III: determination of cracking susceptibility. Acta Agr. Scand. 22:128-136. Christensen, J. V. 1972d. Cracking in cherries, IV: physiological studies ofthe mechanism of cracking. Acta Agr. Scand. 22:153-162.

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Cline, J. and T. Webster. 1994. Cherries under wraps. Grower 24:16-17. Cobianchi, D., and L. Rivalta. 1974. L'impiego dell' Alar contro la "spaccatura" delle mele Stayman. L'Inf. Agr. 14729-14731. Cobianchi, D., W. Faedi, and M. Biguzzi. 1984. The effect of clonal rootstocks, a seedling, and of interstocks on the vegetative and productive behaviour of Stayman Winesap. Ann. dell'Istituto Sperimentale Frutticoltura 15:115-136. Coit, J. E. 1917. Citrus fruits, an account of the citrus fruit industry, with special reference to California requirements and practices and similar conditions. The Macmillan Co., New York. Comai, M., and 1. Widman. 1979. Esperienze per contenere la spaccatura dei frutti di "Stayman Red". Esperienze e Ricerhe. Staz. Spero Agr. Forest. S. Michele all'Adige. 9:55-59. Comai, M., and L. Widman. 1981. Comparison of Stayman clones on various rootstocks. Esperienze e Ricerhe. Staz. Spero Agr. Forest. S. Michele all' Adige 11:33-45.

Considine, J. A. 1979. Biophysics and histochemistry of fruit growth and development. Thesis, Univ. Melbourne, Australia. Considine, J. A. 1982. Physical aspects of fruit growth: cuticular fracture and fracture patterns in relation to fruit structure in Vitis vinifera. J. Hort. Sci. 57:79-91. Considine, J. A., and K. Brown. 1981. Physical aspects of fruit growth: theoretical analysis of distribution of surface growth forces in fruit in relation to cracking and splitting. Plant Physiol. 68:371-376. Considine, J. A., and P. E. Kriedman. 1972. Fruit splitting in grapes: determination of the critical turgor pressure. Australian J. Agr. Res. 23:17-24. Considine, J. A., J. F. Williams, and K. C. Brown. 1974. A model of studies on stress in dermal tissues of mature fruit of Vitis vinifera : criteria for producing fruit resistant to cracking. p. 611-617. In: R. L. Bieleski et al. (eds.), Mechanisms of regulation of plant growth, Bull. 12. The Royal Society of New Zealand, Wellington. Costa, G., C. Giulivo, and A. Ramina. 1983. Influence of growth regulators on apple fruit cracking (cv "Stayman Red"). Acta Hort. 137:366-369. Cortner, S. D., E. E. Burns, and P. W. Leeper. 1969. Pericarp anatomy of crack-resistant and susceptible tomato fruit. J. Am. Soc. Hort. Sci. 94:136-137. Cropley, R. 1963. Apple star crack. In: A. F. Posnette (ed.), Virus diseases of apples and pears. Tech. Commun. 30. Commonwealth Bureaux Hort. Plantation Crops. Cropley, R. 1968. Varietal reactions to viruses causing star crack and russet rings on apple fruits. J. Hort. Sci. 43:157-165. Cunningham, G. H. 1925. Fungous diseases of fruit trees (Blister-Disease, Coniothecum chomatosporum Corda). Auckland, New Zealand. p. 137-143. Davenport, D. C., K. Uriu, and R. M. Hagan. 1972. Antitranspirant film: curtailing intake of external water by cherry fruit to reduce cracking. HortScience 7:507508.

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DeEll, J. R., and R. K. Prange. 1993. Postharvest physiological disorders, diseases and mineral concentrations of organically and conventionally grown McIntosh and Cortland apples. Can. J. Plant Sci. 73:223-230. Dickinson, D. B., and J. P. McCollum. 1964. The effect of calcium on cracking on tomato fruits. Proc. Am. Soc. Hort. Sci. 84:485-490. Dube, S. D., J. D. Tewari, and C. B. Ram. 1969. Boron deficiency in Rymer apple. Prog. Hort. 1:33-36. Evans, 1. B. P. 1907. Notes on plant diseases. I: An apple disease (Coniothecum chomatosporum, Corda). Transvaal Agric. J. Vol. V. no. 19:680-681. Fawcett, H. S., and H. A. Lee. 1926. Citrus diseases and their control. McGraw-Hill Book Co., New York. Fischer, H. 1955. Unusual russeting and cracking in Boskoop, Glockenapfel and other apple varieties, a virus disease? Schweiz. Z. Obst-u. Weinb., 64:125-131. [Hort. Abstr. 25:2609.] Fisher, D. F. 1937a. York skin crack, hydrochloric acid injury and heat cracking. Am. Fruit Grower 57:11-16. Fisher, D. F. 1937b. Present equipment and methods for effective and safe washing of eastern apples. Proc. Maryland Sta. Hort. Soc. 31:8-14. Flore, J. A., and F. G. Dennis. 1990. Disorders caused by environmental factors. p. 84-86. In: A. L. Jones and H. S. Aldwinckle (eds.), Compendium of apple and pear diseases. APS Press, St. Paul, MN Am. Phytopath. Soc. Frazier, W. A. 1934. A study of some factors associated with the occurrence of cracks in the tomato fruit. Proc. Am. Soc. Hort. Sci. 32:519-523. Frazier, W. A. 1947. A final report on studies of tomato fruit cracking in Maryland. Proc. Am. Soc. Hort. Sci. 49:241-255. Frazier, W. A. 1951. A stock with wide calyx base and resistant to cracking. TGC Report No.1, p. 5. Frazier, W. A. 1958. Resistance to cracking. TGC Report No.8, p. 15. Frazier, W. A. 1959. Resistance to cracking. p. 32-33. TGC Rep. 9. Frazier, W. A., and J. L. Bowers. 1947. A final report on studies of tomato fruit cracking in Maryland. Proc. Am. Soc. Hort. Sci. 49:241-255. Garcia-Luis, A., A. M. M. Duarte, 1. Porras, A. Garcia-Lidon, and J. L. Guardiola. 1994. Fruit splitting in 'Nova' hybrid mandarin in relation to the anatomy of the fruit and fruit set treatments. Sci. Hort. 57:215-231. Gardner, R. G. 1993. 'Mountain Gold' tomato. HortScience 28:348-349. Gardner, V. R., and K G. Christ. 1953. Studies on cracking in the Stayman apple. Hort. News 34:2701, 2710-12. Gardner, V. R., F. C. Bradford and H. D. Hooker. 1927. Orcharding. McGraw-Hill Book Co., New York. Gilmer, R. M. and J. Einset. 1959. Some bud mutations of apple with virus-like symptoms. Plant Dis. Reptr., 43:264-269. Goldschmidt, K 1962. The anatomical-morphological causes of cracking in apples (in Swedish). Sver. pomol. Foren Arsskr 63:67-72. [Hort. Abstr. 33:4523.] Goode, J. K, M. M. Fuller, and K. J. Hyrycz. 1975. Skin-cracking of Cox's Orange Pippin apples in relation to water stress. J. Hort. Sci. 50:265-269. Goodwin, B. G. 1929. Blister disease or cracking of apples. Successful remedial measures in Nelson District. New Zealand J. Agr. p. 305-307. Gorini, F. L., A. Sozzi, and G. Spada. 1982. The effects of the growth regulators SADH and CEPA on the quality of fresh and dried Stanley plums. Annali dell

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Magness, J. R., and H. C. DiehL 1924. Physical studies on apples in storage. J. Agr. Res. 27:1-38. Malhotra, V. K., H. N. Khajuria, and J. S. Jawanda. 1983. Punjab Hart. J. 23:153-157. Maotani, T., A. Suzuki, K. Tanaka, K. Kimura, T. Sugiura, O. Kumamoto, T. Nishimura, K. Oshima, and T. Masada. 1990. Control of fruit cracking ofJapanese pear 'Kosui' and 'Niitaka' using gibberellin tape. J. Jpn. Soc. Hart. Sci. 58:859-863. Marini, RP. 1991. Mid-Atlantic apple cultivars. Fruit Var. J. 45:95-97. Marias, J. J., A. M. Bledsoe, R. M. Bostock, and W. D. Gubler. 1987. Effects of spray adjuvants on development of Botrytis cinerea on Vitis vinifera berries. Phytopathology 77:1148-1152. McGarry, A. 1993. Influence of water status on carrot (Daucus carota L.) fracture properties. J. Hort. Sci. 68:431-437. Mendoza, H. A., and D. T. Ortiz. 1984. Identification and evaluation of disease problems in pear (Pyrus communis) in EI Ejido Ocoxaltepec, Ocuituco, and Morelos. Agrociencia (Mexico) 56:9-18. Metcalf, J. G., A. A. Reyes, and W. P. Mohr. 1985. 'Earlirouge', and early fresh market red tomato. HortScience 20:788. Meyer. A. 1944. A study of the skin structure of Golden Delicious apples. Proc. Am. Soc. Hart. Sci. 45:105-110. Meynhardt, J. T. 1957. Does spray irrigation cause berry cracking in grapes? Farming S. Afr. 32:6-9. Meynhardt, J. T. 1964a. A histological study of berry-splitting in some grape cultivars. S. Afr. J. Agr. Sci. 7:707-716. Meynhardt, J. T. 1964b. Some studies on berry-splitting of Queen of the Vineyard grapes. S. Afr. J. Agr. Sci. 7:179-186. Mezzetti, A. 1959. Considerazioni suI meccanismo ch generale spaccature delle mele senescenti. (Observations on the causal mechanism of cracking in over-ripe apples.) Frutticoltura 21:6311-6333. [Hart. Abstr. 30:1770.] Milad, R E., and K. A. ShackeL 1992. Water relations of fruit end cracking in French prune (Prunus domestica 1.cv. French). J. Am. Soc. Hort. Sci. 117:824-828. Mohsenin, N. N. 1972. Mechanical properties of fruits and vegetables: review of a decade of research applications and needs. Trans. Am. Soc. Agr. Eng. 15:10641070. Montgomery, H. B. S. 1959. Russeting and cracking of Cox's Orange Pippin apples. p. 163-164. In: Rep. East MalL Res. Sta. for 1958. Moore, M. H. 1931. Investigations on Coniothecum. A progress report. p. 150-156. In: Annual Report for 1928, 1929, and 1930 II supplement. East Mall. Res. Sta. Mrozek, R F., and T. H. Burkhardt. 1973. Factors causing prune side cracking. Trans. Am. Soc. Agr. Eng. 16:686-692. Niiuchi, K., F. Honda, and S. Ota. 1960. Studies on cracking in tomato fruit, 1: mechanisms of fruit cracking. J. Hart. Assoc. Jpn. 29:287-293. [Hart. Abstr. 31:6464.] Nikitina, K. V. 1959. The keeping quality of apples (in Russian). Sadi i Ogorod 9: 56-9. [Hart. Abstr. 30:1769.] Nilsson, F., and B. Bjurman. 1958. Sprickor pa Ingrid Marie. (Fruit cracking in Ingrid Marie.) Sver. pomoL Foren. Arsskr. [Hort. Abstr. 29:2266.] Nilsson, F., and 1. Fernqvist. 1956. Sprickbildning hos Ingrid Marie. (Fruit cracking in Ingrid Marie.) Sver. pomoL Foren. Arsskr. [Hart. Abstr. 27:2251.] Noga, G. J., and M. J. Bukovac. 1986. Impact of surfactants an fruit quality of 'Schatten Morelle' sour cherries and 'Golden Delicious' apples. Acta Hart. 179:771-777. Noga, G., and M. Wolter. 1990. Surfactants as a cause ofrusseting in apples. Gartenbau wissenscaft 55(1):20-26. [Hart. Abstr. 60:9586.]

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5

Origin and Dissemination of Cherry Miklos Faust Fruit Laboratory Beltsville Agricultural Research Center Agricultural Research Service Beltsville, Maryland, USA Dezso Suranyi Fruit Research Station Cegled, Hungary

I. II.

Introduction Classification of Cultivated Cherries A. Classification of Sweet Cherries B. Classification of Sour Cherries C. Classification of Ornamental Cherries III. The Native Home of Sweet and Sour Cherries A. Sweet Cherry B. Sour Cherry IV. Early Records of Cherry Cultivars A. Archeological Findings B. Period Before the Sixteenth Century C. Period From 1600 to 1800 D. Cherries in America V. The Modern Era, Cherries in the Twentieth Century VI. Cultivar Improvement VII. Rootstock Improvement VIII. Japanese Cherries and Their Movement Into America IX. Worldwide Dissemination and Production of Cherries X. Conclusions Literature Cited

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I. INTRODUCTION Cherry is the common name of several species of the genus Prunus. Among cheries, the sweet cherry, sour cherry, flowering ornamental cherry species, and a few other Prunus species used as rootstocks for cherries are important. The cherry is revered for its fruit and its blossoms. It is an elegant fruit. Some are eaten fresh, others in the form of soup, tart, pie, candied, or covered with chocolate. Liqueurs or wines are made from cherries called kirschwasser in Germany, maraschino in Dalmatia, or cherry kijafa in Danmark. If we consider that cherries are consumed largely in the country of their production, then the consumption ranges from about 1.5 kg per person in the United States to 6 kg per person in Hungary. All other major cherry-producing countries fall within this range. Even the lowest rate of consumption is quite high for a fruit considered as a gourmet item. Cherries are also a source of beauty, being one of the most spectacular flowering trees. Cultivars with double flowers are adored for their grace. The single-flowered ones, with flowers lasting only for a short time, suggest the transient nature of life. In Japan, cherry flowers have an exalted place in the gardens, in public parks, in villages, and in the countryside. Although, we value cherries as a gourmet fruit, an outstanding drink, or a source of beauty, we know relatively little about their origin, development, and dissemination during the course of history. The only comprehensive review of the history of cherries was done by Hedrick in 1915. Since then many new archeological and historical facts were uncovered that justify this review on the origin and dissemination of cherry. A brief review was also published by Webster in 1995. II. CLASSIFICATION OF CULTIVATED CHERRIES Throughout the years botanists have continually changed the classification of plants, including those belonging to the genus Prunus. In 1912, Koehne, the most recent monographer of Prunus, described 119 species. L. H. Bailey (1927) remarked that the number of species in the genus Prunus is probably around 175, but described only 81 in his Standard Cyclopedia of Horticulture. Rehder (1958) reduced the number of species even further, recognizing only 71 species. The number of species belonging to the subgenus Cerasus, the cherries, is relatively small. For clarity, the classification of genus Prunus, simplified after L. H. Bailey (1927) and Rehder (1958), is repeated here.

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The four subgenera in Prunus are determined basically by how the leaves are rolled up in the bud, if the fruit are hairy or not, and if the flowers are in cymes or in racemes: Leaves are convolute (rolled up) in the bud. I. Prunophora, plums, prunes, and apricots. AA. Leaves are conduplicate (folded lengthwise along the midrib) in the bud. B. Fruit is normally hairy, stone is normally furrowed. Flowers apear before the leaves. II. Amygdalus, almonds and peaches. BB. Fruit is normally glabrous or very slightly hairy, stone is smooth. Flowers appear with the leaves: Flowers are in racemes of few flowers, fascicles or cymes. III. Cerasus, cherries. Flowers are in racemes. IV. Padus, racemose cherries. A.

Our concern here is with the subgenus Cerasus. This subgenus is further subdivided into sections. The sections and selected species important in horticulture are given below: Section 1. Microcerasus Koehne. Flowers are solitary or in short fewflowered racemes. Leafaxils with 3 buds. Microcerasus is considered intermediate between cherries (Cerasus), plums (Prunophora), and apricots (Amygdalus). They can be crossed with cherries and used in breeding programs as sources of stress resistance. Cherries are also graftable on Microcerasus. Prunus pumila 1. (Eastern North America), sand cherry. Prunus besseyi Bailey (Eastern North America), western sand cherry. Prunus tomentosa Thunb. Downy Cherry (Northern China and Manchuria, the Himalayas.); sprawling shrub, used mostly as dwarfing rootstock for cherries. Section 2. Pseudocerasus Koehne. As section Microcerasus but sepals upright or spreading and buds are solitary. Flowers are in bunches of few-flowered short racemes. A high proportion of the ornamental flowering cherries are derived from the Chinese and Japanese species of this section or from their hybrids. Prunus serrulata Lind!. (China), Japanese flowering cherry. There are many botanical varieties of this species.

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Prunus pseudocerasus Lind!. (China) grown for its fruit, which is similar but smaller than P. Canescens. Hybridized with P. avium to produce rootstocks, such as 'Colt' for sweet and sour cherries. Prunus donarium Siebold (Japan), Japanese flowering cherry. Bailey considered it a variant of P. lannesiana, but Jefferson and Wein (1984) accepted it as a separate species. Prunus lannesiana Wilson (Japan), Japanese flowering cherry. Prunus sieboldii Wittm. (Japan), Japanese flowering cherry. Section 3. Eurocerasus Koehne. Sepals are upright or spreading, buds are solitary. Flowers are usually in sessile umbels with persistent budscales present at base. Prunus fruticosa Pallas (Europe to Siberia), ground cherry used as edible fruit in limited quantities. Prunus cerasus 1. (Europe, Western Asia), sour cherry. Tree is suckering. Prunus avium 1. ((Europe, Western Asia), sweet cherry. There are many ornamental forms of P. avium: var. pyramidalis Hort., var. pendula Hort., var. asplenifolia Kirchn., var. plena Hort., var. salicifolia Dipp. and others. Tree is not suckering. (Hort. means common usage by horticulturists) Section 4. Mabaleb Koehne Prunus mabaleb L. (Europe, Western Asia), mahaleb cherry. Used as rootstocks for cherries. Several classifiers distinguished P. canescens Bois (Central and Western China) in Eurocerasus as a separate species. P. canescens appears similar to P. cerasus but with smaller fruit. Rehder (1958) did not recognize it as a separate species and listed only three species under Eurocerasus. Watkins (1981) followed this classification. According to Schmidt (Iezzoni et al. 1992), P. canescens behaves as P. cerasus in breeding and should not be a separate species. There are several other Prunus species, listed by Iezzoni et al. (1992), belonging to the above sections, that are potentially useful in rootstock development or development of cultivars with stress tolerance. They are not considered here because their limited utilization.

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A. Classification of Sweet Cherries The sweet cherry, Prunus avium, was named by Linneaus in 1755. It is commonly called the sweet cherry, Mazzard, or Gean cherry. Although avium means birds, De Candolle was the first to use the name bird cherry. Sweet cherries are subdivided by horticulturists into two groups based on the firmness of the flesh of the fruit: 1.

2.

Flesh is tender. This group is known as the Heart cherries, the French Guigne, or the English Gean. The soft-fruited cherries can be subdivided even further into dark-colored cultivars with reddish juice and light-colored cultivars with colorless juice. It is the light-colored group that Linnaeus gave the botanical variety name Juliana. Augustin Pyramus De Candolle elevated the Heart cherries to species status and named them Cerasus Juliana. The flesh is firm. These cherries are called Bigarreau in several languages. The name originally referred to the diverse colors of the fruit (Hedrick 1915), stemming from bigarr(e), meaning variegated.

Several forms of P. avium are used as ornamentals and many latinized common names are used to designate these ornamental variants. One of the major points of controversy was the genus into which sweet cherry was placed. Some used the genus Prunus as an inclusive genus name, other used Cerasus as a restrictive genus designation. Botanists described variations in flesh firmness and leaf shape and gave each form a name, which led to the development of a large number of synonyms. Linneaus called these variations Prunus avium duracina. The first name change came in 1768 when Phillip Miller, an English botanist, changed the name of the sweet cherry to Cerasus nigra. In 1790, Friedrich Erhardt, a German botanist, changed the name and split the species into P. nigricans and P. varia. In 1794, Konrad Moench of Germany called it C. avium. In 1796, Moritz Balthasar Borkhausen, a German botanist, used the name C. varia. The heart cherries were classified by Linneaus as P. cerasus var. Juliana, and Augustin Pyramus De Candolle changed the name to C. Juliana. In 1805, Heinrich Gottlieb Ludwig Reichenbach, a German botanist, introduced P. Juliana, and L.H. Bailey returned it to botanical variety status as P. avium var. Juliana. In 1805, Augustin Pyramus De Candolle also distinguished the Bigarreau cherries and named them C. duracina. In 1807, Christian Hendrick Persoon, a German botanist, termed the sweet cherry P. silvestris, and in 1812, Nicolaus

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Thomas Host, a German botanist, designated it C. intermedia. In 1927, Robert Sweet, an English botanist, called it C. macrophylla, and in 1832, Heinrich Gottlieb Ludwig Reichenbach introduced the name P. dulcis. In 1840, Ernst Gottlieb Steudel used the name C. dulcis. In 1847, M.J. Roemer designated C. bigarella and C. padilla, and in 1869, Karl Koch described three horticultural variants: C. heteropylla, C. asplenifolia, and C. salicifolia. B. Classification of Sour Cherries

The sour cherry, Prunus cerasus, was named by Linneaus in 1753. He thought that the two common groups of sour cherries, those with colorless juice and those with colored juice, were sufficiently different to be botanical varieties and designated them Prunus cerasus caproniana and Prunus cerasus austera, respectively. Other botanists started to change the name of sour cherry soon after Linneaus classified it. The botanical designation of sour cherries, similarly to sweet cherries, expressed the Prunus/Ceras us point of views. Added to the confusion was that, although various types of sour cherries exist, they mayor may not deserve to have species status in the opinion of the classifier. Among sour cherries the commonly recognized types are as follows: 1.

2.

3.

4.

Trees are moderate to small in stature, with hanging branches. They develop many suckers, fruit is acidic, with highly colored, staining juice. They are often designated as acida, frutescens, or collina. Trees are larger, fruit is light colored, juice is relatively light colored or colorless and nonstaining. They are called the Amarelles, from the Latin word for bitter. The English call them Kentish cherries, and the French call them Ceriser Commun. They are also designated as vulgaris, cerasus, or caproniana. Tree is relatively large, fruits are relatively sweet and located on long slender peduncules, juice is dark red. They are the Morellos or the Griottes of the French. Morello means blackish in Italian and Griotte is probably derived from agriotte, from aigre, meaning sharp, in reference to the acidity of the cherries (Hedrick 1915). This type is often designated as morello, austera, or austeza. Trees are small, branching is upright, fruits are high quality, bitterish, and located on short peduncules. Designated as

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marasca. Marasca is a derivative of amaro or the Latin amarus, meaning bitter. It is not surprising, that with such variation in fruit, botanical clas-

sifiers used many combinations of names. After the original designation by Linnaeus in 1753 as Prunus cerasus, the name changed in almost every possible way. In 1768, Phillip Miller, an English botanist, called the sour cherry types Cerasus vulgaris and C. hortenses. In 1790, Friedrich Erhardt, a German botanist, named them P. acida and P. austera. In 1796, Richard Anthony Salisbury, an English botanist, called the sour cherry P. aestiva and in the same year Moritz Balthasar Borkhausen, a German botanist, named them C. aeida and C. austera. In 1804, Jean Louis Marie Poiret, a French botanist, used the names P. plena and P. rosea. In 1805, Augustin Pyramus De Candolle termed the sour cherry C. caproniana. In 1827, Barthelemy Charles Dumortier, a Belgian botanist, used the name C. bigarella, and in 1831, Nicolaus Thomas Host, a German botanist, designated two sour cherries, C. effusa and C. Marasca. In 1832, Heinrich Gottlieb Ludwig Reichenbach introduced the name P. marasca. In 1843, Bechstein called the sour cherry P. oxycarpa, Wilhelm Gerhard Walpers named it C. bungei and in 1847, M. J. Roemer, a Swiss botanist, used the two names C. heaumiana and C. tridentia. In 1866, Philipp Johann Ferdinand Schur, a German botanist, named it P. vulgaris and in 1868, Louis Van Houtte used the name C. rhexii. In 1869, Karl Koch, a German botanist, designated it as C. cucullata. Hybrids of P. avium x P. cerasus, called Duke cherries, are classified as C. regalis Poit. & Turp. 1. H. Bailey (1927) believed the Duke cherry to be a botanical variety and classified it as P. avium var. regalisBailey. The name was changed by Rehder to Prunus x gondouinni Rehd. Duke cherries are tetraploids and presumed to arise from pollination of sour cherry by an unreduced (2n) gamete of sweet cherry (Iezzoni et a1. 1992). Although there is great variation among Duke cherries, they resemble the sweet cherry in appearance but have a tart flavor inherited from sour cherry. Sterility is a commonly associated with the hybrid nature of these cherries. There are dark-colored Dukes with red juice and light-colored Dukes with colorless juice.

C. Classification of Ornamental Cherries The ornamental Japanese cherries represent a separate group and are mostly natives of Eastern Asia. The basic species of Japanese cherries, P. serrulata, was classified by John Lindley (1799-1865),

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an English botanist. Weitch in 1906 and Koehne in 1909 attempted to clarify the classification of Japanese cherries. Hedrick (1915) listed the partial classification of Koehne, giving 61 names that can be regarded as synonyms or variants under P. serrulata. P. serrulata was also called C. serrulata by Elie Abel Carriere (1816-1896),P. mutabilis by Miyoshi, and by many horticulturists as a partially described species P. pseudocerasus Hort. Koidzumi in 1913, Miyoshi, and separately Wilson in 1916 and Miyoshi again in 1921 attempted to clarify the nomenclature of this difficult-to-classify group of cherries. L.H. Bailey (1927) included many previously described species as botanical varieties in P. serrulata, viz. var. spontanea, Wilson, with several forms that include f. Shidare-Sakura Koehne, f. humilis, Wilson, f. Kosioyama, Wilson, and f. praecox, Wilson. He abolished a number of species, P. tenuiflora, P. Levelliana, P. mesadenia, P. Veitchii, P. verecunda, and P. quelpaertensis, and included them in P. serrulata as var. pubescent. Earnest H. Wilson, a collector of oriental plants, also recognized several forms of the botanical variety pubescence: f. sancta, f. Shibayama, and f. Taizanfunkun. Bailey also accepted the determination of Makimo that P. sachaliensis, previously named P. pseudocerasus var. sachaliensis, Schmidt, P. sachaliensis, Koidz., P. Sargentii, Rhed., and P. floribunda, Koehne, should be a botanical variety within the species of P. serrulata. There were further classification attempts, in 1934 by Russel, in 1948 by Ingram, in 1950 by Hara, in 1961 by Sano, in 1963 by Makino, in 1973 by Ohwi and Ohta, in 1974 by Honda and Hayaski, and in 1976 by Gashu, who undertook the placement of various cultivars into the right species. For these classifications see Jefferson and Wain (1984). There are other basic species of Japanese cherries into which cultivars can be classified, which confuses the situation even further. One such species is P. lannesiana Wilson. This species also was named C. lannesiana by Elie Abel Carriere, P. serrulata lannesiana by Koehne, P. pseudocerasus var. hortensis by Karl Johann Maximovitz (1827-1891) and P. donarium by Philipp Franz von Siebold (1796-1866). There are many forms in P. lannesiana listed by Wilson similar to those found in other species. P. sieboldii is another species of Japanese cherry originally described by Max Karl Ludwig Wittmack, editor of Gartenflora and a professor at Berlin. This species also was described as C. sieboldii, by Elie Abel Carriere and as P. pseudocerasus var. sieboldii by Karl Johann Maximovitz. Jefferson and Wain (1984) recognized a serious nomenclature problem among the Japanese cherries. They realized that the taxonomic

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assignment of cultivars to wild Japanese species is inappropriate. Several of the Japanese cherries are indigenous to Japan or escaped from early cultivation. Many of the cherries that escaped from cultivation in Japan are the progenies or hybrids of species planted in mountainous areas for the horticultural interest of "cherry viewing" that started around 800. The earliest references on "cherry viewing" date back to 720 (Jefferson and Wain 1984). Japanese horticulturists selected ornamental mutations and perhaps hybridized desirable trees to improve their ornamental values. Between 1600 and 1867, Japanese gardeners selected many variants from the "wild" with ornamental merit. Mizuno in 1681 and 1716 in Kadan Komuko listed 40 cultivars, many of which are still in existence today (Jefferson and Wain 1984). Honda and Hayashi (1974) went even further and placed the origin of at least 150 ornamental cultivars between 794 and 1192 when many cherry trees were planted in the gardens. The Japanese established two terms to differentiate among cherries: mountain cherries (Yama-zakura) and village cherries (Sato-zakura). Jefferson and Wain (1984) proposed that the Japanese ornamental cherries are obviously horticultural mixtures of species. They should not be assigned to anyone species, but handled as the" Sato-zakura group," signifying that these are cultivated ornamental cherries. Placing the flowering cherries in a group clearly sets them apart from all other botanical taxa of Prunus. They proposed that the designation for each form should be Prunus (Sato-zakura group) cv. Fugenz6, as an example. III. THE NATIVE HOME OF SWEET AND SOUR CHERRIES The earliest description of the "cherry" comes from Theophrastus in ca. 300 B.C. He described a large tree with round, red fruit. From this description De Candolle (1986) concluded that Theophrastus described the sweet cherry. The name Theophrastus used for the cherry was kerasos. The Greek kerasia may had originated from the name of the town Kerasun, Pontus, on the Black Sea (possibly Giresun, Turkey, today) and became the species name of cerasus. (Pontus, a territory in the times of Alexander the Great, was located in what is presently north-eastern Turkey.) This implied that the cherry originated in Pontus. Hedrick (1915) expressed a contrary opinion and agreed with those botanists who stated that the town received its name from the cherries grown in the area rather than vice versa. Thus, the name

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Kerasun may have signified that high-quality cherries were grown in Pontus, and Lucullus, among others, obtained prized specimens from there. Another outstanding cultivar that originated from Pontus is the 'Black Tartarian' (described later) brought to Russia by Prince Potemkin. The name of the cherry, in old English, ciris, chiri or cyrs, is cognate to the Old High German chirsa or chersa. The name stems from classical Latin or Greek and varies little among the different languages. The classical Latin words cerasus (cherry tree) and cerasum (cherry) correspond to the Greek kerasea (cherry tree) and kerasiov (cherry), respectively. In West Germanic it was keresja, kerisja, kirisja, or kirissa, in Middle High German kirse or kerse, and Modern German kirsche. In Latin it was transformed into ceresia and ceresea, which were also the progenitors of the Romanic forms ciriega in Old Italian, cereza in Spanish, cereja in Portugese, cereisa or cereira in Provencal, and cerise in French. Chery or chiri in Middle English was not known until the fourteenth century (Murray 1893).

A. Sweet Cherry De Candolle (1886) observed cherry (P. avium) in the forest of Ghilan (north of Persia, south of the Caucasus) and in Armenia; in Europe in south Russia; and generally from south of Sweden to the mountainous parts of Greece, Italy, and Spain. He concluded that sweet cherry had originated in an area south of the Caucasian mountains and a secondary dissemination took place into Europe. The area where De Candolle placed the origin of the cherry escaped the Ice Age during the Quaternary period. Therefore, it is possible that a secondary migration of cherry took place to Europe. However, areas of northern Italy, the Balkan Peninsula, the Carpathian Basin, and most of France also escaped the Ice Age and cherry could have survived this period in parts of Europe as well. Therefore, there was no need for a secondary dissemination to explain the presence of cherry in this area. If a secondary migration took place it had to occur very early. For all practical purposes, Europe should be considered as native territory for modern horticulturists who are looking for native characteristics in cherries. Discorides (first century A.D.) mentioned cherries as kerasia, a dietary component. His concerns were only medical and although his writings confirm that he knew about cherries, his records do not give any evidence for their origin. He wrote: "Cerasia, if they be taken while they are new, are good for the belly, but being dried they stop

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the belly," according to the English translator J. Goodyear in 1655 (Gunther 1934). Terrentius Varro (117-27 B.C.) described when to graft cherries in a manner that conveyed the impression that cherries were common in Italy in his time. Nearly 150 years later, Pliny the Elder described the cherries of Rome in A.D. 79, including the cherry cultivar: 'Apronian', 'Lutatian', 'Caecilian', a duracinus cultivar known in Campania as 'Plinian', 'Junian', the 'Lusitanian' cherry, a cherry that grows on the banks of Rhenus (Rhein), and Macedonian cherry, which was a small tree, and a Chamaecerasus, which was a small shrub, both of which were probably sour cherries. Some of these cherries were named after Romans and in all likelihood they originated in Italy. 'Apronius' was named after Apronius, a Roman magistrate, and 'Lutatian' was named after Lutatius Catulus, a contemporary of Lucullus, who rebuilt Rome after it was destroyed by fire. 'Caecilian' commemorates the Caecilian family, rich, powerful friends of Lucullus, and 'Plinian' was named, naturally, after Pliny himself. The description of 'Junian' fits characteristics of the French "Guigne" or the English "Gean" group. Hedrick (1915) proposed that "Guigne" may be a version of "Junian." As an ancient Lusitania in the modern Portugal, 'Lusitanian', in Hedrick's (1915) opinion, may have been the cultivar 'Griotte' of Portugal which was grown from ancient times in that country. Pliny mentioned that 'Lusitanian' was highly valued in Belgica (Belgium). Thus, Pliny established that Prunus avium, the sweet cherry, was grown in his time in Italy, Portugal, Belgium, and along the Rhine. Even though the sweet cherry was common in Europe and in southern Asia (Fig. 5.1) botanists suspected that cherry is not indigenous to Britain and Western Europe. That there is no Celtic or Teutonic native name for cherry seems to confirm this observation (Murray 1893).

Pliny the Elder stated that Lucullus, a Roman general stationed in Asia Minor, a territory that included Pontus, brought back cherries upon his retirement in 67 B.C. from the region of the Black Sea. De Candolle (1886) was of the opinion that Lucullus may have brought cherries to Italy, but cherries were native in Europe as well as in northwestern Asia and were cultivated in Italy before Lucullus. Thus, Pliny's description of Lucullus bringing cherries back from Asia Minor may signify that cherries were cultivated in those days in northern Persia, but Pliny's comments cannot be construed as a proof for the origin of cherries. Regardless of the origin of cherry, Pontus is a location where excellent cherry cultivars were originated. There-

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Fig. 5.1. Illustration of the native area of sweet cherry (striped area), sour cherry (solid line), and the ground cherry (broken line). (Illustration is redrawn after Terpo [1974]; sour cherry area and the area for P. fruticosa was added by Faust and SuranyL)

fore, it is not surprising that Lucullus, a connoisseur of fruit, brought back excellent cherries from Pontus. It is recorded from a later period, in al-Biruni's list of plants, that both sweet and sour cherries were present in northern Persia in 1050 (Harvey 1975). Sweet cherries were grown in southern Russia north of the Caucasian mountains. This is probable because the Hungarians learned the Russian name for cherries, cberesbnia, which became the Hungarian cseresznye during the period between 500 and 800 when the Hungarians passed through south Russia coming from the east. The name cseresznye (cherry) can be found in early deeds of Hungarian land grants beginning in 1256 when the earliest Hungarian language documents were produced (documents were previously written in Latin). The name of cherry can also be found in geographical descriptions in Hungary. Cseresznyet6 (cherry lake), cseresznyeszer (cherry lodge) cseresznyes tet6 (cherry mesa) all refer to various locations. Fekete cseresznye (black cherry) was mentioned in 1217, referring to the fruit itself (Suranyi 1992).

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B. Sour Cherry The Macedonian cherry and the Chamaecerasus, mentioned above in Pliny's description, were probably identical, and both were probably Prunus fruticosa Pallas, a synonym of Prunus chamaecerasus Jaquin. Nicolaus Joseph Jaquin (1727-1817), an Austrian botanist, kept the name chamaecerasus, perpetuating it because this name was used by Pliny (Hedrick 1915). The ground cherry, P. fruticosa, is native to Europe. De Candolle (1886) described the native habitat of sour cherry as being from the Caspian Sea to the environs of Constantinople. He described sightings of sour cherries by botanists in the wild in Bithynia (area near Constantinople in Turkey on the Black Sea), at Mount Olympus, and on the plains of Macedonia. De Candolle (1886) also described P. cerasus as a wild tree from Italy and France. In Dalmatia, on the Adriatic coast of Croatia in the region of Zara (presently called Zadar), a liqueur, or cordial, called maraschino, has been made from small, black, sour, bitter, 'Marasca' cherries from prior to 1700. Even though this process is relatively recent, the sour cherries used for the process are unimproved and abundant in the region, indicating that they are likely to be native there. The so called Gypsy cherries are native in the Carpathian Basin. Hedrick (1915) assigned a much larger area than did De Candolle as the native habitat of sour cherry. Hedrick (1915) contemplated that sour cherry is native to an area bordered by Switzerland to the Adriatic Sea on the west, Germany on the north, and the Caspian Sea on the southeast. Sour cherries were common in Italy in antiquity. Pliny the Younger described the "woods of ancient trees," surrounding his villa near Rome between 97-107, that were full with sweet chestnut, ash, poplar, linden, elm, and many suckering (certainly sour) cherries (Hobhouse 1992). De Candolle (1886) also quotes Virgil (Publius Vergilius Mara 70-19 B.C.), the great Roman poet, to say that cherries were in the woods of Italy. His comments apply to P. cerasus and not to P. avium: "Pullulat ab radice aliis densissima silva Ut cerasis ulmisque" (The most dense forest puts forth from root cherries and elms) (Virgil, Georgics 2:17). As Hedrick (1915) stated, the development of sour cherry had to happen farther west or north from southern Russia. This can be substantiated by the location of land races. Land races usually develop in native areas of any given plant species. Land races can be characterized by specific descriptions under which many slightly differing clones can be grouped (Iezzoni et al. 1992). Kolesnikova (1975) suggested that such land races of cherries developed in response to

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environmental factors in Asia and Europe. Such land races are Cigany (Gypsy, Zigeuner) and Pandy (Crisana, Karaser Wechsel), predominantly developed in Hungary; Oblacinska is a land race in Serbia; Mocanesti (Shepards's cherry) is a land race in Romania; Strauchweichseln, and Weinweichseln are land races in Germany; Stevensbaer a land race in Denmark; and Vladimirskaya is a land race in Moldavia. Thus land races indicate a large area as native sour cherry territory. The development of Pandy as a land race is notable. It is a very high-quality sour cherry "discovered" about 1848. In the beginning, some believed that it was a cultivar that originated along the river Karas (Hungarian name) or Crisana (Romanian name), which flows from the Carpathian Mountains of Romania to the Hungarian river Tisza. Hence its name 'Karaser Wechsel' in German and 'Crisana' in Romanian. "Pandy" is thought to be the Hungarian who first recognized the excellent quality of this sour cherry. Others suggest that a Turkish military officer brought it to Szentes, a city along the river Karas, as a gift (the Turks occupied Hungary between 1526 and 1680). Yet others believe that it was found in a garden in the village of Pando Today very few cherries are grown in Pand but they are abundant in the surrounding area. There are other versions of its origin. The truth is that exactly where and when Pandy first was recogized for its excellence is unknown (Suranyi 1985). That sour cherry had to be native in the Carpathian Basin is indicated by the Hungarian word for sour cherry. The Hungarians did not learn the Russian name, visbnya, for sour cherries as they learned cseresznye for sweet cherries. The Hungarians use the word meggy for sour cherry, which is an original Finn-Ugor (the language family to which belongs the Hungarian language) word. There was no sour cherry in the original Asian home of Hungarians. Meggyoriginated from the word mol, a berry from a mountain plant, or from ire-mol, blood and berry. The word has underwent transformations. The letter 1 became gyand the a or the more closely sounding a to e, transforming mol to meggy. Thus, the word usage "blood-berry" was transferred to a new fruit, the sour cherry, when the Hungarians found it in the Carpathian Basin (A magyar nyelv sz6tara 1967-1970). The Hungarians used the name "meggy" for locations with P. cerasus groves. Meggy, the sour cherry, was mentioned more often than cseresznye, the sweet cherry, among the Hungarian location designations beginning in the thirteenth century when written records became more widespread. Names of locations listed since 1233 include Meggyes, Meggyespatak, Meggyestelek, Meggyesmezo,

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Meggyeshalom, Meggyeskovacsi, Nyirmeggyes, Sam ogymeggyes, Zalameggyes, and Meggyespuszta, and in an older spelling as Medgyes, Medgyesbodzas, and Medgyeshaza (Suranyi 1992). In the location combination names, the suffix refers to creek, patch, heath, or knoll, or in "Medgyesbodzas," an area where sour cherry ("meggy") and elder ("bodza") were common, indicating that sour cherries were abundant and probably native in that particular area. The large number of locations with sour cherry in their name also may indicate that sour cherries were widespred, and likely to be native in the Carpathian Basin. De Candolle (1886) justified his idea of secondary dissemination of the cherry with the argument that the Albanians, descendants of the Pelasgians (very early inhabitants of ancient Greece), called the sour cherry vyssine, an ancient name that reappears in the German Weichsel and the Italian visciolo. He estimates the use of the name "vyssine" before 600 B.C. It is notable that the Russian word for sour cherries is vishnya. Therefore, if the original word for sour cherries was vyssine and the Russians learned it later, then the development of the sour cherry must be clearly further to the north and the west than the Caucasian Mountains. This also easily explains why the Hungarians did not learn the Russian term for sour cherries and eventually used their old original term when they found sour cherries in the Carpathian Basin. De Candolle (1886) considered the two cultivated cherries to be distinct species, but close in their characteristics. He stated that sour cherry is derived from sweet cherry. Later workers considered another species, P. fruticosa Pall. (2n = 32), the ground cherry, as a probable parent of P. cerasus (Fogle 1975). Crosses in Sweden between P. fruticosa and either of the diploid or tetraploid sweet cherries (P. avium) have given progenies resembling sour cherries (Olden and Nybom 1968). Close examination of morphological traits strengthened the conclusion that P. fruticosa may be involved in the development of sour cherry. Families of hardy Russian cultivars show greater resemblance to the ground cherry than do families of less hardy cultivars (Hillig and Iezzoni 1988), and chloroplast DNA polymorphism studies suggest that the ground cherry is the maternal parent of sour cherry (Iezzoni et al. 1989) Watkins (1976) used the argument that cherries, particularly the tetraploid sour cherry, reproduce fairly true from seed as a proof that the species belonging to Eurocerasus developed in isolation from species of subsection Pseudocerasus. They developed to the west of the central Asian center of origin while most other Prunus species developed to the east and this may account for the differences

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Fig. 5.2. Locations of P. avium (stars) and P. [ruticosa (dots) in Turkey where Suranyi collected cherries in 1970 and 1988.

between the fruit-producing and the ornamental species. He does not distinguish between sweet and sour cherries. Terpo (1974), a Hungarian botanist specializing in fruit-producing plants, designated a native area for sweet cherries from midFrance to the Volga River and from Germany to southern Italy and Greece (Fig. 5.1). He designated a much narrower area for P. fruticosa, from the Danube River to northeastern Russia (Fig. 5.1), which is a further indication of the northern origin of sour cherry. If the sour cherry is a hybrid between the ground cherry and sweet cherry/then the hybridization should have occurred where the territory of the two species were common (Fig. 5.1). However, P. fruticosa may occur further south than Terpo contemplated it. One of us (D.S.) found P. fruticosa in two collecting trips made in 1970 and 1988 in Turkey and south of the Caucasian Mountains (Fig. 5.2). IV. EARLY RECORDS OF CHERRY CULTIVARS A. Archeological Findings Archeological evidence places cherry into the Neolithic Period (about 4,000-5,000 years ago). De Candolle (1886) described P. avium seeds

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found in Stone Age caves or dwellings of western Switzerland, Bourget, France, and Parma, Italy. Three stones of 7 mm in diameter were uncovered at Kempen an Niederrheinen (Bertsch and Kand 1949). Two broken stones were also uncovered from the burned layer at Siimeg-Mogyor6sdomb in Hungary from the same period (Hartyanyi et al. 1968; Dombay 1960). According to Hartyanyi and Novaki (1975) the domestication of cherry in the Danube Valley clearly dates to the late Neolithic Period (4000 years ago). Neolithic cherry seeds were unearthed in Kempen (lower Rhine Valley) and in Stuttgart (Renfrew 1973). There is also evidence from this period of the presence of sour cherry in England at Nympsfield (Roach 1985). Late Iron Age remains of P. avium were unearthed at Maiden Castle in Dorset, England (Roach 1985). From the early Bronze Age (3500 years ago in Europe), important information was obtained for the presence of cherry at the Austrian Mondsee (Werneck 1955), at the Swiss Pfaffkerse-Robenhausen digs (Heer, cited by Ermenyi [1978]), and at Haugh Head, England (Roach 1985). Seeds of cultivated cherries were found from pre-Lucullus times in Switzerland in Basel, Aalen, and Xanten. The seeds were 8 to 9 mm long (Renfrew 1973). Cherry stones from the early Roman period (400 B.C.) were found in Tac-Gorsium (Pannonia, the present day Hungary). At the same location mahaleb seeds also were discovered (Kocztur, cited by Ermenyi [1978]). Cherry seeds were found from the late Roman period at Nussdorf, Penzerdorf, and Linz, Austria (Werneck 1955). These seeds match the seeds of the local cherry land race (Werneck 1956). Archeological excavations in Germany along the Rhine revealed that the Romans used cherries in their diet and that they collected them in neighboring forests (Knorzer 1970a). One seed was found at Xanten-Colonia Ulpia (along the Rhine) dating to the first century (Knorzer 1967); four intact and 11 broken seeds were unearthed at Aachen from the same period (Knorzer 1967), and at Neuss the excavation of an ancient camp of Roman legionaries provided 64 burned seeds (Knorzer 1970). Excavated Roman wells also provided cherry seeds at Irel, Saalburg (Roach 1985), Rottweilben and Butzbach, Germany (Schroeder 1971; Knorzer 1970b). Figure 5.3, a mosaic found in the region, shows one of the earliest illustrations of cherries. Roman soldiers who manned the fort at Caerws in Wales ate cherries and blackberries. Cherry stones were excavated at Silchester, Seley, West Witterring, and also from the waterlogged sites near the Thames in London (Roach 1985). Stones from these sites resemble the stones of the modern cultivated sour cherry. Excavation of a

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Fig. 5.3. Early illustration of cherries. Detail of a floor mosaic from the dining room of a Roman villa built in Cologne, Germany, in the first century. The mosaic was more likely installed during renovations carried out around 230. Chronological analysis is from G. Hellenkemper Salies in the Bonner Jahrbuch 184, 1984, p. 76.

second century site under St. Thomas Street, in Southwark, England, revealed the remains of fruits, including cherries (Roach 1985). Archeological finds of cherry seeds from the Middle Ages are more numerous. Evidence of cherry use in the Czech Republic from the seven to tenth centuries is provided by cherry and sour cherry seeds found in fireplaces, waste pits, and grave fillings. At Mikulcic parts of two cherry trees were also found (Opravil 1972). In the city of Uhersky Brad, during the archeological excavations of 1962, 2497 sweet and sour cherry seeds were discovered (Opravil1966); in Opava 962 sweet and sour cherry seeds were found (Opravil 1964, 1965, 1969), and in Ostrova, Olomuec, and Pilsen, 51, 183, and 291 cherry seeds, respectively, were found in archeological excavations, indicating a considerable production of cherries in the area during the

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turn of the first millennium (Opravil1964; Tempir 1962; Burian and Opravil1970). The German evidence provides a similar picture. At Haus-MeerNiederungsburg during the excavation of a tenth-to eleventh-century castle among various seeds of fruits were 1148 cherry seeds (Janssen and Knorzer 1970), and in an excavation dating to the same period in Neuss 1012, burned cherry seeds were discovered (Knorzer 1970a). The Polish archeological evidence for the presence of cherries is from a somewhat later period. Cherry seeds from the tenth to the twelfth century were found at Opole and Wolin (Moldenhawer 1955). Cherry seeds from the twelfth to the fifteenth century were discovered in several Polish cities. At Posnan there were 54 cherry seeds and at Plock, 11 sweet and 24 sour cherry seeds, were found (Moldenhaver 1955). At Gdansk were 824 cherry seeds were unearthed and at Szczecin, 130 (Lechnicki 1955). From two wells at Budapest, Disz Square nos. 8 and 10, 66 cherry and 100 sour cherry seeds were discovered and 3 seeds were found in a clay pot at Hunyadi street 22 (Hartyanyi and Novaki 1975). Confirmed wild cherry seeds from the thirteenth to the fourteenth century were unearthed from the fort of Kereki-Feherko, Hungary (Hartyanyi et al. 1968). It is notable that relatively few seeds of cultivated cherries were found in Poland, Litvania, and Latvia, and from the southern edges of Europe. It is possible that the first improvement of cherries occurred in middle Europe during the Neolithic period. B. Period Before the Sixteenth Century The references to cherry by Theophrastus and Terrentius Varro have been mentioned in Section III. Hedrick (1915) quotes references to cherry by third-and fourth-century writers Athenaeus, Ammianus, Tertullian, and St. Jerome. Cherries were in cultivation relatively early in Spain and in Italy. Ibn al-Awwam in his book Kitab ab-filaha, mentioned that P. mahaleb was used as the rootstock described in the eleventh through the thirteenth centuries Spanish-Arabic documents (Hobhouse 1992). In England, the cherry was regularly grown in the gardens and orchards of the monasteries. At Norwich, beside the appleyard there was a cherruzerd or orto cersor, the cherry garden. At Ely, the records of sale for 1302 show that cherry trees were grown in the vineyard area (Roach 1985).

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The 1295-1296 accounts for the Holborn gardens of Henry de Lacy, Earl of Lincoln, included an item for cherries, and cherries were commonly sold near the S1. Paul churchyard. The churchyard had been the subject of much dispute in 1355, and cherries were specifically mentioned in the records of this incident (Roach 1985). In Italy, Giovanni Boccaccio (1313-1375) mentioned cherries planted in a Florentine terraced garden (Hobhouse 1992). Pierandrea Mattioli or Matthiolus (1501-1577), a Tuscan from Italy, in translating the work of Dioscorides listed the fruits of Italy and included cherries. Pietro de Crescenzi (1478) in Liber ruralium commodorum recommended middle size gardens (Fig. 5.4) for those who could afford this size, and larger ones, 4.8 ha including orchards, for wealthier people. He also recommended that trees should be planted in their rows spaced 6.5 m apart and include pears, apples, mulberries, cherries, plums, and such noble trees as figs, nuts, almonds, and quinces (Hobhouse 1992). The German herbal, German Herbarius, printed in Mainz in 1485, does not name cultivars but groups cherries into two groups, sweets and sours, commenting that the sours make the mouth fresh (frisch). A woodcut in this herbal illustrates the sour cherry. Apparently the Germans until 1569 refrained from giving names of the cultivars when describing them in a medical herbal (Hedrick 1915). Keeping with this tradition, in Gart der Gesundheit the cherries were divided into four groups: (1) the Amarellen, sour,darkred cherries with long stems; (2) the Weichselkirschen, red, probably sour cherries with white juice and short stems; (3) the Sii sskirch en, red or black sweet cherries with long stems; and (4) additional undefined types distinguished by their shape and the province in which they were grown (Hedrick 1915). In 1526, in England, appeared The Grete Herball, which was the Le Grant Herbier or Arbolayre translated "out of ye Frensshe into Engllysshe" using the same medieval figures. The illustrations were very general, some of them were made to serve for two different plants. In the Arbolayre, the illustration for cherry served also for Atropa belladonna, while in The Grete Herball it represented the cherry and the Potentilla tormentilla (Blunt and Raphael 1994). From the illustration it is impossible to tell just what plant it should illustrate (Fig. 5.5). A herbal, De Historia Stirpium, written by Leonhart Fuchs, a medical doctor, and published by Isingrin of Basel, Switzerland, in 1542 and a year later in German translation entitled New Kreuterbuch, also illustrates cherries. In Fuch's work, flowers and green, semi-mature, and mature fruits are all painted on the same plate (Fig. 5.6).

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Fig. 5.4. Cherry trees are tended in this late fifteenth century miniature. The miniature illustrates the translation of Crescenzi's work.

In England, cherry is described in the New Herball of William Turner (1510-1568), a physician, published in part in 1551 in London, the second part (together with the first) in Cologne in 1562, and the whole work in three parts in Cologne in 1568, and in the Herball of John Gerard published in 1597 and greatly improved by Johnson

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IJPttetnefre ben talleb bamatmeS/8nb dJe otbetagtPOtes. ([lJetpe~ ben goolJ to tttl Fig. 5.5. Cherry, in the Grete Herball: this woodblock was used for more than one plant.

in 1633. Gerard obtained information from Matthiolus and from Rembert Dodoens (1517-1585) who published Cruydeboeck in Antwerp in 1554, and also owed much to Fuchs. Although Gerard may have adapted some of his material from others, he compiled a system, especially concerning cherries, that gives a good insight of his own thinking. Hedrick (1915) carefully interpreted Gerard's classification of cherries and somewhat modernized his descriptions. According to Gerard (1597), the ancient herbalists considered four kinds of cherries: (1) the great and wild; (2) those tame or of the garden; (3) those that had sour fruit; and (4) those that are called in Latin Chamaecerasus or the dwarfe cherry tree. Gerard described the English Cherry tree as a high, great tree with large leaves and round cherries hanging on long stems. He contrasted this with the Flanders cherry which "brings forth his fruit sooner and greater than the other" and called it Cerasus praecox, Belgica. Hedrick (1915) interpreted these descriptions to mean that the English Cherry was

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Fig. 5.6. Cherry, an illustration from Fuchs' De Historia Stirpium (1542).

the wild cherry (P. avium) and the Belgian Cherry was an early-ripening type of P. avium. However, it is unclear whether wild cherries extended into England. It is more likely, perhaps, that cherries were carried to England by the Romans and, if Gerard's wild type was

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indeed widespread, it may have escaped from cultivation. Gerard also described the Spanish Cherry as a large tree with white flowers and large, light-colored fruit that was also white inside, and the Gascoin Cherry as having great fruit with occasional strikes of purple color. Hedrick (1915) equated the Spanish Cherry with the Yellow Spanish type of the Bigarreaus, and the Gascoin Cherry with 'Bleeding Heart', a cultivar that Joan of Kent brought back to Enland when her husband was in Guienne and Gascony in France. Gerard also described two sour cherries-the Cluster Cherry and the Morello, which he called Morell Cherry. Gerald's description of cherries was quite extensive. He described ornamental double-flowering types and a dwarf cherry which in all likelihood belonged to P. fruticosa. He mentioned the 'Heart Cherry', which is probably one of the heart cherries, and the 'Luke Wardes' cherry, which is one of the oldest named sweet cherries known in England. He described the Agriot Cherry which Hedrick (1915) equated with Griotte Commune, a sort that was supposedly brought back from Syria by the crusaders and recorded in France as early as 1485. Parkinson's Paradisi in sole paradisus terrestris, published in 1629, described 33 cultivars. He described many of the same cultivars as did Gerard and included cultivars such as the 'Naples Cherry', the name of which may indicate that this cultivar was from Italy. Cherries were planted in the garden at Versailles. The map of the Le Jardin Potager Du Roy a Versailles planted by La Quintinye (16261688) clearly identifies cherries in one block near to the royal entrance (Tukey 1964). In addition to description and illustration in pomological and botanical books, cherries were the subject of various paintings. A group of five illuminated manuscripts, called Tacuinum Sanitatis, that originated in Italy in the late fourteenth and early fifteenth centuries were derived from Arab medical treatises. These are known as medieval health books and their text is illustrated with paintings. Plants depicted in these book have some medicinal values. A harvest of cherry tree is illustrated (Fig. 5.7). A cherry tree also plays an important role in a painting known as the Garden of Paradise by an unknown Rhenish artist made between 1410 and 1420 (Fig. 5.8). In this complex painting, S1. Dorothy picks fruit from the Tree of Life depicted as a cherry tree (Hobhouse 1992). One of the known artists of his time, Giuseppe Arcimboldo (15271593) of Italy painted portraits composed of fruits and other plants. In 1590, he completed Vertumnus, the portrait of Rudolf II (15521612), Holy Roman Emperor from 1576 to 1612. Arcimboldo used

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Fig. 5.7. Cherry harvesting probably for medical purposes. Miniature from Tacuinum Sanitatis, an Italian medical book from the late fourteenth to early fifteenth century.

several cherries in this portrait (Fig. 5.9), heart cherries for the lower lips, a dark sour cherry for the left eye, and lighter colored cherries as ornaments in the hair (Pieyre de Mandiargues 1977). Joachim Benckelaer's painting Women With Vegetables, painted in 1573, depicts a basket of cherries (Fig. 5.10). A Dutch work, Madonna at a Table With Child, painted during the first half of the sixteenth century, has cherries on a fruit plate on the table. Andrea Montegral's fresco, painted between 1426 and 1459, has a garland of fruit containing cherries. And, finally, inlayed tables of the Italian Renaissance are decorated with cherries along with other flowers (Fig. 5.11)

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Fig. 5.8. St. Dorothy picks cherries from the Tree of Life. Detail from Garden in Paradise by an unknown Rhenish painter, painted between 1410 and 1420.

(Rossi 1979). Cherries also entered into the folklore. Cherries were depicted in Transylvania on easter eggs (Malonyai 1909), as single fruits (~ avium or ~ cerasus) or in umbels, perhaps picturing ~ fruticosa (Fig. 5.12).

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Fig. 5.9. Cherries as lips, eyes, and decoration in the hair. Detail from Vertumnus by G. Arcimboldo, a humorous portrait of Rudolph II, Holy Roman Emperor. Painted in 1590.

C. Period From 1600 to 1800

John Tradescant the Elder made a special trip to the Low Countries (Holland) in 1611 to collect new plants for the gardens being constructed at Hatfield where he was the head gardener for King Charles I of England. As the result of his trip he brought back a cherry that was named 'Tradescant's Black Heart Cherry' (Hobhouse 1992) or, as Parkinson termed it in 1629, John Tradescant Cherrie, also called 'Elkhorn' in the United States (Hedrick 1915). Thomas Skip Dyot Bucknell, a landowner in Sittingsbourne, Kent, who published the The Orchardist, carried out experiments in the 1740s in a 6-acre orchard planted with apples and cherries (Henrey 1975). 'Black Tartarian', a Russian cultivar, originated in Pontus and was brought to Russia by Prince Potemkin, the chevalier of Catherine the Great, in 1783 (Roach 1985). 'Black Tartarian' was introduced into England in 1794 and again in 1796. In 1796, 'Tartarian' was bought to England by the plant collector John Fraser of Sloan Square, Chelsea, who purchased it from a German who grew it in his garden in St. Petersburg (Roach 1985). It was introduced into America as 'Black Tartarian' by William Prince of Flushing, Long Island, during the early part of the nineteenth century. By the beginning of the twenti-

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Fig. 5.10. Detail from the Joachim Beuckelaeras painting, Woman With Vegetables, painted in 1573.

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Fig. 5.11. Inlayed tables from Florence from the Renaissance period.

291

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Fig. 5.12. Easter eggs from Transylvania from about 1900 (Malonyai 1909).

eth century it became the favorite home-garden cherry east of the Mississippi (Hedrick 1915). Janos Lippai, the brother of Archbishop Lippai of Pozsony (Bratislava), and the gardener of the Archbishop's garden, described several cherry cultivars and growing techniques in 1667. He was aware of the high rate of flower production and the relatively low rate of fruit set among cherry cultivars. Self-incompatibility in cherry was not recognized until 250 years later. Cultivars of cherries also were described in pomological terms in Italy beginning in the sixteenth century. A. Del Ricco in 1595 described' Aquaiole', 'Moscadelle', 'Duracine', 'Agriotte', 'Visciole con gambo lungo', Ciriegie a grappoli', 'Ciriegie visciole palobine con gambo corto', Ciriegie dette Turche', 'Ciriegie Amarenne 0 Marasche

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dette', 'Ciriege del Frate', 'Marchianne', 'Duracine Moraiole minute', 'Moraiole grosse', 'Ciriege San Giovanni', 'Ciriegie viscioline minutissime', and 'Ciriegie bianche'. It is obvious from this list that the Italians by this time were familiar with "duracine," the Bigarreautype cultivars as well as well as "bianche," or white cultivars in sweet cherries and also with sour cherries. Some cultivars had small fruit, others had large fruit (Basso 1982). This listing soon was followed by Iconogia Plantarum (Tomi X) of Aldrovani (1522-1605), painted by a team of artists, which depicts a great number of fruit trees of the sixteenth century, including six illustrations of cherries (Baldini 1990). Aldrovani's illustrations include pictures of cerasa and ciriegie, both meaning cherries (the current Italian word is ciliegie). During the seventeenth and eighteenth centuries, the Medici family commissioned paintings of fruits, including cherries. The most important paintings in this series were painted by Bartolomeo Bimbi (1648-1724) who produced two 116 x 155-cm canvasses illustrating cherry cultivars. Basso (1982) identified 34 and 32 cultivars, respectively, in the two paintings and could identify all but 3 of 37 types. All types of cherries were represented, from very dark to bright red to almost white in color, similarly to those described about 100 years earlier by Del Ricco. Three additional Italian works are worthy of mention. One is the two-volume treatise written by P.A. Micheli (undated, c. 1700) in which he described some 25 cultivars cultivars (Basso 1982). The second is G. Gallesio's work, produced in 1817, that describes and illustrates 6 sweet and 8 sour cherry cultivars and describes a plum x cherry hybrid, 'Ciliego Susine', which may be the first description of this kind of fruit (Baldini and Tosi 1994). The third is by O. Targioni Tozetti in 1858, describing 16 types of cherry, including Prunus mahaleb and also a plum-cherry (Basso, 1982), which mayor may not be the same as that described by Gallesio 41 years earlier. Henry Louise Duhamel Du Monceau (1700-1782), a respected French horticulturist, included cherries (Fig. 5.13) in his multivolumed Traite des Arbes Fruitiers (Raphael 1990). Prior to producing this book, Duhamel, in 1768, described a sour cherry, the first to be widely grown in Europe, which was known particularly among the French, as 'Montmorency a Longue Queue' or 'Cerise de Montmorency'. This cherry originated in the Montmorency Valley of France before the seventeenth century, probably as a seedling of 'Cerise Hative' or of 'Cerise Commune'. When 'Montmorency' arrived in America is not known, but it was grown much before William

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Fig. 5.13. 'Cerise de Hollande', a cherry cultivar in H. L. Duhamel Traite des Arbres fruitiers, 1768. Volume 1, plate X.

Prince spoke of it in 1832 (Hedrick 1915). It soon became very popular as a sour cherry and today is by far the most predominant sour cherry cultivar grown in North America. In 1688 Ray described a new cherry, 'May Duke', which became well known worldwide. This is considered the first description of a Duke cherry, which turned out to be a hybrid between the sweet and sour cherries. The name 'May Duke' may be a corruption of Medoc, a district in Geronde, France, from where this cultivar was originated (Hedrick 1915). Nearly 60 years earlier, Parkinson (1629) described

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Fig. 5.14. Illustration of cherries in Parkinson's Paradisi in Sale from 1629: 1, the May Cherry; 2, the Flanders Cherry; 3, the White Cherry; 4, the great leafed Cherry; 5, Luke Wards Cherry; 6, the Naples Cherry; 7, the Heart Cherry; 8, the bignarre or sported Cherry; 9, the wild cluster Cherry; 10, the Flanders cluster Cherry; 11, the Archduke Cherry; 12, the dwarfe Cherry. Illustration nos. 9 and 10 may be P. padus and no. 12 may be P. fmUcosa.

a cherry as "one of the fairest and best of cherries" named 'Archduke' (spelling of Parkinson) (Fig. 5.14). Thus the Duke name was mentioned before Ray described 'May Duke'. 'Arch Duke' (spelling of Hedrick) was scarce for unknown reasons for about 150 years af-

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ter its description. It was rediscovered in 1847 in the nurseries of Thomas Rivers (England), where it has been grown for nearly a century. Close examination of this cultivar revealed that it differed from 'May Duke' (Hedrick 1915). Several dukes were known in the seventeenth century that, in the opinion of Roach (1985), originated in England since they were known in France as 'Anglais'. This means that they represented the few cultivars that originated in Britain; most other cherries grown in Britain in the sixteenth and seventeenth centuries had been imported from the Continent. This suggests that the Duke name may have a different origin than the transliteration of Medoc, as Hedrick proposed. D. Cherries in America Cherry growing started in America with the first settlers and became widespread throughout the land. In 1954, Marshall remarked that although cherries were less popular in the United States than in Europe, cherries in the early days were planted everywhere in the United States, around farm houses, in gardens, and along the roadside. However, even by the time Marshall commented on widespread cherry production, the rapid increase in the use of processed foods resulted in profound changes in the pattern of production as well as consumption of cherries. U.S. cherry production increased from 45,000 tin 1890 to 315,000 tin 1980, while the number of trees decreased (Childers 1983). This resulted in the concentration of the industry in a few districts most suitable for cherries and a large portion of the crop was processed. By 1993, 74% of the estimated 376,000 t of U.S. production was processed and utilized frozen, canned, or brined. Sweet cherries were brought as seeds to New York by the early Dutch settlers. In 1641, George Fenwick of Saybrook, Connecticut, wrote to George Winthrop, the governor of Massachusetts Bay Colony, about the plentiful supply of cherries and peaches (Hobhouse 1992). Peter Stuyvesant, the last Duch governor of New York, distributed cherries and other fruits up the Hudson Valley to homesteaders (Hedrick 1915). Sturtevant noted (Hedrick 1919) that cherry stones were among the seeds mentioned in 1629 to be distributed by the Massachusetts Company, and cherries were planted at Yonkers, New York, in about 1650 and in Rhode Island in 1669. Cherries also were cultivated in Virginia and Maryland about the same time. In the 1700s, the French planted cherries in Nova Scotia, Prince Edward Island, and in early settlements along the St. Lawrance River (Iezzoni

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et al. 1992). About 20 cultivars were advertised by the Prince Nursery in 1767. Marshall (1954) interpreted this advertisement as to suggest that trees offered for sale prior to this time may have been propagated from seeds. Cherries were planted by the colonists in Pennsylvania, New Jersey, Delaware, Virginia, and North Carolina (Marshall 1954). Even as early as 1676, cherries were reported to have been grown in "notable abundance" in Virginia (Hedrick 1915). As settlers moved westward so did the cherries. An important step in the westward movement was when Henderson Lewelling traveled from Iowa to Oregon in 1847 with 300 trees in tubs loaded on the wagons drawn by oxen (Marshall 1954). Among his cherry trees were a 'Bigarreau' and an 'English Morello'. The label of one of the trees was lost and it was renamed 'Royal Ann'. The cultivar was in reality 'Napoleon', and has been grown since 1820, but the name 'Royal Ann' persisted on the Pacific Coast (Marshall 1954). In 1850, Seth Lewelling (18191897) joined his brother Henderson in Milwaukee, Oregon, where Henderson had established a nursery under the name of Meek & Lewelling. Henderson Levelling moved to California in 1853 and the partnership with Meek was dissolved in 1857. As the sole proprietor of the nursery, Seth Lewelling started to develop new cultivars of cherries. In 1860 he introduced a dark cherry, the 'Republican', called by him 'Black Republican'. Five years later he introduced 'Lincoln'. In 1875 he originated two cultivars, both grown from the seeds of 'Republican', and named them 'Bing' and 'Yan' after two Chinese workmen working for him (Hedrick 1915). 'Bing', with a long, mahagony-colored fruit, has become the most important cherry cultivar of the United States. A few small orchards were planted in California and plantings increased as more settlers arrived. The first order for named cultivars was placed by W. H. Nash and R. 1. Kilburn of Calistoga, Napa County, to a New York nursery. The trees arrived after a long voyage around the Horn in the spring of 1850. The shipment included among other trees 'Napoleon' and 'Black Tartarian'. More cherry trees were available when Lewelling arrived from Oregon in the spring of 1851 and started to sell trees, including cherries (Olmo 1976). As the interest increased in cherry production, the search for new and better cultivars also expanded. 'Centennial', raised by Henry Chapman in Napa Valley, fruited first in 1876. The 'Oregon' was introduced by H. W. Prettyman of East Portland, Oregon, in 1888. 'Lambert' was introduced by J. H. Lambert, Milwaukee, Oregon, in 1887. 'Andrews' was fruited first in about 1896 by C. N. Andrews in a mountain val-

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ley near Redlands, California. 'Paul', found by E. V. D. Paul on a place purchased by him in Ukiah, California, was exhibited first in 1907. 'Nonpareil' originated in an orchard owned by the Earl Fruit Company at Vacaville and was not distributed. 'Early Burbank', originated by Luther Burbank, was sold in 1903 to a group of Vacaville growers and was valued for its earliness (Wickson 1914). V. THE MODERN ERA, CHERRIES IN THE TWENTIETH CENTURY

After the turn of the century, the sweet cherry industry expanded even further, especially in the Pacific Coast states. Gardner (1913) noted that cherry production in Oregon was low because of insufficient pollination. Orchards had been small and planted with mixed cultivars. With the beginning of the cherry industry and larger plantings, fewer cultivars reduced cross pollination, causing poor fruit set. 'Napoleon' planted in solid blocks or interplanted with 'Bing' was unproductive. However, 'Napoleon' and 'Lambert' in combination gave satisfactory results. This was the beginning of the recognition of self-and cross-incompatibility in cherries, but it took almost another 20 years before horticulturists knew enough about the use of pollinizers, planting distances, and bee requirements to recommend orchard designs for the optimum pollination (Claypool et al. 1931). About 750,000 cherry trees were grown in Oregon in 1900. Tree number changed little until 1930 when the count was about 800,000. Cherry plantings increased after World War II. By 1950 the census recorded one million trees. Brining became a major use of Oregon cherries. Brining was established in the 1930s following research by professor E. H. Wiegand at Oregon State College and, by the 1970s, utilized half of Oregon's output (Zielinski 1976). Brining initiated the start of the maraschino cherry industry. Maraschino, or glace cherries, are bleached, cooked in colored syrup, and flavored by imitation maraschino flavor, for use in garnishing deserts or in cordials. The cherry industry also expanded in California. The industry shifted to the Vaca Valley, then to Stockton and Lodi in the Central Valley east of San Francisco Bay (Olmo 1976). In 1911, about 5500 t of canned cherries were marketed, and in 1912, 244 carloads of fresh cherries were shipped to the eastern markets (Wickson 1927). Sweet cherry planting steadily increased in California, reaching 6000 ha by 1970 (Olmo 1976).

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In Washington, early plantings of both sweet and sour cherries were mainly in the west coast counties where the climate was favorable for tree growth. As time progressed, more sweet cherries were planted in the dry regions of the state, while sour cherries remained west of the Cascades. From 1920 to 1930, sour cherry planting doubled and planting peaked in 1940. Since then, sour cherry production decreased gradually (Luce 1976) and presently it is a minor crop in Washington State. Sweet cherries were planted first in the western counties around farm houses and roadways. From 1920 on, larger blocks of cherries were planted in the central counties, where fruit splitting caused by rains was less frequent. Planting of sweet cherries steadily increased and in 1960 it was the third most important fruit crop in Washington State after apples and pears. Sweet cherry production steadily increased, and in 1993 Washington was the largest producing state in the United States. In Wisconsin, the early success of orchards in Door County encouraged planting of sour cherries. In 1910 a single order of 41,000 cherry trees was placed for the spring of 1911. By 1950 there were over 1 million cherry trees in Door county. Economic considerations and yield variability reduced the size of the cherry industry so that in 1973 there were only 1600 ha of productive cherry trees in the county (Klingbeil 1976). A sweet cherry industry developed in Montana around 1920. Metcalf (1976) estimated that about 1000 ha of sweet cherries were grown in Lake and Flathead counties. In 1910, cherry trees were numerous in Utah and their number slightly increased until 1964 when 1300 and 420 ha were planted with sweet cherries and sour cherries, respectively (Stark 1976). Sour cherry plantings increased even further and today Utah is one of the leading sour cherry producing states. New York is not a large producer of sweet cherries, but apparently New Yorkers like cherry ice cream. A large percentage of the New York sweet cherry crop (85%) was used in ice cream mixes in the 1960s (Slate 1976). For more than 70 years Michigan has led the country in the production of "red tart" sour cherries. In the mid-1800s farmers of Grand Traverse County relied on the potato as their primary cash crop, but every farm also had its orchard of mixed fruits, which often produced more cash (especially cherries) than did potatoes (Lupton 1964). A leading citizen and banker of Traverse City, Berney J. Morgan, foresaw the potential opportunities in cherry growing and planted 5 ha of sour cherries in the 1890s. Another cherry enthusiast, John Kroupa, planted the first orchard (4 hal on the Old Mission

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Fig. 5.15. Location of sweet cherry industry in California, Oregon, and Washington and the sour cherry industry in Michigan. Note that in California the cherry industry is located in the Santa Clara Valley near the coast where temperature is cool and in the Upper San Joaquim Valley where cool air gets into the valley through the opening through the mountains at San Francisco. The inset in Michigan shows the Old Mission Peninsula. Arrows indicate the location on the map and on the inset.

Peninsula while working for the Ridgewood Farms in 1893 (Kessler 1971), a location exceptionally favorable for sour cherries. The Old Mission Peninsula is a small tongue of land that averages 1.5 km (1 mile) in width but extends 35 km (22 miles) out into Grand Traverse Bay (Fig. 5.15). Ace Johnson, the captain of the steamship J. T. Westcott, transported ore from Escanaba to Elk Rapids. Captain Johnson came across

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the bay to Old Mission to pick up apples or other fruits and take them to Escanaba (M.L. Hilt, undated). After a few years of transporting fruit, Captain Johnson purchased a farm and employed John Kroupa to plant another cherry orchard (3-4 hal in 1896. Eventually, John Kroupa established a cherry planting on his own farm and became one of the most knowledgeable cherry growers in the area. At the beginning of the twentieth century there were only a few growers growing cherries commercially in the Traverse City area. The sour cherry industry of Michigan developed to a commercial processing scale during the second decade of the twentieth century with the establishment of the first canning factory in 1912 owned by Birney J. Morgan, Perry Hannah, and C. J. Kneeland, and the invention of the Dunkley cherry pitter in 1917 (Kessler 1971). In 1922 the Grand Traverse Packing Company of Traverse City froze the first cherries at the point of production (M. L. Hilt, undated). The number of trees increased dramatically within a few years from less than 500,000 in 1911, to more than 1 million in 1920, and to 2 million by 1933. The size of the cherry industry increased further after World War II. By 1959 there were 3.5 million trees and by 1968 tree number reached the 4,350,000 mark. By 1926, Michigan produced 2200 t of frozen cherries, which increased to 2900 t in 1930 and 52,200 t in 1969 (Kessler 1971). In 1924 the "Blessing of the Blossoms", a one-day event, was conceived by Jay P. Smith, a newspaperman, which was converted to a Cherry Festival in 1926 and subsequently designated by the Michigan State Legistrature as the National Cherry Festival in 1928. The Michigan Association of Cherry Producers was organized in 1938 with A. J. Rogers, a grower and processor, as its first chairman. An important step, especially in sour cherry production, was the development of mechanized harvesting. Experiments on adapting mechanical tree shaking to sour cherries in Michigan were initiated in 1956 by H. P. Gaston, a horticulturist of Michigan State College, and Jordan Levin, a mechanical engineer of the U.S. Department of Agriculture. Mechanical harvesting was adapted rapidly. By 1966 approximately 50% of all sour cherries in the state were harvested with mechanical shakers (Kessler 1976) and by 1969 the number increased to 75% (Kessler 1971). All commercial sour cherries are now harvested mechanically. Another step forward in producing highquality, processed sour cherries was the improvement in fruit handling after machine harvesting. For example, cherry quality can be maintained if cherries are precooled in water-filled tanks attached to the harvesting machines and carried cooled to the pitting plants.

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302 Table 5.1.

Cherry production and utilization in the United States, 1992. Production (t)

State

Sweet

Sour

Total

Michigan Washington Oregon California Utah New York Wisconsin Pennsylvania Idaho Colorado Montana Total

18,000 97,000 55,000 31,000 3,200 1,100

122,500

140,500 97,000 59,750 31,000 19,700 16,600 4,550 4,100 1,200 750 800 375,950

1,100 1,200

4,750 16,500 15,500 4,550 3,000 750

800 208,400

167,550

Note. Utilization of sweet cherries: 95,420 t fresh, 33,500 t canned, 66, 080 t brined; sour cherries 4400 t fresh, 45,000 t canned, 107,000 t frozen. Source. Agricultural Statistics (1993).

The growth of the Michigan sweet cherry industry is more difficult to follow. The rapid development in canning stimulated sweet cherry planting. In 1929 there were 79,000 sweet cherry trees in Michigan. Tree numbers increased to 112,482 by 1931, to 196,000 by 1940, and to 1 million in 1968. In 1930, 48% of the trees were planted in northwestern, 31 % in central-western, and 21 % in southwestern Michigan. Between 1930 and 1960, the sweet cherry industry shifted north, with 72% of the trees located north of Manistee, 21 % in the central-west and only 8% in the southwest (Kessler 1971). After brining started in the 1930s, a greater portion of the Michigan sweet cherry crop was brined and by 1969 the bulk of the cherry crop went to brining (Kessler 1971). The brine market accepts immature cherries, enabling the grower to harvest before fruit becomes full ripe and susceptible to cracking. Marketing of fresh Michigan sweet cherries started in the mid-1960s (Kessler 1971). Throughout the modern production era beginning in the 1970s, cherry production became more concentrated in area as production increased (Table 5.1, Fig. 5.15). Four states presently produce 96% of the U.S. sweet cherry crop and three states produce 92% of the sour cherry crop, and even in these states cherries are concentrated in small geographical areas that are exceptionally favorable for production.

5.

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CULTIVAR IMPROVEMENT

Conscientious improvement of cherry cultivars started in North America by Luther Burbank about 1900. Breeding of sweet cherries started at the New York Agricultural Experimental Station, Geneva, New York, in 1911 by R. Wellington and was continued by R. D. Way, R. C. Lamb, S. K. Brown, and R. Andersen; at the Horticultural Experimental Station, Vineland, Ontario, Canada, in 1915 by G. H. Dickson and continued by G. Tehrani; at the Research Station, Summerland, British Columbia, Canada, in 1924 by A. J. Mann and continued by K. O. Lapins and W. D. Lane; at the Idaho Agricultural Experimental Station in 1934 by L. Verner; at the California Agricultural Experimental Station, Davis, in 1935 by R. M. Brooks and continued by P. E. Hansche; and at the Irrigation Experimental Station of Washington State at Prosser in 1950 by H. Fogle, continued by T. Toyama. In Oregon, Q. B. Zielinski introduced two cherry cultivars, 'Corum' and 'Macmar', from chance seedlings, but did not initiate cherry breeding. Several cherry cultivars were introduced from the Utah Agricultural Experimental Station by B. N. Wadley and S. V. Thomson from 1970 to 1982. Several private breeders introduced sweet cherry cultivars in California: F. W. Anderson introduced 'Bing d'Andy' (Bingandy); Marvin Nies introduced 'Ruby' and 'Garnet'; Floyd Zaiger introduced 'Starkrimson' and 'Zaiwite-Sweet'; and in Michigan, the Hilltop Nursery introduced 'Cavalier'. The cultivars introduced by each of these programs and the name of the breeders are listed by Brown et al. (1996). Sweet cherry breeding began in 1925 in England by M. B. Crane (Crane 1947) and continued by P. Methews in the 1960s; in Germany at the Max Planck Institute beginning in about 1955 by M. Zwintzscher, at the Fruit Research Station, Jork, starting in 1953 by E. 1. Loewel (Zahn 1985), and at Naumburg starting in 1961 by H. Michatsch and continued by M. Fischer; in Switzerland, at Wadenswil, in 1960 by E. Schaer and continued by R. TheilerHedtrich (1985); in the Chech Republic sweet cherry breeding started in 1972 by J. Blazek and continued by J. Blaikova; in Romania, at Bistrita, beginning in 1950 by 1. Ivan and, at Pitesti in 1971 by V. Cociu (Cociu and Gozob 1985); in Bulgaria, at Kustendil, in 1953 (St. Georgiev 1985); in Bulgaria at Plovdiv in 1988 by A. Jivondov and P. Gercheva; in Italy, at Verona, in 1965 by G. Bargioni, and, at Bologna and Rome, ionizing radiation was conducted to produce short internode dwarf cultivars beginning in 1963 (Sansavini and Lugli 1988).

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Cultivars were introduced in Italy also from Piacenca in 1972, Florence in 1983, and Rome in 1983. At the Ukranian Institute of Irrigated Horticulture at Melitopol, cherry breeding probably started in the 1950s by Oratovskiy and at Kiev, also in the 1950s, by S. H. Duka. In Australia, at the New South Wales Research Station, breeding started in about 1925 and was continued by F. T. Bauman. U. P. Hedrick (1915), with the assistance of G. H. Howe, O. M. Taylor, C. B. Tubingen, and R. Wellington, summarized the existing knowledge on cherries and described 1145 cultivars. Origins of most of these cultivars were unknown. Hedrick (1915) remarked that to his knowledge none of the cultivars described in his book, The Cherries of New York, had originated by mutations, indicating their genetic stability. He contrasted this with the ornamental cherries, several of which are double flowered, weeping or fastigiate in growth habit, or have abnormally colored foliage that originated as mutations. Mutations, however, do occur in cherry. Most of the introduction in sour cherries were clonal selections of the 'Montmorency' cultivar. At least 10 such strains were introduced between 1925 and 1956 (Fogle 1975). In Hungary, a large number of clonal selections were produced from 'Pandy' and 'Cigany' sour cherries by S. Brozik (Tomcsanyi 1979). In Italy and in Canada, ionizing radiation experiments that began in 1963 produced artificial mutations of sweet cherries with short internodes (Lapins 1971; Sansavini and Lugli 1988). Sour cherry cultivar development was preceded by identification of synonomy. In 1921, George Howe of the New York Agricultural Experimental Station compared 'King', 'Stark', 'Monarch', 'Sweet', and 'Large' Montmorency strains and found them to be identical. V. R. Gardner in Michigan initiated a program for improving 'Montmorency' by finding more vigorous and productive strains in 1930s and collected these variants, mutants, or strains. A sour cherry breeding program was started in Michigan by R. Andersen in 1971 and continued by A. Iezzoni. Sour cherry improvement started at the University of Minnesota in 1933. Sour cherry breeding commenced in Hungary at Budapest in 1948 by P. Maliga, and was continued by S. Brozik and J. Apostol; in the Ukraine at Melitopol, probably in the mid 1950s; in Romania at Pitesti in 1972 (Cociu and Gozob 1985); in and Germany in 1965 by M. Zwintscher. Cultivars introduced by each program are listed by Brown et al. (1996). The German program concentrated on disease-resistant cultivars based mostly on 'Shattenmorelle' and Koroser. In Hungary, P. Maliga, S. Brozik, and J. Apostol succeeded in producing high-quality self-compatible cultivars that can replace the outstanding 'Pandy', which is self-incom-

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patible. The Romanian program is based on their own land races and the Russian program released cold-tolerant cultivars that are hybrids of P. fruticosa x P. cerasus. The American western (P. bessey) and eastern (P pumila) sand cherries have been utilized in the improvement of fruits in the North Central and Great Plains States of the United States and the Prairie Provinces of Canada. Work with sand cherries started in the Dominion Experimental Station at Morden, Manitoba, in 1896. The South Dakota Agricultural Experimental Station named at least 6 cultivars, and for a while the North Dakota and Minnesota Experimental Stations and the United States Department of Agriculture at Mandan, North Dakota, and Cheyenne, Wyoming, used sand cherries in their research, but have not released selections. In 1895, N. E. Hansen of the South Dakota Experimental Station started to use sand cherries to produce cherry-plums. The prime motivation for this work was the cold and drought resistance of sand cherries, which the investigators tried to transmit into high fruit quality cultivars. These cultivars never became popular. Their introductions are listed by Fogle (1975).

The outstanding breeder, Paul Maliga of Hungary, was able to solve most problems needed to improve sour cherries. He and J. Apostol succeeded in developing very high-quality, productive cultivars adapted to central European conditions, which overcame the incompatibility problems, poor flesh color, and limited size characteristic of most sour chery cultivars. The outstanding cultivars released included 'Meteor korai' 'Erdi nagygyiimolcsii', 'Favorit', 'Erdi jubileum', 'Erdi botermo', 'Korai pipacsmeggy', 'Maliga emleke', and 'Csengodi'. VII.

ROOTSTOCK IMPROVEMENT

The earliest cherry orchards were seedlings. When grafting started, P. avium (mazzard) was used as the rootstock, although P. mahaleb (mahaleb) was mentioned as rootstock for cherries in the elevenththirteenth centuries in Spanish-Arabic documents (Hobhouse 1992). Miller, in his Gardener's Dictionary (1754), described the Mahaleb Cherry as a cultivated specimen, but does not mention it as a rootstock (Hedrick 1915). In the eighteenth century the French started to use 'St. Lucie', the perfumed cherry (P. mahaleb), because of its limited suckering (Duhamel 1768). Targioni Tozetti mentioned mahaleb in 1858 as a rootstock in Italy (Basso 1982). In North America, mazzard was used first as a rootstock and this changed in favor of

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mahaleb when Charles Downing (1802-1885) proposed the use of the Mahaleb cherry as a superior rootstock in 1845. Soon after this, in 1851, Thomas also recommended it as a dwarfing stock for cherries (Hedrick 1915). Mahaleb came into general use after 1860, and by 1890 it almost entirely replaced mazzard. Eighty years later, rootstock trials in the 1970s indicated that mazzard was a better rootstock for cherries and the use of mazzard increased again. There are many requirements for a good cherry rootstock. Rootstocks need to be adapted to the soil, relatively resistant to soil diseases, and have dwarfing characteristics especially for fresh market sweet cherries, which are harvested by hand. About 1930, N. H. Grubb exerted efforts to develop better clones of mazzard cherries at the East MaIling Research Station, England. As a result, clone F12/1 was introduced in 1933, and later 'Colt' aP. avium x P. pseudocerasus hybrid, was produced, and 'Charger', another mazzard rootstock was released (Tydeman and Garner 1966). Increased activity in developing cherry rootsocks was apparent in the 1970s. A series of P.avium o P.mahaleb rootstocks clones were selected from 30,000 open-pollinated seedlings of P. mahaleb by Lyle Brooks in Oregon (Stebbins et a1. 1978). In 1979, Schimmelpfeng and Liebster reported P. cerasus 'Weihroot' selections for rootstock of cherries. 'Weihroot' is a wild Bavarian genotype of P. cerasus and the selections were dwarfing to an extent of 20-30%. In 1980, Webster reported the use in England of two sour cherries, 'Stockton Morello' and 'Kentish', as dwarfing rootstocks. In the 1980s, several other groups attempted to improve rootstocks for cherries in Belgium, France, Germany, Romania, and Russia. The Belgians used species hybrids for their rootstocks and produced 'Damil', 'Camil', 'Inmil', and other GM numbers (Trefois 1985). The French used the 'St Lucie' cherry as the basis of their program (Perry 1987; Lichou et a1. 1990). The German program started in 1965 (Gruppe 1985) and useful rootstock hybrids were selected from the intercrossing of various species within the subgenus Eucerasus: (P. avium, P. canescens, P. cerasus' Schattenmorelle', and P. fruticosa). The German program introduced the various Gisela numbers (Schmidt and Gruppe 1988). In contrast, crosses with subgenus Pseudocerasus species (P. concinna, P. incisa, P. nipponica, P. subhirtella, and P. pseudocerasus) did not yield useful rootstocks (Schmidt 1985). In Romania dwarfing of sweet cherries was important (Cireasa and Surdu 1985). In Russia, at Oryol, the program focused on frost tolerance, using exotic parents such as Padus maackii (Kolesnikova et a1. 1985). In the United States in a limited way 'Vladimir' was used as a rootstock. Vladimir is the generic name of a group of morello cherries originating in Russia and introduced to

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America in about 1900. Details of activities of each breeding group can be found in the descriptions by Perry (1987). Despite all the effort exerted thus far, the rootstock problem of cherries has not been solved. Breeders were successful in producing dwarfing rootstocks that decrease tree size 25-30% compared to a tree grown on F12/1 root. The most dwarfing rootstocks are 'Damil', a P. fruticosa selection, a P. fruticosa x P. avium selection, and the P. cerasus 'Weihroot' selections (Perry 1987). Thus, size reduction in sweet cherries by rootstock should be attainable.

VIII. JAPANESE CHERRIES AND THEIR MOVEMENT INTO AMERICA In Japan, the flowering cherry tree, Sakura, or zakura in Japanese, is one of the most exalted of all flowering plants. As early as the fifth century the Japanese Emperor and his Court paid homage to the Sakura. During the sixth century, the 26th Emperor of Japan, Keitai Tenno, who lived in Neo-mura (village ofNeo), planted a cherry tree in commemoration of his reign. The tree is still alive today (Fig. 5.16) and is called 'Usuzumi-no-sakura', which means "cherry tree of gray blossoms." 'Usuzumi' belongs to the species P. spachiana f. ascendens based on traditional classification (White 1992). Reverence toward cherries in Japan has increased over the centuries. By 1800, a collection of approximately 1000 cherry trees containing nearly 80 different selections had been planted in Kyoto. The number of types increased to 130 by 1990. The earliest records of Japanese cherries being introduced into the United States are probably those listed in the 1846 and 1847 catalogs of Ellwanger and Barry Co. of Rochester, New York, and Parsons and Co. of Flushing, Long Island, New York. The introduction of a wild species of Japanese cherries into the United States probably did not occur until 1876 when William S. Clark, first President of the Agricultural College, Sapporo, Japan, sent home some seeds of Prunus sargentii Rehd., native to the mountains of northern Japan and southern Sakhalin (Jefferson and Fusonie 1977). In 1903, through the efforts of David Fairchild and Barbour Lathrop, the Office of Foreign Plant Introduction of the Bureau of Plant Industry, U.S. Department of Agriculture, imported 30 named cultivars of cherry trees into the United States. David Fairchild who traveled to Japan in 1902 was impressed by the picturesque beauty of the Japanese cherry trees and in 1905 ordered 75 flowering cherry trees and 25 single-flowered weeping-type trees from Japan, which

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Fig. 5.16. 'Usuzuni-no-Sakura', a 1400-year-old ornamental cherry tree in Neo village, Japan, in 1992.

arrived in May 1906 and were planted on the Fairchild estate in Chevy Chase. Subsequently, in 1907, the Chevy Chase Land Company ordered 300 trees to be planted in the community of Chevy Chase. Publicity about the Japanese cherry trees caused the wife of President William Howard Taft to become interested in this beautification project. Through her efforts, Colonel Spencer Cosby, the Superintendent of Public Buildings and Grounds, ordered 90 double-flowering Japanese cherry trees (P. serrulata 'Fugenzo') from Hoopes Brothers and Thomas Co., West Chester, Pennsylvania, in April 1909, to be planted on public grounds. By June, the Washington newspapers carried stories of a possible donation of cherry trees by the Mayor of Tokyo to Mrs. Taft. In August 1909, the Japanese Embassy in Washington, DC, officially informed the Department of

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State that the city of Tokyo intended to donate 2000 cherry trees to the United States. The cherry trees were shipped and arrived on 10 December, 1909 at Seattle and were taken by train on 6 January, 1910 to Washington, DC. Then, they were immediately transported to the Department of Agriculture's Garden Storehouse on the Monument Grounds to be examined by scientists from the Bureau of Entomology and the Bureau of Plant Industry. The team found the large trees seriously infested with insects and diseases and ordered destroyed them (Jefferson and Fusoni 1977). Mayor Yukio Ozaki of Tokyo heard of the destruction of the gift of trees and made steps to replace the shipment. Scions were collected from 12 selections from the Ekita-mura area (village of Ekita) along the banks of the Arakawa River, and the scions were fumigated and grafted to selected rootstock. The following December the trees were dug, refumigated, and by the end of January 1912, 6000 Japanese cherry trees were shipped to the United States. Half of the trees were destined to Washington, DC, as a gift from the people of Tokyo and the rest to New York City as a gift from the Japanese Society of Tokyo. The trees arrived at Washington, DC, in mid-March and were found pest free. On 27 March, 1912, Mrs. Taft participated in a planting ceremony in West Potomac Park with the wife of the Japanese Ambassador, Viscountess Chinda. Eighteen trees of the greenish-yellow flowered P. serrulata Lindl. 'Gyokio' were planted on the White House grounds.The cherry tree became an important symbol in Washington, DC, and their bloom is commemorated with parades and an annual election of a cherry queen. IX. WORLDWIDE OF CHERRIES

DISSEMINATION AND

PRODUCTION

Although sweet and sour cherries are ubiquitous in the temperate zone, there has been little effort to take them farther south into subtropical regions. Breeding programs to develop low-chilling-requiring cherries are very recent (W. B. Sherman, personal communication). Although there are low-chilling-requiring Prunus species among the cherry genotypes, the existing high quality cherry cultivars all have high-chilling requirements. Cool conditions may be required for another reason. Commercial production of sweet cherries are limited by rain falling during the ripening period, which causes cracking of Bigarreau-type sweet cherries and the subsequent brown-rot infection destroys the usefulness

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of the fruit. Moderately wet and cool conditions of Europe limit the development of rotting organisms. In North America, sweet cherries are grown in arid irrigated areas where cracking is limited. In hot climates cherries develop double fruit, an undesirable characteristic, that limits production. Sour cherries are not subject to cracking, nevertheless they are also better adapted to cool climates. In the North American Continent, 75% of sour cherries are grown in Michigan and the rest in New York and in Utah. Production in Europe is centered in the states of the former Soviet Union, Poland, Hungary, the Czech Republic, and Romania, all with relatively cool climates. As early as 1667, Lippai commented in his book, The Garden of Pozsony, that both the sweet and sour cherries prefer a cool location. Why cherries prefer a relatively cool climate also puzzled Chandler (1957), who noted that the trees of common sour cherry cultivars have rather long chilling requirements. At low elevations in southern California, bloom of the trees is greatly delayed and very few fruit set. The southern limit of sour cherry growing in North America, however, is not fixed by lack of winter chilling. Chandler (1957) thought that some other influence fixes the limit further north than where chilling would be inadequate and considered the absolute limit in North America for sour cherry the 36° latitude. He noticed that sweet cherry developed good flavor in districts where the summers are cool and was much better in the cool coastal areas of California than in the hot interior districts. Lupton (1964) thought that the reason Traverse City area of Michigan is superbly adapted for cherry production was due to the broad expanse of water around it. "In early spring, the Bay's cool temperature keeps the cherry buds closed and in late spring the warmth of the water keeps temperature stable for good flower bud formation and continued growth." Others also have tried to explain the localization of cherry production. H.V. Taylor (1949) remarked that "the cherry is a notoriously difficult crop to grow; the tree is fastidious of soils, and the management has to be skillful otherwise disappointment follows. That may be the reason why the growers of Kent (England) have obtained almost a monopoly of production and why fruit growers of other counties are hesitant to take risk of engaging in cherry production". Despite Taylor's remarks, cherry production in Kent has steeply declined (Tobutt 1985). Most of the world cherry production is in Europe, its native home. North America, where cherries developed a secondary center of production, still accounts for only 16% of world production (Table 5.2).

5.

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Table 5.2.

311

Cherry production worldwide (sweet and sour combined). Country

Production (1000 t)

"Native" area (Europe and Asia Minor) Former Soviet Union Germany Poland Former Yugoslavia Italy Turkey France Hungary Spain Czech Rep. + Slovakia Romania Bulgaria Portugal Greece Austria Belgium

470 312 185 179 160 105 102 98 76 65 65 60 45 29 28 15

Adapted area (Americas, Asia, Oceania) USA India Canada Japan New Zealand Australia Chile Argentina

376 21 15 11 10 10 8 5

Source: Westwood (1988), Agricultural Statistics (1993), J. Apostol (personal communication).

This is in great contrast to the peach, where production in adapted areas is overwhelmingly greater than that in China, where it originated (Faust and Timon 1995). While the peach moved from the Orient to Europe; the cherry has never made the reciprocal trip to China. X. CONCLUSION

The history of the cherry is very uneven. For almost 2000 years, from 300 B.C. to 1700, the cherry was a mediocre fruit harvested from native material. Suddenly, around 1700 outstanding cultivars appeared on the scene. During the 200 years between the discovery of 'May

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Duke' in 1688 and the introduction of 'Bing' in 1875, many outstanding cultivars were selected, most of which are still outstanding today. After a century of relative quiescence, purposeful breeding activity started at several locations worldwide, which undoubtedly will produce new cultivars in the future. In addition to its uneven development there are several paradoxes in cherries. Sweet cherry trees are very large and need to be reduced in size. There are dwarf types in sour cherries, but because they are harvested by machines, a reasonably large tree is required under which the harvester can be maneuvered. Thus, the desired dwarf genes are located in the wrong species and it is difficult to transmit the dwarf size by conventional techniques. Sour cherries are tetraploids (2n = 32), sweet cherries are diploid (2n = 16), although tetraploid sweet-cherry types occasionally occur. Thus, there is a possibility to intercross sour cherries with tetraploid cultivars carrying the dwarf character. We have yet to succeed in this effort and the goal of a small sweet cherry trees still eludes the industry. The effect of gibberellic-acid-synthesis inhibitors is dramatic in sweet cherries, but they have not been exploited to produce trees small in stature. The paradoxes continue. The market prefers firm fruit in sweet cherries, but unfortunately they crack when rain occurs close to harvest. The soft-fruited heart cherries do not crack, but are undesired. Thus, we have a dilemma in solving the cracking problem. The strongly nonelastic cell wall of Bigarreau cherries provides the desired firmness, but does not allow elasticity of cells and the fruit cracks severely. The strongly nonelastic fixed cell wall creates more paradoxes. In most large fruit development, cells separate and the resulting intercellular spaces greatly enlarge fruit size by allowing air in the fruit. The strongly fixed nonelastic cell wall of cherry prevents this. Thus, even though large cherries are preferred, it is likely that the cherry will remain small. There are other difficulties. Sour cherry harvesting has become totally mechanized. Trunk shakers clamp onto the tree at the time when the bark is fully hydrated and separates easily from the woody elements. Even the most gentle shaker moves this bark around and the tree suffers as a result. What loosens and tightens the bark? Nurserymen have known this phenomenon for ages and bud trees when the bark slips, yet horticulturists do not have the first clue to the physiology involved. The ultimate paradox is that while both sweet and sour cherries are ancient fruits, we still know very little about them. Although

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cherry is a high-value crop, production is small compared to other agricultural commodities. Thus, the number of researchers working with sweet or sour cherries is small. Nevertheless, results are forthcoming and improvements are in the pipeline. We may have better rootstocks in the near future, cultivars will be more disease resistant, and self-compatibility and fruit quality of sour cherries will be improved. However, as our predecessors have discovered, it may be difficult to improve the quality of sweet cherries over the existing outstanding cultivars. LITERATURE CITED A magyar nyelv t6rteneti sz6tara. 1967-1970. (Historical dictionary of the Hungarian language), Vol.3. Magyar Akademia, Budapest. Agricultural Statistics. 1993. U.S. Government Printing Office, Washington, DC. Baas, J. 1951. Die Obstarten aus der Zeit des R6merkastells Saalburg im Taunus bei Hamburg. Saalburg Jahrbuch 10:14-29. Bailey, L. H. 1927. The standard cyclopedia of horticulture. Rev. ed. Macmillan, London. Baldini, K 1990. Fruits and fruit trees in Aldrovandi's Iconographia Plantarum. Adv. Hort. Sci. 4:61-73. Baldini, K, and A. Tosi. 1994. Scienza e arte nella Pomona Italiana di Giorgio Gallesio. Acad. dei Georgofili, Firenze. Basso, M. 1982. Ciliege. In: K Baldini (ed.), Agrumi frutta e uve nella Firenze di Bartolomeo Bimbi pittore mediceo. Paretti Grafiche', Firenze. Bertsch, K. and F. Kand 1949. Gesichte unserer kulturpflanzen. Wissenschaft v. Stuttgart. Bertsch,V. and K Opravil. 1970. Stredoveke nalezy v Olomouci. Archeology 12:150158 Blunt, W. and S. Raphael. 1994. The illustrated herbal. Thames & Hudson, New York. Brown, S. K., A. F. Iezzoni, and H. W. Fogle. 1996. Cherries. In: J. Janick and J. N. Moore (eds.), Fruit breeding, Vol. 1. Wiley, New York. Chandler, W. H. 1957. Deciduous orchards, 3d. ed. Lea & Febiger, Philadelphia. Childers, N. F. 1983. Modern fruit science. Horticultural Publ., Gainesville, FL. Cireasa, V., and V. Surdu. 1985. Researches regarding the reduction of the sweet cherry height. Acta Hort. 169:163-168. Claypool, L. L., F. 1. Overly, and E. L. Overholser. 1931. Sweet cherry pollination in Washington for 1931. Proc. Am. Soc. Hort. Sci. 28:67-70. Cociou, V., and T. Gozob. 1979. Tag. Ber., Acad. Landwirtsch. Wiss. Berlin, #St 174:151-158. Transl. 30111, Natl. Agr. Library, Beltsville, MD. Cociou, V., and T. Gozob. 1985. The sour cherry breeding program in Romania. Acta Hort. 169:91-96. Crane, M. B. 1947. Cherry breeding. p.l1. In: Report of John Innes Institute for 1946. Crescenzi (P. de Crescentius, Crescentio) 1478. n libro della agricultura, Florence. De Candolle, A. 1886. Origin of cultivated plants. Hafner, New York. (Reprint 1967.)

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Dombay, J. 1960. Die Siedlung und das Graberfeld in Zengovarkony. Arch. Hung. 33:30-48

Ermenyi, P. 1978. Forrastanulmany a regeszeti korokbol szarmaz6 csonthejas gyumolcsleletekrol Kozep-Europaban. Magyar Mezogazd. p. 135-165. In: Muz. Evkonyve 1975-1977 Faust, M., and B. Timon. 1995. Origin and dissemination of peach. Hort. Rev. 16: (in press). Fogle, H. W. 1975. Cherries. In: J.Janick and J.N. Moore (eds.) Advances in fruit breeding. Purdue Univ. Press, West Lafayette, IN. Fuchs, L. 1542. De historia stirpium. Insigrin, Basel (Translated into German as New Kreuterbuch) (facsimile edition published in Munich in 1964). Ganzinger, K. 1959. Ein Krauterbuchmanuscript des Leonhart Fuchs in der Wiener Natinalbibliotek. Siidhoffs Archiv fur geshichte der Medizin. 43:213-224. Gardner, V. R. 1913. Pollination ofthe sweet cherry. Oregon Agr. Exp. Sta. Bull. 116. Gerard, J. 1597. The herball or general historie of plants. London. Gruppe, W., and H. Schmidt. 1972. Ergebnisse non Artkreuzungen von Kirschen. Gartenbauwiss. 37:73-84. Gruppe, W. 1985. Overview of cherry rootstock breeding program at Giessen 19651984. Acta Hort. 169:189-198. Gunther, R. T. 1934. The Greek herbal of Dioscorides (Translated by J. Goodyear, 1655). Hafner, London. Hartyanyi, P., Gy. Novaki, and A. Patay. 1968. Noveny es termesleletek Magyaroszagon az ujkort6l a XVIII szazadik. p. 5-81. In: Magyar Mezogazd. Muz. Evkonyve 1967-1968. Hartyanyi, P. and Gy. Novaki. 1975. Novenyi mag-es termesleletek Magyarorszagon az ujkort61 a XVIII szazadig. p. 23-71. In: Magyar Mezogazd. Muz. Evkonyve 19731974.

Harvey, J. 1975. Gardening books and plant lists of Moorish Spain. Garden History 3:10-21.

Hedrick, U. P. 1915. The cherries of New York. New York Agr. Exp. Sta., Geneva. Hedrick, U. P. 1919. Sturtevant's notes of edible plants. New York Agr. Exp. Sta., Geneva. Henrey, B. 1975. British botanical and horticultural literature before 1800. Oxford Univ. Press, London. Hillig, K. W., and A. F. Iezzoni. 1988. Multivariate analysis of sour cherry germplasm collection. J. Am. Soc. Hort. Sci. 113:928-934. Hilt, M. L. Undated. Early days of the cherry industry in the Grand Traverse region. Mimeographed memoir. Hobhouse, P. 1992. Gardening through ages. Simon & Schuster, New York. Honda, M., and Y. Hayashi. 1974. Nihon no sakura. Sheibundo Shinkosha, Tokyo. Iezzoni, A., H., Hancock, and K. Krahl. 1989. Evolution of sour cherry: clues from isoenzyme and chloroplast DNA polymophism. p. 132. In: Am. Soc. Hort. Sci. 1989 Prog. & Abstr. Annu. Mtg. Iezzoni, A., H. Schmidt, and A. Albertini. 1992. In: J. N. Moore and J. R. Ballington (eds.), Genetic resources of temperate fruit and nut crops. Int. Soc. Hort. Sci., Wageningen. Janssen, W., and K. H. Knorzer. 1970. Die fruhmittelalterliche bei Haus-Meer Stadt Meerbusch. Kr. Grevenbroich, 1968-1968. Jefferson, R. M., and A. E. Fusonie. 1977. The Japanese flowering cherry trees of Washington DC. Agr. Res. Servo Washington, DC.

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Jefferson, R. M., and K. K. Wain. 1984. The nomenclature of cultivated Japanese flowering cherries (Prunus): the Sato-zakura group. U.S. Dept. Agr. Nat. Arbor. Contr. Washington, DC. Kessler, G. M. 1971. Annu. Rep. Michigan State. Hort. Soc. for 1970. 100:114-147. Kessler, G. M. 1976. Michigan. In: D. V. Fisher and W. H. Upshall (eds.), History of fruit growing and handling in United States of America and Canada, 1860-1972. Am. Porn. Soc., University Park, PA. Klingbeil, G. C. 1976. Wisconsin. In: D. V. Fisher and W. H. Upshall (eds.), History of fruit growing and handling in United States of America and Canada, 18601972. Am. Porn. Soc., University Park, PA. Knorzer, K. H. 1967. Romerzeitliche Pflanzenfunde aus Aachen. ArcheooPhysica 2:39-64 Knorzer, K. H. 1970a. Romerzeitliche Pflanzenfunde aus Neuss Novaesium, IV: limes. Forshungen 10, Berlin. Knorzer, K. H. 1970b. Romerzeitliche Pflanzenreste aus einem Brunnen in Butzbach (Hessen) Saalburg Jarbuh. Berich. Saalburg Mus. Kolesnikova, A. F. 1975. Selection of some biological characteristics of sour cherry in Central Russia, III: phenotypic and genotypic potential of types of cherries which are used in selection. Orloskoe otd-nie priokskogo knizhnogo izd-va p. 52-105. Translation 34900, Nat. Agr. Library, Beltsville, MD. Kolesnikova, A F., Y. U. V. Ossipov, and A. 1. Kolesnikov. 1985. New hybrid rootstock for cherries. Acta Hort. 169:159-162. Lapins, K. O. 1971. Mutation frequencies in vegetative shoots derived from two zones of irradiated buds of sweet cherry, Prunus avium L. Rad. Bot. 11 :197-200. Lechnicki, G. 1955. Szczatki roslinne z wykopalisk Gdanskich w latach 1950-1952. p. 252-259. Studia Wczesnosred. III, Warsaw, Poland. Lichou, J., M. Edin, C. Tronel, and R. Saunier. 1990. Cherry production: sweet cherry for the fresh market. Ctifl, Paris. Lippai, J. 1667. A Pozsonyi Kert. (The garden of Pozsony) Facsimile ed. Akademiai Kiad6, Budapest, 1966. Luce, W. A. 1976. Washington. In: D. V. Fisher and W. H. Upshall (eds.), History of fruit growing and handling in United States of America and Canada 1860-1972. Am. Porn. Soc., University Park, PA. Lupton, T. W. 1964. Origin of cherry industry in Grand Traverse County. Eastern Fruit Grower. May: 24. Malonyai, D. 1909. A Magyar nep miiveszete, Vol. 2. Franklin, Budapest. Marshall, R. E. 1954. Cherries and cherry products. Interscience, New York. Metcalf, H. N. 1976. Montana. In: D. V. Fisher and W. H. Upshall (eds.), History of fruit growing and handling in United States of America and Canada, 1860-1972. Am. Porn. Soc., University Park, PA. Micheli, P. A. Undated manuscript, c. 1700a. Enumeratio quarundam plantarum sibi per Italia, et Germaniam observatorum in acta Tournefortii methodum dispositarum. Tomus IX. Bibl. Dip. Bot. Univ., Florence. Micheli, P. A. Undated Manuscript, c. 1700b. Lista di tutte Ie frutteche giorno per giorno dentro Il, anno sono poste alIa mensa dell' A.R.e del Ser.mo Gran Duca di Toscana. Bibl. Dip. Bot. Univ., Florence. Moldenhawer, K. 1955. Jadalne owoce pestkowe i wloskie w wykopaliskach polskich z okresu wczesnosredniowiecznego. Wiadmosc. Archeol. 22:171-180 Murray, J. A. H. 1893. A new English dictionary on historical priniles. Clarendon, Oxford, UK.

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J., and N. Nybom. 1968. On the origin of Prunus cerasus L. Hereditas 59:327-345. Olmo, H. P. 1976. California. In: D. V. Fisher and W. H. Upshall (eds.), History of fruit growing and handling in United States of America and Canada, 1860-1972. Olden, E.

Am. Porn. Soc., University Park, PA. Opravil, E. 1964. Rostliny ze Stredovekych nalezu v. Ostrove. Zvlast. otisk CSM Ser. B. Cedy Hist. 13:9-12. Opravil, E. 1965. Rostlinne nalezy z archeologickeho vyzkumu stredoveke Opavy provadeneho v roce 1962. CSM Acta Mus. Sil. ser. A 14:77-83. Opravil, E. 1966. Rostliny ze Stredovekych objectu v. Plzni Solni ulice. Cas. Narodn. Mus. odd. prirod. 135:84-88. Opravil, E. 1969. Rostlinne nelezy z archeologickeho wyxkumu stredove Opravy provedeneho v roce 1967. CSM Acta Mus. Sil. Ser. A. 18:175-181. Opravil, E. 1972. Rostliy z welkomorawskeho hradiste v Mikulcicick: vixkum z let 1945-1965. Archeol. Brne 2. Parkinson, J. 1629. Paradisi in Sole: Paradisus Terrestris. Republished in 1976 (A garden of pleasant flowers). Dover, New York. Perry, R. L. 1987. Cherry rootstocks. p. 217-268. In: R. C. Rom and R. F. Carlson (eds.), Rootstocks for fruit crops. Wiley, New York. Pieyre de Mandiargues, A. 1977. Arcimboldo the marvelous. Abrams, New York. Raphael, S. 1990. An oak epring pomona. Oak Spring Garden Library, Upperville, VA. Rehder, A. 1958. Manual of cultivated trees and shrubs: hardy in North America. Macmillan, New York. Renfrew, J. M. 1973. Paleoethnobotany. The prehistoric food plants of the Near East and Europe. Columbia Univ. Press, New York. Roach, F. A. 1985. Cultivated fruits of Britain: their origin and history. Blackwell, New York. Rossi, F. 1979. Mosaiken und Steinintarsien. Kohlhammer, Stuttgart. Sansavini, S., and S. Lugli. 1988. Abilitato a Vignola mutante compatto di Durone nero II. Frutticoltura 9:53-59. Schimmelpfeng, H., and G. Liebster. 1979. Prunus cerasus als unterlage selection arbeiten, vermerung eignung fUr sauerkirschen. Gartenbauwissenshaft 44:55-59. Schmidt, H. 1985. First results from a trial with new cherry hybrid rootstock candidates at Ahrensburg. Acta Hort. 169:235-243. Schmidt, H., and W. Gruppe. 1988. Breeding for dwarfing rootstocks for sweet cherries. HortScience 23:112-114. Schroeder, K. 1971. Geologish-paleobotanische Untersuchung eines romerzeitlichen Brunnens by Irrel Kreis Bitburg-Priim. Gesch. u. Kinst des Trierer Land. u.s. Nachbargebiete 34:97-117. Slate, G. 1. 1976. New York. In: D. V. Fisher and W. H. Upshall (eds.), History of fruit growing and handling in United States of America and Canada, 1860-1972. Am. Porn., Soc. University Park, PA. St. Georgiev, V. 1985. Some results with cherry breeding in the Research Institute for Fruit Growing in Kustendil, Bulgaria. Acta Hort. 169:73-78. Stark, A. L. 1976. Utah. In: D. V. Fisher and W. H. Upshall (eds.), History of fruit growing and handling in United States of America and Canada, 1860-1972. Am. Porn. Soc., University Park, PA. Stebbins, R. L., J. R. Tjienes, and H. R. Cameron. 1978. Performance of sweet cherry cultivars on several clonally propagated understock. Fruit Var. J. 32:31-37. Suranyi, D. 1985. Kerti novenyek regenye. Mezogazdasagi Kiado, Budapest.

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Suranyi, D. 1992. Magyar gyiimolcs multban es jelenben. Kerteszeti Egyetem, Budapest. Taylor, H. V. 1949. Foreword. In: N. H. Grubb, Cherries. Lockwood, London. Tempir, Z. 1962. Nalez pecek a skorapek z plodu ovochych drevin v Opatovicich nad Labem. Archeoi. 14:497-516. Terpo, A. 1974. Gyiimolcstermo novenyek helye a fejlOdestOrteneti rendszerben. In F. Gyuro (ed.), A gyiimolcstermesztes alapjai. Mezogazdasagi Kiado. Budapest. Theiler-Hedtrich, R. 1985. Sweet cherry breeding programe at the Swiss Federal Research Station, I: results of fruit characters and flowering period inheritance. Acta Hort. 169:51-62. Theophrastus. 300 B.C. De causis plantarum. Book III, Chapter 13. English translation B. Einarson and G. K. KLink, 1976. Heineman, Cambridge, UK. Tobutt, K. R. 1985. New approaches to breeding sweet cherry scion varieties at East Malling, with particular reference to small tree size. Acta Hort. "169: 43-50. Tomcsanyi, P. 1979. GyiimolesfajUlink. Mezogazdasagi Kiad6, Budapest. Trefois, R. 1985. Dwarfing rootstocks for cherries. Acta Hort. 169:147-156. Tukey, H. B. 1964. Dwarfed fruit trees. Macmillan, New York. Tydeman, H. M., and R. J. Garner. 1966. Rootstocks Colt, Cob and Charger. p. 130134. In: Reports for East MaIling Res. Sta. for 1965. Watkins, R. 1976. Cherry, plum, apricot, and almond. p. 242-247. In: N. W. Simmonds (ed.), Evolution of crop plants. Longman, London. Watkins, R. 1981. Plums, apricots, almonds, peaches and cherries. In: G. Bateman (ed.), The Oxford encyclopedia of trees of the world. Oxford Univ. Press, Oxford, UK. Webster, A.D. 1980. Dwarfing rootstocks for plums and cherries. Acta Hort. 114:103. Webster, A. D. 1995. The taxonomic classification of sweet and sour cherries and a brief history of their cultivation. In: A. D. Webster and N. E. Looney (eds.), Cherries: crop physiology, production and uses. CAB Intern, Wellingford. Werneck, H. L. 1955. Die Obstweihefund im Vorraum des Mithraeums zu Linz Donau, Oberosterreich. Naturkundi. Jahrb. Stadt Linz. p. 41-54. Werneck, H. L. 1956. Romischer und vorromischer Wein- and Obstbau in Ostereichischer Donauraum. Verhandi. Zoolog. Botanischen Ges., Vienna. Westwood, M. N. 1988. Temperate-zone pomology. Timber Press, Portland. White, G. A. 1992. Neo villagers of Japan donate ancient flowering cherry to the United States. Diversity 8:26-27. Wickson, E. J. 1914. The California fruits and how to grow them. Pacific Rural, San Francisco. Wickson, E. J. 1927. Cherries in California. In: L. H. Bailey (ed.), The standard cyclopedia of horticulture. Macmillan, London. Zahn, F. G. 1985. The cultivation of sweet cherries in Jork, Germany. Acta Hart. 169:85-90.

Zielinski, Q. B. 1976. Oregon. In: D. V. Fisher and W. H. Upshall (eds.), History of fruit growing and handling in United States of America and Canada, 1860-1972. Am. Porn. Soc., University Park, PA.

6 Artemisia annua: Botany, Horticulture, Pharmacology Jorge F. S. Ferreira, James E. Simon, and Jules Janick Center for New Crops and Plant Products Department of Horticulture Purdue University West Lafayette, Indiana, USA I. II.

III.

IV.

Introduction Botany A. Taxonomy B. Floral Biology C. Glandular Trichomes D. Secondary Metabolites 1. Aromatic Volatiles 2. Nonvolatile Sesquiterpenes 3. Chemotaxonomy Horticulture A. Field Cultivation 1. Germplasm 2. Crop Culture 3. Harvest 4. Postharvest Handling B. In Vitro Culture C. Genetic Improvement Pharmacology of Artemisinin and Derivatives A. The Challenge of Malaria B. Chemistry C. Isolation and Synthesis D. Detection and Quantification 1. Sample Preparation 2. Chemical Detection 3. Immunoassays

Horticultural Reviews, Volume 19, Edited by Jules Janick ISBN 0-471-16529-8 © 1997 John Wiley & Sons, Inc. 319

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J. F. S. FERREIRA, J. E. SIMON, AND J. JANICK Biosynthesis Mode of Action 1. Plant Growth Regulator 2. Antimalarial G. Drug Development 1. Drug Availability 2. The Future of Artemisinin as an Antimalarial Conclusions Literature Cited E.

F.

V.

I. INTRODUCTION

Artemisia annua L. (Asteraceae, formerly Compositae), also known as qinghao (Chinese), annual or sweet wormwood, or sweet Annie, is an annual herb native to Asia, most probably China (McVaugh 1984). Artemisia annua occurs naturally as part of a steppe vegetation in the northern parts of Chahar and Suiyuan provinces (40 0 N, 109 0 E) in China, at 1000 to 1500 m above sea level (Wang 1961). The plant now grows wild in many countries, such as Argentina, Bulgaria, France, Hungary, Romania (where it is cultivated for its essential oil), Italy, Spain, the United States, and the former Yugoslavia (Klayman 1989, 1993). Although it is unclear how or when A. annua was brought to the United States, it was naturalized around Nashville, Tennessee, in the nineteenth century (Gray 1884) and now occurs in waste areas in east and central North America (Bailey and Bailey 1976). Artemisia annua is used for the crafting of aromatic wreaths, as a flavoring for spirits such as vermouth, as a source of essential oils, and most recently as a source of artemisinin, the most important natural antimalarial after quinine. The generic name Artemisia refers to Artemis (Greek name for Diana), goddess of maternity, because in antiquity, plants of this genus, probably A. absinthium (wormwood), were used to control the pangs of childbirth, to regulate women's menstrual disorders, and as an abortifacient (Riddle and Estes 1992). Many Artemisia species are cited by early herbalists including Theophrastus in the third century B.C., (Einarson and Link 1976), Pliny (Bostock and Riley 18551857), and Dioscorides (Gunther 1959) in the first century, and Gerard (1597). Wormwood, probably A. judaica, is mentioned in the Bible (Rev. 8:10, 11). In A.D. 340, Ge Hong prescribed aerial parts of Artemisia for treatment of fevers in the Chinese Handbook of Prescriptions for Emergency Treatments, and in 1527, Li Shi-Zhen, a Chinese herbalist/pharmacologist, mentioned the use of huang hua hao (or yellow flower, later identified as A. annua) for children's fever, and qinghao (A. apiacea) as a treatment for the disease now known

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as malaria (Klayman 1993). Today, most references to qinghao refer to A. annua. In 1967, Chinese researchers evaluated the effectiveness of several traditional herbal remedies against malaria and A. annua was one of the plants selected. While extractions of A. annua with hot water or ethanol had no antimalarial effect (Klayman et a1. 1984) and a hot infusion made of the dried plant did not cure malariainfected mice (Klayman 1989), in 1971, a low-temperature extraction of A. annua with diethyl ether yielded a complex with antimalarial activity on both infected mice and monkeys. The main active principle, named artemisinin, was isolated and its structure defined in 1972 in China (Anon. 1979). Artemisinin, formerly referred to as arteannuin, also known as qinghaosu (in Chinese: su extract from, qing hao = green herb) is a rare sesquiterpene lactone endoperoxide belonging to the cadinane series (Brown 1993a). Although priority for the isolation of artemisinin is usually attributed to Chinese workers in 1972, the late D. L. Klayman (1993) reported that D. Jeremic'" first isolated artemisinin but reported an incorrect ozonide structure for the compound (see Jeremic'" et a1. 1973). By the end of 1972, artemisinin and derivatives were tested in 10 regions of China with ca. 6000 patients (Klayman 1993). The isolation and characterization of artemisinin has increased the interest in A. annua worldwide and, along with taxol, is considered one of the most novel discoveries in recent medicinal plant research. Artemisinin is the base compound for the synthesis of more potent antimalarial drugs with reduced toxicity for humans. Artemisinin is effective against both Plasmodium vivax and P. falciparum, two of the four species that cause human malaria, with p. falciparum responsible for the often fatal cerebral malaria, an advanced stage of the disease. Although total de novo synthesis of artemisinin has been achieved (Schmidt and Hofheinz 1983; Xu et a1. 1986; Ravindranathan et a1. 1990; Avery et a1. 1992), the low yield and complexity of synthesis required make it apparent thatA. annua is currently the more economically viable source of artemisinin. General reviews on A. annua as a source of artemisinin have been made by Klayman (1985,1989,1993), Woerdenbag et a1. (1990), Trigg (1990), and Ferreira and Janick (1995b). Early studies on the chemistry and clinical aspects of artemisinin have been reviewed (Anon. 1982; Luo and Shen 1987; Zhou and Xu 1989; Zaman and Sharma 1991). In 1993, an international symposium on artemisinin was convened in London (Baker 1994) and published information on artemisinin-related antimalarials was updated by Woerdenbag et a1. (1994b). In this paper, we review the botany and horticulture of A.

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J. JANICK

annua, and the pharmacology of artemisinin, including chemistry, isolation and synthesis, detection, biosynthesis, mode of action and toxicity, and development of artemisinin-derived drugs. II. BOTANY A. Taxonomy The Asteraceae is the largest dicotyledenous family of flowering plants with members widely distributed. The former name, Compositae, is derived from the many blossoms (or florets) combined into a flower head or capitulum. The plants in this family are mostly annual and perennial herbs, with a few woody species but not usually true trees. The family is composed of about 800 genera and 20,000 species, many highly aromatic and others extremely bitter due to the accumulation of terpenoids (Bailey 1951). Although sesquiterpene lactones occur in other families, the largest number of these compounds isolated in the last 30 years are from the Asteraceae (Fisher 1990). The Asteraceae is divided into 2 subfamilies: Cichorioideae (Lactucoideae), composed of 8 tribes, and Asteroideae, composed of 9 tribes. Artemisia belongs to the tribe Anthemideae of the Asteroideae. Based principally on floral morphology, Hall and Clements (1923) divided the genus into 4 sections: Abrotanum Bess., Absinthium D.C., Dracunculus Bess., and Seriphidium Bess. and placed A. annua in section Abrotanum. Rydberg (1927) and Beetle and Young (1965) added a fifth section, Tridentatae, segregating from the section Seriphidium, all North American taxa with affinities to A. tridentata. Poljakov (1961) combined the sections Abrotanum and Absinthium into a new merged section Artemisia, which places it in conflict with the Flora Europaea, which chose instead to combine the sections Absinthium and Seriphidium into a single section Artemisia. The section Dracunculus is clearly separated from other subgenera by their sterile central disk florets and other morphological characters, such as hemispheric involucre of flower heads (Greger 1982). Recent taxonomical treatment by Yeou-ruenn (1994) provides yet another classification scheme in which the genus Artemisia contains 5 sections, including Absinthium, Abrotanum, Artemisia, plus 2 newer ones Viscidipubes Y. R. Ling, and Albibractea Y. R. Ling. Seriphidium, now elevated to a genus, rather than a section of Artemisia; contains 3 sections (Seriphidium, Minchunensa Y. R. Ling, and ]uncea (Poljak., Y. R. Ling & C. J. Humphries).

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The absence of hairs on the receptacle was the only morphological characteristic separating the sections Abrotanllm from Absinthium. Recent studies by Ferreira and Janick (1995a) established that the trichomes on the capitulum of A. annua are the 10-cell biseriate type. There are no distinct chemical characteristics segregating the species into these 2 subgeneric sections, since both produce similar sesquiterpenes belonging to the eudesmanolides and guaianolides class. This may be the reason for Poljakov's (1961) decision to combine the sections Abrotanum and Absinthium. The genus Artemisia includes ca. 400 species (Heywood and Humphries 1977) worldwide and 65 species north of Mexico in the Western Hemisphere. Species of Artemisia, usually shrubs, often with divided leaves and inconspicuous flowers, are known for their bioactive secondary compounds and essential oils used for flavorings, fragrances, and medicinals. Several artemisias had been used by Greek, Roman, Persian, and Arabic physicians as antihelmintics and stomachics (Mehrotra et a1. 1990) and were the source of santonin, once a valuable antihelmintic drug, now obsolete due to its toxicity to humans. In addition to A. annua, there are several other widely recognized and related herbal species in this genus, including A. abrotanum (southernwood), A. absinthium (wormwood), A. dracunculus (French tarragon), and A. vulgaris (mugwort). A brief description of these well-known herbs based on various sources (e.g., Grieve 1971; Stuart 1979, and Simon et a1. 1984) follows. Artemisia annua is named for its annual cycle; other species, with the exception of A. klotzschiana Bess., are either biennial or perennial (Hall and Clements 1923). The chromosome number is 2n = 18 (Bennet et a1. 1982). Description: A. annua is a large shrub often reaching more than 2 m in height. It is usually single-stemmed with alternate branches. The aromatic leaves are deeply dissected and range from 2.5 to 5 cm in length. Distribution: The plant is native to China but is currently naturalized in many countries, including the United States (see Introduction). Constituents: At least 40 volatiles have been isolated from the essential oil of A. annlla (see Section II.D), with the main aromatic volatiles including artemisia ketone, 1,8-cineole, camphor, and f3-caryophyllene (Charles et al. 1991). The plant also contains several nonvolatile sesquiterpenes of interest, includingartemisinic acid, arteannuin B, and artemisinin (Fig. 6.1). Artemisinin has been isolated from leaves and flowers of field-grown (Klayman et a1. 1984; Ferreira et a1. 1995a) and in vitro-grown plants (He et a1. 1983; Nair et a1. 1986; Martinez and Staba 1988; Whipkey et a1. 1992; Ferreira et a1. 1995b; Ferreira and Janick 1996b).

J. F. S. FERREIRA, J. E. SIMON, AND J. JANICK

324

Deoxoartemisinin

R

R= R= R= R= R= R=

=

-

-

-

0 Artemisinin (qinghaosu)* OH Dihydroartemisinin* OCH] Artemether* OCH z CH] Arteether* OCOHzCHzCONa Sodium artesunate* ·OCHZC6 H 6CO zH Artelinic acid*

o Artemisitene

o Arteannuin B

o Deoxyartemisinin

-C2?COOH z

Arteannuic (artemisinic) acid

Fig, 6.1. Artemisinin and derivatives (antimalarial compounds indicated by asterisks),

Artemisinin has been detected in leaves, small green stems, buds, flowers, and seeds (Acton et al. 1985; Zhao and Zeng 1985; Liersch et al. 1986; Martinez and Staba 1988; Singh et al. 1988; Madhusudanan 1989; Ferreira et al. 1995a). Artemisinin has not been reported in roots of field-grown plants (Pras et al. 1991; Klayman 1993; Ferreira et al. 1995a) or pollen (Ferreira et al. 1995a). The high-

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est concentration of artemisinin is found in the inflorescence, which at anthesis may contain more than 10 times as much artemisinin as leaves (Ferreira et al. 1995a) (Fig. 6.2). The detection of artemisinin from seeds appears to be due to the presence of floral debris (Ferreira et al. 1995a). Uses: Used traditionally to treat fevers and hemorrhoids and currently as the source of the antimalarial artemisinin, which is the base compound for more stable, soluble, and potent antimalarial drugs (see Section IV.G); also used in aromatic wreaths. 0.30

2 0.25 ,-...

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0.20

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180 190 200 210 220 230 240 250 260 270 280 290 300 310 320

Jut

Aug.

Sep.

Oct.

Nov.

Time (Julian days) Fig. 6.2. Artemisinin content (% dry wt) of field-grown A. annua from leaves (0), inflorescences (.6.), and whole artemisia plants, in a weighed average basis (A), sampled from 1 July to November 1992. Development stages, starting from flowering: 1, flower buds visible; 2, pollen shed; 3, immature seeds; and 4, mature seeds. (Source: Ferreira et a1. [1995a].)

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A. abrotanum L. (southernwood) is a popular ornamental and highly aromatic herb known as garde robe by the French due to its ability to repel insects such as moths. Field southernwood refers to a different species, A. campestris 1., native to Europe and Asia. The name southernwood derives from the old English therne-wudu meaning a woody plant from the south (southern Europe origin). At one time, herbalists considered the plant to be an aphrodisiac, which might have been responsible for the change in common name from "old man" to "boy's love or lad's love." Description: The plant is a perennial erect shrub, 90 cm high, multibranched, with feathery graygreen leaves, 6 cm long, finely divided (thread-like), and somewhat downy, with a sweet, strong fragrance with a hint of lemon. Flowers are very small, inconspicuous, greenish-yellow, in loose panicles, and appear from late summer to early autumn. Distribution: The plant is native from Southern Europe but has been introduced in temperate zones as a garden plant, becoming naturalized in the eastern and central portions of North America and now widespread. Constituents: Essential oils, mainly absinthol; also an alkaloid called abrotine (Glasby 1991). Traditional medicinal uses (dried whole plant): Emmenagogue, febrifuge, antiseptic, antihelmintic, stimulant, and stomachic; used in aromatic baths and poultices for skin conditions. The leaves have been used in aromatic vinegars and can be rubbed on the skin as an insect repellent. Today, this plant is grown as an ornamental garden plant. A. absinthium L. (wormwood) is referred to in old herbals as a medicinal to counteract the effects of poisoning by hemlock and toadstools, to treat dyspeptic diseases, fever, and gastrointestinal worms (Wilbert 1991), and was used as an antifertility drug in antiquity (Riddle and Estes 1992). It also produces methyl jasmonate, a senescence-promoting substance (Ueda and Kato 1980). Wormwood is one of the bitterest herbs known ("as bitter as wormwood" is a very ancient proverb), and has been used as a principal ingredient in antiseptic fomentations. Roman wormwood (A. pontica 1.) or small absinthe refers to another distinct herb. Description: Perennial, erect-growing underbrush 0.75-1.50 m high; multibranched, hairy stems bearing highly aromatic bipinnate and trip innate gray-green leaves covered in down. Capitula are 3-4 mm in diameter, with graygreen bracts, and numerous yellow florets appearing from late summer to late autumn. Distribution: Central Europe, North America, and Asia. Widely introduced garden plant. Found native on waste ground, especially near the sea, in warm regions; adaptable to a wide range of soil types and soil pH. Constituents: Bitter principle is due to absinthin and anabsinthin; volatile oil (0.5-1.00/0 oil/fresh wt)

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stimulates secretions and promotes appetite, contains a- and f3thujone, phellandrene, thyjyl alcohol, cadinene, and azulene; also a glucoside; resins and starch; antihelmintic action due to santonin, a toxic sesquiterpene. Also found in this species are lirioresinol A; 3,6- and 5,6-dihydrochamazulene; the diterpenoid absinthin; and the sesquiterpenoids anabsin, anabsinin, artabsin, artabsinolides A, B, C, and D, artemolin A and B (Glasby 1991). Traditional medicinal uses: Whole flowering plant and leaves used as antihelmintic, antipyretic, antiseptic, antispasmodic, carminative, stimulant, tonic, and stomachic. The tincture was formerly used to treat nervous diseases, and the crushed plants in liniments. Wormwood was also used as a pain reliever for women during labor and as a cardiac stimulant. Despite its toxicity in high doses, wormwood had been used by brewers in place of hops (Humulus lupulus), as a flavoring for vermouth, bitters, and liqueurs, and, until 1912, as the basis for absinthe, a liquor containing oil of wormwood, anise, and other aromatics, now banned because of its toxicity. Side effects: A central nervous system depressant; habitual use causes convulsions, stupor, nervousness, restlessness, and vomiting; overdose cause vertigo, cramps, intoxication, delirium and eventually death. The plant can cause contact dermatitis and is recognized as an insect (moth) repellent. A. dracunculus 1. cv. sativa (French tarragon) 1. is used as a spice in French cuisine. Plants of the "true" French tarragon, or estragon, are difficult to obtain and maintain. Even under ideal circumstances, its delicate flavor may revert to the coarser flavor of Russian tarragon (Erichsen-Brown 1989). French and Russian tarragon both originated in Russia. Russian tarragon is hardier and the seeds are fertile. French tarragon rarely produces fertile seeds and is considered a sterile derivative of the wild Russian tarragon. Both tarragons are used on the commercial market as a condiment herb and as a culinary garden herb, but the French tarragon is considered to be the true tarragon and is the desired marketed type. Russian tarragon is continually introduced into commerce inadvertently as growers obtain seeds from seed companies marketing it as French tarragon. Description: Perennial, multibranched woody plant that can reach up to 1.5 m in height; the plant is slightly hairy, with narrow lanceolate leaves, 3 to 8 cm long and 2 to 10 mm broad. Leaves are green and smooth on both sides with 1 or 2 basal lobes in each side, but can also be finely pubescent. The flowers are greenish-white and 4 mm in size, numerous, brownish on drooping stems. The disk flowers are sterile, the ovaries abortive. Distribution: The plant is cultivated extensively in Southern Europe, Israel, and the United States. Constituents: Essential oils, sometimes referred to as estragole oil,

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the fresh herb yields 0.5-2.5% oil, with dry tarragon yielding about 0.5-0.8% oil. The pale-yellow to amber oil contains aromatic volatiles with methyl chavicol (or estragole) as the principal component (>50% of the total oil). An oleoresin of tarragon is also commercially available. Traditional medicinal uses: Dried or fresh herb has no modern medicinal use, but it was once referred to as a "chief medicine" (Erichsen-Brown 1989). The root was formerly prescribed for toothaches and to stimulate the appetite; it was also considered a diuretic and emmenagogue. For culinary use, its delicate anise-flavored leaves are widely used as flavorings in vinegars, salads, beef, chicken, fish, stew, preserves and pickles, shellfish, herb butter, and eggs, and as an ingredient in mustards, perfumes, soaps, and liquors. Tarragon may also act as an antioxidant in foods. A. vulgaris 1. (mugwort or St. John's herb) is an ancient plant, deeply respected throughout Europe and Asia, and once known as the "mother of herbs" (mater herbarum). It was one of the 9 herbs employed to repel demons and venoms in pre-Christian times. Although used to flavor drinks, particularly beer, the common name derived from the old Saxon muggia wort, meaning midge root, after its ability to repel insects. Japanese mugwort comes from another species, A. princeps Pamp. Description: Erect, sparsely pubescent, perennial; stems grooved and reddish-purple, angular, reaching 1. 75 m; leaves 2.5-5 cm long, pinnate or bipinnate with toothed leaflets, dark green on the adaxial surface and whitish and downy on the abaxial surface. Flowers brownish-yellow to red, numerous and small, arranged on panicles and appearing late summer to mid-autumn. According to Hall and Clements (1923), no other Artemisia is as variable in its morphology as A. vulgaris. Distribution: Asia and Europe. Naturalized in North America. Found in many soil types, along hedgerows, rivers, and streams. Constituents: Volatile oils, resin, tannin, and a bitter principle, absinthin, which stimulates digestion. Traditional medicinal uses: Dried flowering shoots, leaves, roots used as a diuretic, emmenagogue; stimulates the appetite and helps digestion. The Chinese use the heated leaves for rheumatism. Used also as tea, for stuffing fowl and meat or fish. Repels flies and moths. Leaves have been used to flavor tobacco. Formerly used in flavoring and clarification of beer. Side effects: Large dosage and prolonged use injures the nervous system. B. Floral Biology

A. annua is a determinate short-day plant. Nonjuvenile plants are very responsive to photoperiodic stimulus and flower 2 weeks after

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induction (Fig. 6.3). In greenhouse studies, nonjuvenile plants flowered 2 weeks after being moved to 8-, 10-, or 12-h photoperiods, while plants under 16-, 20-, and 24-h photoperiods remained vegetative. In field studies in the northern temperate zone, the first flower buds were visible on 4 September when daylength was 12:57 h at Lafayette, Indiana (40 0 21' N). Greenhouse data suggested that induction occurred 2 weeks earlier when the daylength was 13:31 h (Ferreira et al. 1995a). However, temperature x photoperiod interactions have not been investigated. The nodding flowers (capitula) are greenish-yellow and 2 to 3 mm in diameter, with calyces composed of numerous, imbricated bracts

Fig. 6.3. Vegetative (A) and flowering (B) shoot of Artemisia annua. Bar size em. (Source: Ferreira and Janick [1995a].)

=1

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(Fig. 6.4A-C). These capitula are displayed in loose panicles containing numerous bisexual flowers (florets) in the center and pistillate marginal florets, the latter extruding stigmas prior to the central flowers (Fig. 6.4B). Both flowers have a synpetalous tubular corolla with the top split into 5 lobes in the hermaphroditic florets and into 2-3 lobes in the pistillate florets. The receptacle is glabrous, not chaffy, and triangular in shape. The stigma is bifid (Fig. 6.4F) with modified acuminate epidermal cells that assist in the capture of the pollen and is referred to as a pollen presenter. The pollen presenter in the Asteraceae is considered to be of the active type, where the growth of the style pushes the pollen presenter past the anthers, causing the pollen to be collected and then extruded for presentation and dispersal by wind action (Ladd 1994). The 5 stamens have bilocular anthers, turned to the center of the floret (introrse), and with the connective attached to the bottom part of the corolla, inferior to the top of the style (Fig. 6.4F). Each stamen has a lanceolate appendix at the top, which alternates with the lobes of the corolla (Ferreira and Janick 1995a). Ovaries are inferior and unilocular and each generates one achene (Bailey 1951) ca.l mm in length and faintly nerved. The pollen is tricolpate and relatively smooth, typical of anemophylous species (Fig. 6.4G,H). Pollen grains have an internal columellae-tecta complex configuration in the exine, which is common to all taxa of the tribe Anthemideae and seems to vary from 23 layers in A. annua (Skvarla and Larson 1965). The pollen is extremely allergenic as in other species of Artemisia (Mitchell 1975; Arora and Gangal 1991; Rantio-Lehtimaki et a1. 1992). The allergenic protein has been shown to be present on the surface of the exine (Park et a1. 1993). The plant is naturally cross-pollinated by insects and wind action, which is unusual in the Asteraceae (McVaugh 1984). The floral morphology of A. annua, described on the basis of both light and scanning electron microscopy (Ferreira and Janick 1995a), indicate that the capitula is adapted to self-pollination. Experimental data, however, indicates that self-pollination is not only rare but difficult to achieve by bagging (Peter-Blanc 1992), which infers the presence of self-incompatibility in this species as in other members of the Asteraceae (North 1979). C. Glandular Trichomes Trichomes and glands, which contain volatile monoterpenes and sesquiterpenes, are common to the Asteraceae (Mehrotra et a1. 1990).

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(E)

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(F)

500 IJrn 10 IJrn (G)

(H)

5IJrn

Fig. 6.4. Floral morphology of A. annua. (A) Nodding capitulum. (B) Expanded capitulum showing calyx with imbricated bracts (b), receptacle (r), marginal pistillate floret (p), and internal hermaphroditic (h) florets. Glandular trichomes are found abundantly on the receptacle, bracts, and florets of the capitulum. (C) Cross section of the involucre showing imbrication of bracts. (D) Unexpanded floret showing orientation of glandular trichomes. (E) Fully developed, turgid, glandular trichome, based on SEM. (F) Details of hermaphroditic floret with lobed anthers attached to basal portion of the corolla (c), pistil with bifid stigma (s), style (st), and ovary (o). Note that in a hermaphroditic floret, the stigma reaches this state of development only after pollen shed. (G) Tricolpate pollen grain with vestigial spines, characteristic of wind-pollinated species, and germination pores (gp) bulging from the furrows. (H) Pollen crosssection based on light microscopy shows details of bulging germination pores. (Source: Ferreira and Janick [1995a].)

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These terpenoids are localized in the heart-shaped glandular trichome found in species of this family (Kelsey and Shafizadeh 1980; Rodriguez et a1. 1976). Illustrations in floras indicate glandular trichomes on the florets of A. paniculata (Besser 1843), A. mauiensis (Degener 1939), A. annua (Makino 1960), and various other species (Hall and Clements 1923). Biseriate glandular trichomes have been reported on both leaf surfaces of A. nova (Kelsey and Shafizadeh 1980), in floral stalks of A. tridentata (Slone and Kelsey 1985), on both leaf surfaces and ovary surfaces of A. umbelliformis (Cappelletti et a1. 1986), and in the adaxial leaf surface of A. campestris ssp. maritima (Ascensao and Pais 1987). Biseriate glands were reported to be isolated and collected from various Artemisia species by Slone and Kelsey (1985). Biseriate glandular trichomes were observed in the leaves of A. annua (Duke and Paul 1993) at the earliest stage of development, but later became obscured by filamentous T-trichomes. Both biseriate and filamentous trichomes are arranged in two rows, in troughs, along either side of the leaf midrib, but are arranged randomly on the abaxial surface of the leaf and stems. Duke and Paul (1993) described the origin of biseriate glands in leaves, based on light, scanning, and transmission microscopy. The earliest stage of gland formation observed is the l-cell stage, in which a single epidermal cell enlarges and protrudes above the leaf surface. After considerable expansion, this cell divides anticlinally, and then both of the resulting cells divide periclinally. In the 1- to 4-cell stage, vacuoles are relatively small and plastids are proplastids with only a few unstacked thylakoids. The 6-cell stage contains chloroplasts with few stacked thylakoids and no starch grains; lack of starch grains is the only distinguishing feature between chloroplasts of glands and mesophyll tissues. The final 10-cell stage is the result of further periclinal cell division of the 2 apical cell layers. After all 10 cells form, the cuticular surface of the gland begins to separate from the cell wall, near the tip of the gland. The onset of cuticular detachment was considered to be associated with the onset of secretory activity. Subcuticular space borders the 6 apical cells of the gland. Cytoplasm is denser at this stage than earlier and, at this point, the two basal cells contain chloroplasts and relatively large vacuoles. The apical cell pairs generally have no chloroplasts and the subapical 2- cell pairs (4 cells) contain large, amorphous chloroplasts without starch grains. Transmission and scanning electron photomicrographs of bisseriate glandular trichomes are shown in Fig. 6.5. Ten-celled biseriate glandular trichomes are abundant in the bracts, receptacles, and florets of the capitulum in A. annua, as shown in

Fig. 6.5. Biseriate glandular trichomes of A. annua. (A) Scanning electron micrograph (SEM) of glandular trichome at the basal portion of the corolla with apical cells collapsed due to fixation process. (Source: Ferreira and Janick [1995a].) (B) Transmission electron micrograph (TEM) of a lO-cell stage gland. The arrows indicate the dense layer of osmiophilic material that comprises a portion of the cell walls of the two apical cells. The apical cell pair has no chloroplasts, but the subapical two cell pairs (4 cells) contain large, amorphous chloroplasts without starch grains. Bar size = 5 pm. (Source: Duke and Paul [1993].) w w w

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Fig. 6.4B,D,E (Ferreira and Janick 1995a). These glands are easily ruptured, releasing the volatile oils when florets are pressed, and occur in both pistillate and hermaphroditic florets from their earliest developmental stages. Five-celled T-shaped trichomes with an elongated top cell occur abundantly in stems, pedicels, and stipules but rarely in bracts of the capitulum. The floral biseriate trichomes originate in the same manner as leaf glandular trichomes. During floral development, the subcuticular space at the apex of glandular trichomes enlarges to a sac-like structure that surrounds 4 to 6 apical cells. These apical cells discharge fluids into the sac-like structure, which expands and bursts during trichome disintegration. Intact glandular trichomes were seldom observed in florets after anthesis, but a few reached the final stage of flower development intact. Duke et al. (1994) concluded that the subcuticular spaces of the biseriate glandular trichomes in leaves of A. annua are the sites of sequestration of artemisinin and artemisitene because both compounds were lacking in a glandless biotype, and virtually all artemisinin and artemisitene could be extracted by a 5-s leaf dip in chloroform, without visible damage to other leaf epidermal cells. Artemisinin was also extracted by a i-min dip of inflorescences in petroleum ether or acetonitrile (Ferreira and Janick 1995a). Artemisinin appears to be sequestered in biseriate foliar and floral glandular trichomes of A. annua. Although glandular trichomes are present in the early stages of flower formation, artemisinin content peaks when flowers approach full bloom (anthesis). This suggests that artemisinin must be produced or sequestered in these glands as they mature. Immunocytochemical localization of artemisinin will be necessary to confirm these glands as the sites of artemisinin synthesis. D. Secondary Metabolites 1. Aromatic Volatiles. The highly aromatic volatile or essential oils

of A. annua, composed primarily of terpenoids, with some phenylpropanoids and nonvolatile fatty acids, are abundant in the leaves and flowers, with only trace amounts found in the main or side stems and roots (Charles et al. 1991). Highest yields of essential oils have been reported at flowering (Simon et al. 1990). The volatiles of A. annua are extracted via hydrodistillation or steam distillation and have been chemically characterized by gas chromatography and GC/mass spectroscopy (Libbey and Sturtz 1989; Lawrence 1990;

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Simon et al. 1990; Charles et al. 1991; Woerdenbag et al. 1993a; Ahmad and Misra 1994; Woerdenbag et al. 1994a). More than 60 constituents are reported in the distilled oil (Table 6.1), and significant variation in both oil content and oil composition within this species has been noted (Charles et al. 1991; Woerdenbag et al. 1993a, 1994a). The major volatile constitutents (>5 % of the total essential oil, and highest reported values for each compound) include artemisia ketone (6S.5%, Charles et al. 1991), 1,S-cineole (31.5%, Libbey and Sturtz 19S9), camphor (27.5%, Charles et al. 1991); germacrene D (lS.90/0, Woerdenbag et al. 1994a), camphene hydrate (12%, Charles

Table 6.1.

Essential oil constituents in Artemisia annua.

Allo-aromadendrene a-Copaene a-Humulene a-Pinene a-Terpinene a-Terpineol Limonene a-Thujene a-Thujone Artemisia alcohol Artemisia ketone Benzyl isovalerate Benzyl 3-methyl butyrate Borneol (3-Bisabolene (3-Cadinene (3-Caryophyllene (3-Cubebene (3-Farnesene (3-Pinene cadinene Camphene Camphene hydrate Camphor Caryophyllene oxide Chrysantheone cis-Chrysanthenol cis-Chrysanthenol acetate cis-Sabinene hydrate (E)-6-Methyl-3,5-heptadien-2-oney Fenchol

y-Cadinene y-Elemene y-Muurolene y-Terpinene Germacrene-D Isoeugenol Isopinocamphone Linalool Longipinene Myrcene Myrtenal Myrtenol n-Nonacosane n-Pntacosane p-Cymene p-Ethylcumene Pinocarvone Sabina ketone Sabinene Sabinol Santolinatriene Terpinen-4-ol trans-2, 7-Dimethyl-4,6-octadien-2-ol trans-Pinocarveol trans-Sabinene hydrate 4-Tetramethyl ether 3,7,7-Trimethylbicyclo[3.1.1]-2-heptene Unknown (MW 220) Yomogi alcohol 1,8-Cineole

Source: Anon (1982), Libbey and Sturtz (1989), Lawrence (1990), Charles et al. (1991), Woerdenbag 1993a, 1994a, and Ahmad and Misra (1994).

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et al. 1991), a-pinene (16%, Charles et al. 1991), ~-caryophyllene (8.6%, Woerdenbag et al. 1994a), myrcene (8.5%, Woerdenbag et al. 1994a); and artemisia alcohol (7.5%, Woerdenbag et al. 1993a). Borneol was erroneously reported as artemisia ketone in earlier literature reports (Ahmad and Misra 1994). 2. Nonvolatile Sesquiterpenes. Many of the sesquiterpenes of A.

annua are nonvolatile, and, thus, not released upon water or steam distillation, and have little role in aroma or fragrance. These compounds can be denatured by the process of oil extraction. These nonvolatile sesquiterpenes can be recovered from the plant by solvent extraction, and some show high antimalarial activity. At least 20 known sesquiterpenes are produced by A. annua, including arteannuin A (artemisinin), arteannuin B, artemisitene (Acton and Klayman 1985), artemisinic acid, artemisilactone, qinghaosu I, II, III, and IV (Glasby 1991), artemisinin G (Wei et al. 1992), annulide (Brown 1993a); deoxyisoartemisinin, deoxyisoartemisinin C, compound 5 (unnamed), 6,7-dehydroartemisinic acid, artemisinin C, qinghaosu VI, and deoxyartemisinin (Ranasinghe et al. 1993), plus 3-isobutyrl cadin-4-en-11-01, cadin-4(7),11-dien-12aI, cadin-4(15),11-dien-9-one (Ahmad and Misra 1994). Almost all belong to the cadinane series (Brown 1993b), and many are bioactive (Duke et al. 1988), except for the last 3 cadinanes, which have not been evaluated. No other cadinanes have been reported outside this species. Further discussion on the chemistry of these compounds is found in Section IV. 3. Chemotaxonomy. The widespread occurrence of volatile and nonvolatile monoterpene and sesquiterpenes in the Asteraceae has led to the use of these compounds as possible taxonomic markers in the classification of this family as well as in the treatment of the genus Artemisia. In the earlier 1923 morphological classification of Hall and Clements, A. annua, A. absinthium, and A. vulgaris were all included in the section Abrotanum (Seaman 1982). Kelsey and Shafizadeh (1979) reviewed the systematics of Artemisia based on its chemical constituents and concluded thatA. annua belongs within the section Abrotanum, because it contains novel nonvolatile sesquiterpenes such as arteannuin-B and artemisinin, but placed A. absinthium in the section Absinthium. Artemisinin and other cadinanes are unique to A. annua, with the possible exception of A. apiacea (Liersch et al. 1986). In contrast, many of the same monoterpenes are common to several species. For example, based on the volatile oils, A. annua could appear to be similar to A. absinthium

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(section Absinthium), but the presence of artemisinin and related derivatives brings A. annua chemotaxomically closer to species belonging to the section Abratanum. High concentrations of artemisia ketone and its derivatives in the volatile oils of A. annua suggest possible systematic significance since these compounds have not been found outside tribes of the Asteraceae, but some chemotypes of A. annua are devoid of this compound. Because of the widespread occurrence of both monoterpenes, and in particular certain sesquiterpenes in the Asteraceae, Seigler (1981) argued that the use of these chemical constituents are of limited value in establishing the phylogeny of higher plants. Greger (1982) suggested that the distribution of different polyacetylenes, coumarin-sesquiterpene ethers, and sesamin-type lignans, rather than the aboveground monoterpenes, sesquiterpene lactones, or flavonoids, is a better chemical marker for differentiating species within the section Artemisia. The sections Abratanum and Absinthium produce the most structurally diverse polyacetylenes and species of A bratan um produce the most structurally diverse and biosynthetically complex sesquiterpene lactones within the Asteraceae (Kelsey and Shafizadeh 1979). The differential accumulation patterns between spiroketalenol ethers, found in sections Artemisia and Absinthium, and that of pontica epoxides, as found in Abratanum (in which A. annua resides), may also be useful markers in separating sections (Greger 1982). Yet, A. annua, which accumulates high epoxides, is also considered phylogenetically close to A. biennis Willd., A. taurnefartiana Reich., and A. klatzschiana, all of which accumulate high concentrations of spiroketalenol ethers (Hall and Clements 1923; Poljakov 1961), making the differential accumulation of these compounds of lesser significance in classification. The sections Drancunculus and Seriphidium, in contrast, accumulate dehydrofalcarinone derivatives. III. HORTICULTURE A. Field Cultivation Field cultivation of A. annua is presently the only commercially viable method to produce artemisinin because the chemical synthesis of this complex molecule, while possible, is uneconomical. Traditionally, A. annua has been collected from wild stands in China, which still provide much of the raw material that is harvested and processed for human clinical trials as a new antimalarial drug in

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southeast Asia (WHO 1994). As interest and use of artemisinin has increased, a number of studies have been carried out to introduce A. annl1a as a cultivated crop, (Simon et al. 1990; Laughlin 1993,1994). Because A. annl1a is a short-day annual, cultivation will be unadapted in the tropics, where plants will flower without achieving sufficient biomass. Trials on A. annl1a as a field crop have been carried out in Australia (Laughlin, 1993, 1994), Brazil (Magalhaes 1994), India (Singh et al. 1986, 1988), Switzerland (Delabays et al. 1992, 1993), the United States (Simon and Cebert 1988; Simon et al. 1990; Morales et al. 1993, Ferreira et al. 1995a), and Vietnam (Woerdenbag et al. 1994a) with encouraging results. Germplasm. Investigations on A. annl1a germplasm for artemisinin in many countries has indicated highly variable artemisinin content, sometimes as low as 0.01 % (Trigg 1990). In the United States, strains have been detected with mean artemisinin concentrations ranging from 0.05 to 0.21 % and individual plants producing up to 0.42 % at the full flowering stage (Ferreira et al. 1995a). Swiss researchers reported clones of Chinese origin that produce 1.1 % artemisinin (Delabays et al. 1993), but these clones have been unavailable to most researchers. Hybrids between these high artemisinin, low vigor, Chinese clones with vigorous, low artemisinin clones of Italian (0.04%), Yugoslavian (0.16%), and Spanish (0.22%) origin contained 0.7 to 0.8% artemisinin combined with high vigor (Delabays et al. 1993). The difficulty in developing high biomasss yielding lines or cultivars rich in artemisinin is due in part to the unavailability of germplasm sources, the absence of methods for hybridization, and the lack of an inexpensive, rapid screen for artemisinin and its derivatives. Evaluation of germplasm of A. annl1a for essential oils has been conducted in the United States, and accessions rich in specific oil constituents such as artemisia ketone, camphor and 1,8-cineole have been identified (Charles et al. 1991). These accessions could be used to develop lines with custom designed oil profiles within the genetic limits of the germplasm. Artemisia annl1a of Chinese origin contains artemisia ketone as a major component (Charles et al. 1991, Ahmad and Misra 1994), while A. annl1a of Vietnamese origin lacked this compound (Woerdenbag et al. 1994a), suggesting large genetic variation in essential oil composition. Given the range of essential oil composition reported in germplasm collections, cultivars of A. annl1a with high oil or distinct volatile oil profiles could be developed for the flavor and fragrance industry. 1.

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2. Crop Culture. Although asexual propagation is easily achieved by cuttings from vegetative plants, clonal propagation from elite clones for field production would require that stock plants be maintained under long days to prevent flowering. Seed propagation, however, is the most practical and economic method for commercial propagation. Artemisinin seeds keep their vigor for at least 3 years if stored dry under cool conditions. In temperate zones the minuscule seeds that self sow, germinate early in the spring in the field of the following year. As such, A. annua has become naturalized as a weedy annual, adapted to both fertile farm land and waste areas. Specialized seed planters can uniformly sow the very small seed. The seeds can be coated or mixed with inert materials for improved sowing and seed distribution. Shallow planting coupled with frequent light irrigations has worked relatively well to achieve good stands. The most important problem to overcome in crop culture is uniform crop establishment and weed control. Most researchers (Acton et al. 1985; Liersh et al. 1986; Singh et al. 1988; Laughlin 1993; Morales et al. 1993) transplant artemisia to the field at the 5-6 leaf stage. This usually requires 4 to 6 weeks of greenhouse growth. In areas with sufficiently long growing seasons, direct seed planting may produce similar dry matter and artemisinin yields (Laughlin 1993), and could be a practical alternative to transplanting with effective preplanting herbicide programs. Although weed control is one of the major production costs, no herbicides are registered for use in the United States on A. annua and the prospects for new registrations appear bleak. Future production strategies must be developed with and without the application of herbicides. Early-season weed control is essential to allow vigorous crop development, since few weeds develop under the canopy. Several herbicides have been identified that could be used in production. Chloramben at 2.2 (a.i.) kg/ha before emergence or trifuralin at 0.6 kg/ha, incorporated before transplanting, followed by fluazifol at 0.2 + 0.2 kg/ha broadcast after emergence, and acifluorfen at 0.6 kg/ha after emergence gave good weed control without reduction of leaf yield or artemisinin content in Mississippi (Bryson and Croom 1991). Preplant application of 2.2 a.i. kg/ha napropamide resulted in good weed control without phytoxicity (Simon and Cebert 1988). Studies on time of planting indicate that in northern temperate zones, late spring or early summer plantings are best to achieve high biomass yields. The key step to maximize artemisinin yields is to achieve high biomass before the onset of floral induction, which is

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initiated when the photoperiod is ca. 13.5 h. Late-season planting of A. annua results in very short flowering plants with low yields. There

is a possibility of fall sowing with the overwintering of young seedlings but little information is available on winter survival in geographical zones in which this technique might be successful. In India, establishment is best achieved by planting in the cool period of mid-December, while in Tasmania, October plantings doubled leaf dry matter as compared to November plantings (Laughlin 1994). In Indiana, plants were field transplanted every month from April through July and sampled biweekly for growth and oil accumulation. Maximum essential oil production occurred at peak flowering. May and June transplanting dates resulted in the highest essential oil yields (Simon et al. 1990). A. annua responds well to balanced fertilizers, and appears to be responsive to nitrogen (relative to growth), but there are few data available on the accumulation of artemisinin relative to fertility. Srivastava and Sharma (1990) reported that micronutrients such as boron may increase artemisinin concentration, but it is unknown whether the soils used were deficient or whether artemisia is responsive to boron. Field experiments in Tasmania (Laughlin 1993) indicated that lime increased dry matter yields in soils of pH 5.0. In greenhouse trials, leaf yields were optimal and remained relatively constant from pH 5.5 to 7.4, and pH had little effect on artemisinin content. Water stress during the 2 weeks before harvest led to reduced leaf artemisinin content (Charles et al. 1993). Shukla et al. (1992) reported that exogenous application of plant growth regulators may increase artemisinin content as well as plant height, but further studies are needed to determine whether growth regulators can be used effectively. Initial artemisinin yield studies in Switzerland were carried out at plant densities of 2.5 plants/m 2 (Delabays et a1. 1993). In Tasmania, dry matter yields increased from 2.0 to 6.8 t/ha as density increased from 1 to 20 plants/m 2 ; plant density did not affect artemisinin or artemisinic acid content (Laughlin 1994). In Indiana, biomass yield increased from 2.7 to 11.1 plants/m 2 at 0,67, and 134 kg N/ha, but optimum biomass was achieved at 67 kg N/ha (Simon et a1. 1990). Hybrids obtained from Switzerland and tested in Campinas, Brazil, between September and November produced 2.15 t of dried leaves/ha, 6 kg/ha of artemisinin, and 14.6 kg/ha of artemisinic acid when spaced 0.3 x 0.5 m (Magalhaes 1994). Increasing density also increased essential oil production per area as a direct result of the increase in total biomass (Simon et a1. 1990).

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For commercial oil production, plants can be field cut, allowed to partially air dry, and then be steam distilled with excellent results. The mechanized systems used in commercial mint production and distillation work well with A. annua. For the herbal trade as a dried aromatic wreath, production recommendations differ, since individual phenotypic characteristics are critical for the drying and shaping of individual plants. Here, greater spacings between plants results in higher number of side or lateral shoots, and an increased dry weight gain per plant, desirable traits for the ornamental industry. Studies examining the influence of topping on subsequent plant growth resulted in a significant reduction in final plant height, biomass yield, and lateral shoot length or spread (Simon and Cebert 1988). Plants have been identified that exhibit different growth habits and flowering times, indicating the potential to select for ornamental traits in a given population of A. annua. 3. Harvest. Studies by Ferreira et al. (1995a) in which artemisinin was evaluated separately in leaves and inflorescences clearly indicate that artemisinin content is highest in inflorescences with maximum concentration close to or at anthesis. This is confirmed in a number of greenhouse and field trials, where peak artemisinin was achieved during full flowering (Singh et al. 1988; Pras et al. 1991; Morales et al. 1993; Ferreira et al. 1995a), although others report artemisinin content highest with harvest just before flowering (Acton et al. 1985; Liersch et al. 1986; EI-Sohly 1990; Woerdenbag et al. 1991, 1994a; Laughlin 1993). Because artemisinin content is relatively low, harvesting is best carried out when artemisinin content per unit area is at a maximum, in order to reduce extraction and processing costs. Thus, the optimum time of harvest must take into consideration maximum artemisinin content as well as biomass yield. Recently, there has been some interest in producing artemisinin from artemisinic acid (arteannuic or qinghao acid), which is 8- to 10-fold more abundant than artemisinin (Roth and Acton 1987; Jung et al. 1990a; Laughlin 1993; Vonwiller et al. 1993). If this technique should prove feasible, optimum harvest will have to take both compounds into consideration. The concept of harvesting artemisia for both essential oils and artemisinin or artemisinin derivatives warrants exploration. If artemisinin is the targeted product this may not be possible, since steam distillation could destroy the peroxide bridge. If artemisinic acid, which does not contain a peroxide bridge but can be converted

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to artemisinin (Sangwan et al. 1993), is the targeted chemical, then dual procurement of essential oils and artemisinic acid may be feasible. The harvest of A. annua for the floral and craft trade is much different than that for the extraction of secondary products. Plants need to be harvested 15-25 cm above the ground and should be taller than 1.5 m. For the craft trade, plants are harvested at full bloom, prior to seed maturation, as the color changes from dark green to a more golden color. For the floral trade, plants are harvested later as the seeds develop. Once the plants are harvested, they are usually dried in a shaded area or under forced heated air, not to exceed 35°C. Quality of this product is based mainly on aroma and visual appearance (color, size, shape of plant, and stage of development). Postharvest Handling. Because of the tremendous bulk of biomass produced by A. annua, plant material has to be at least partially dried before processing. Ferreira et al. (1992) compared artemisinin content by HPLC-EC from freeze-dried, oven-dried (40°C), and indoor air dried A. annua shoots. Highest artemisinin yields were obtained with indoor air drying (0.13%) as compared to oven drying (0.100/0) and freeze drying (0.02%). Time of air drying (2, 4,6, or 8 days) did not affect artemisinin content. Microwave (2 min, 100% power) drastically reduced or eliminated artemisinin from 5.0 g fresh wt leaf samples, and artemisinin was reduced by ca. 50% when the samples were microwave dried for 5 min at 50% power (Ferreira et al. 1992). However, microwaving of standard artemisinin solutions for 5 min at 50% power caused no artemisinin loss as determined by HPLC-EC (J. F. S. Ferreira, unpublished). 4.

B. In Vitro Culture Many investigators have reported successful in vitro propagation of A. annua via shoot cultures (He et al. 1983; Nair et al. 1986; Martinez and Staba 1988; Fulzele et al. 1991; Elhag et al. 1992; Whipkey et al. 1992; Woerdenbag et al. 1993c; Brown 1993b; Ferreira and Janick 1996b). Shoots are easily cultured using standard protocols and cytokinin supplementation. Benzyladenine (BA) and coconut water are effective in inducing shoot formation (Whipkey et al. 1992). In vitrogrown plants readily acclimate to soil. However, many plants derived from tissue culture appear to have cytokinin abnormalities, being highly branched and bushy without apical dominance. Such plants do not flower unless their growth habit reverts to normal (Ferreira 1994).

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Callus can be obtained with media supplemented with combinations of auxin and cytokinins (Martinez and Staba 1988; Kim et al. 1992; Brown 1993; Ferreira and Janick 1996b), but nonfriable callus is usually obtained (Nair et al. 1986). The highest yields of friable callus were obtained with 4.4 /-lM BA and 4.5 f.lM 2,4-D, but only 10% of the clones generated callus (Ferreira et al. 1996). Cell cultures can be obtained with difficulty from callus cultures in liquid callus induction medium. Artemisinin appears only in trace amounts in undifferentiated callus and cell cultures (Nair et al. 1986; Kudakasseril et al. 1987; Liu et al. 1992, Woerdenbag et al. 1992, Ferreira et al. 1996) or not at all (He et al. 1983; Tawfiq et al. 1989; Fulzele et al. 1991; Brown 1993b; Ferreira and Janick 1996b), suggesting that a certain degree of differentiation is required for artemisinin production (Martinez and Staba 1988, Fulzele et al. 1991, Brown 1993b). The medium from cell cultures had no detectable levels of artemisinin (Fulzele et al. 1991; Ferreira and Janick 1996b), but Nair et al. (1986) reported low levels of artemisinin from culture medium from callus. There are inconsistencies in the literature regarding the presence of artemisinin in different organs of in vitro-grown plants. Artemisinin is produced by differentiated shoot cultures, Le., shoots plus roots (Martinez and Staba 1988; Fulzele et al. 1991; Whipkey et al. 1992), but occurs at trace levels, if at all, in shoots without roots (Martinez and Staba 1988; Jha et al. 1988; Fulzele et al. 1991; Woerdenbag et al. 1993b; Paniego and Giulietti 1994, Ferreira and Janick 1996b). Most workers (Martinez and Staba 1988; Tawfig et al. 1989; Fulzele et al. 1991; Kim et al. 1992; Ferreira et al. 1996) did not detect artemisinin in roots in vitro, although Nair et al. (1986) and Jha et al. (1988) reported trace amounts. Recently, hairy root cultures of A. annua have been produced by transformation with Agrobracterium rhizogenes (Weathers et al. 1994; Jaziri et al. 1995). Weathers et al. (1994) reported high artemisinin levels (0.4%), but those hairy root cultures have been unstable (P.J. Weathers, personal communication) and Jaziri (1995) could not confirm the presence of artemisinin in hairy root cultures. Because artemisinin is highly phytotoxic (Duke et al. 1987) and appears to be sequestered only in glandular trichomes (Duke et al. 1994; Ferreira and Janick 1995a), the reports of high artemisinin in root cultures appears suspect, but, if true, could have a profound effect on our understanding of artemisinin biosynthesis. A number of studies have evaluated the effect of growth substances on the production of artemisinin from shoot cultures. Artemisinin increased 7 times in shoot cultures treated with 100 f.lg/mL of

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miconazole for 6 weeks (Kudakasseril et al. 1987). Woerdenbag et al. (1993b) reported 0.16% artemisinin in shoot cultures maintained in Murashige and Skoog medium supplemented with 0.2 mg/L BA, 0.05 mg/L NAA, and 1% sucrose. Artemisinin increased with 10 mg/L GAs (54%), 0.5 giL casein hydrolysate (69%), and 10 or 20 mg/L naftine (40%), while other growth regulators, such as miconazole and terbinafine; elicitors, such as cellulase, chitosan, glutathion, and nigeran; the precursor mevalonic acid; and gene regulators, such as 5-azacytidine and colchicine had a negative effect or no effect on shoot artemisinin production. Whipkey et al. (1992), based on unreplicated data, reported that 6-benzylaminopurine (BA) at 0.4 J.lM (1.0 mg/L) and kinetin at 46.5 J.lM (10 mg/L) increased the yield of artemisinin in shoot cultures about 30%, but this increase was due to an increase in dry matter production, which overcame a decrease in artemisinin content (in mg/g dry wt). Daminozide at 0.6, 6.2, and 62.4 J.lM and chlormequat (CCC) at 0.6,6.3, and 63.4 J.lM were evaluated because growth retardants which influence gibberellins have been shown to increase terpene formation (EI-Keltawi and Croteau1986, 1987). Daminozide and CCC greatly reduced dry weight of shoot cultures at all concentrations, but increased artemisinin content (mg/g dry wt) at all but the lowest concentration of CCC. This experiment was repeated using replicated treatments and an improved HPLC-EC procedure for detection and quantification of artemisinin (Ferreira and Janick 1996a). None of the growth regulators significantly increased artemisinin accumulation, although artemisinin content with CCC at 6.3 J.lM was higher than the control (0.019 vs. 0.014%). Artemisinin was undetected in the presence of BA at 4.4 J.lM and kinetin at 4.6 and 46.5 J.lM.

In this experiment, there was a significant correlation over all treatments (r = .775, P = 1%) between root number and artemisinin content. In a separate study on the effect of BA (0.0, 0.5, 5.0, and 50 J.lM), shoot production was maximal at 5.0 J.lM BA, but rooting and artemisinin decreased as BA increased with rooting and artemisinin highest in BA-free medium. BA also increased shoot vitrification. Martinez and Staba (1988) also reported an increase in artemisinin when plants developed a root system. Roots do not contain artemisinin but evidently enhance its production in cultured shoots. Removal of roots from shoots cultured in hormone-free, liquid medium reduced shoot artemisinin by 53% (Ferreira and Janick 1996b). Shoot cultures in hormone-free medium consistently produced roots and contained artemisinin levels as high as 0.280/0 in some clones (Ferreira et al. 1995b), the highest reported leaf artemisinin derived

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from differentiated shoot cultures. Thus far, observations from in vitro culture of A. annua indicate that this process is unlikely to be feasible for the commerical production of artemisinin. Tissue culture was evaluated by Ferreira et al. (1995b) as a system to store clones and as a system for selection of high artemisinin. Artemisinin content was ca. 40% higher for greenhouse than for tissue culture-grown plants, but the correlation between artemisinin content for the same clones grown in vitro and in the greenhouse was r = .50, (P = 5%), indicating that only 25% of the variability was accounted for by regression. When tissue-cultured plants were reanalyzed after 2 years of culture, artemisinin content was 20% lower and the correlation coefficient between the two analyses was r= .61, (P 1%). These relatively low correlation coefficients indicate that evaluation of artemisinin content from tissue-cultured plants is an unreliable procedure to estimate artemisinin content. The results may be an artifact of tissue culture, possibly due to somaclonal variation or adaptive changes. C. Genetic Improvement Broad-sense heritability for artemisinin production, the ratio of genetic to total variation (genetic + environmental) was estimated from asexually propagated clones derived from a random mating population in greenhouse and field trials (Ferreira et al. 1995b). Heritability estimates varied from 0.910 (greenhouse, individual basis) to 0.985 (combined field and greenhouse, family basis). These high broadsense heritability estimates indicate that artemisinin content in A. annua has a high genetic component, i.e., heritability plays a key role in the trait. Delabays et al. (1993) fertilized a high-yielding (over 1%), low-vigor, Chinese clone of A. annua, which was induced to flower under controlled greenhouse conditions with pollen from Italian, Yugoslavian, and Spanish origins. These hybrids produced on average 0.64,0.73, and 0.95% artemisinin, respectively, from a yield of dry leaves of about 2000 kg/ha. The fact that artemisinin content between high and low lines is intermediate suggests that artemisinin content is controlled by additive genetic factors. Artemisinin content of nonflowering plants grown under long days in the greenhouse was found to be highly correlated (r = .93 to .95, P = 1 0/0) with the same clones grown under the long days in the field (Ferreira et al. 1995b). Thus, greenhouse evaluation of A. annua under long days has the potential to be an efficient system to select for high-artemisinin-yielding clones. Superior clones could be induced

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to flower under short days and could then be intercrossed. Repetition of this cycle on progeny would be expected to lead to genetic gain for artemisinin production. IV. PHARMACOLOGY OF ARTEMISININ AND DERIVATIVES A. The Challenge of Malaria Malaria, a disease known in antiquity, was referred to in Egyptian writings of the sixteenth century B.C., with symptoms of shivering, fever, and spleen enlargement. Followers of Hippocrates in the fifth century B.C. described the recurrence of fevers at regular intervals and observed a connection of the disease to marshes (Klayman 1989). In the seventeenth century, Italians believed that breathing bad air (mal aria) arising from swamps was responsible for the disease, and in the first half of the nineteenth century the term malaria entered the English medical literature. The names of the disease in French (paludisme) and Spanish (paludismo) are derived from the Latin palus, meaning marsh. In 1899, the mystery of malaria transmission was solved independently, by Ronald Ross, an English physician working in India, and Giovanni Battista Grassi, an Italian physician, who proved that the disease was spread by the bite of the female Anopheles mosquito (De Kruif 1939). The existence of malaria in pre-Columbian America is controversial; some suggest that the disease first appeared with the importation of slaves from Africa (Klayman 1989). Human malaria is caused by four major Plasmodium species: falciparum, vivax, malariae, and ovale. Plasmodium falciparum, the prevailing parasite species in most of the world, causes the most severe form of malaria (cerebral), which often is fatal to children (Wirth et al. 1986) and nonimmune adults. Malaria, one of the most devastating diseases in the tropical world, is on the increase (Miller 1992). Over 300 million clinical cases occur worldwide, resulting in up to 2.7 million deaths annually. Most of these cases occur in Africa, but large areas of Asia, Central, and South America have high incidences of the disease (Nussenzweig and Long 1994). Malaria has been essentially eradicated from the United States and Cuba, but about 1000 cases are reported annually in the United States, mainly travelers, migrant workers, and military personnel, and, rarely, in people living around international airports. Malaria poses a major barrier to the economy of developing tropical countries, and its control is an important goal for improved world health (Wirth et al. 1986).

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Immunity to malaria builds up only after several years of recurring infections but is only partially effective. An effective vaccine, if released at affordable price, may offer the best long-term control option for malaria. However, until this occurs, the best approach appears to be the use of quinine-derived drugs, in areas where resistant strains of Plasmodium are not present, or alternative drug therapies, such as semi-synthetic derivatives of artemisinin, with proved efficacy against multidrug-resistant strains of P. falciparum and little or no side effects. B. Chemistry Artemisinin is a sesquiterpene lactone belonging to the cadinane series. In addition to a lactone group, artemisinin contains an endoperoxide bridge, which is rare in secondary metabolites and, unlike other antimalarials, lacks a nitrogen-containing ring. The integrity of the stereo structure of the ring system has also been reported as vital for antimalarial activity (Anon. 1982; Luo and Shen 1987), and artemisinin-related compounds without the peroxide or with only one oxygen in the bridge (deoxyartemisinin and derivatives) are devoid of antimalarial properties. Artemisinin is an odorless and colorless compound and forms crystals with a melting point (mp) of 156-157°C. Its molecular weight, determined by high-resolution mass spectroscopy, is mle 282.1742 M+ (Luo and Shen 1987). It has an empirical formula of C15H2205 and shows no absorption in most of the UV range. In the IR region there is a peak at 1745 cm-1that corresponds to a strong gamma lactone function, and there are other peaks at 831, 881, and 1115 cm-l, which correspond to the peroxide function (Anon. 1982). The lH-NMR and 13C-NMR spectra led to the assignments of three methyl groups (one tertiary and two secondary), and an acetal function (Luo and Shen 1987). Artemisinin is surprisingly stable in neutral solvent heated up to 150°C (mp), or up to 50°C above its melting point (200°C) for 2.5 min in pure form (Lin et al. 1985). The molecule is also light stable (Anon. 1982). The peroxide bridge is considered the most chemically reactive moiety of artemisinin. Catalytic hydrogenation of the molecule produces deoxyartemisinin, an epoxide devoid of antimalarial activity (Anon. 1982). When reduced with sodium borohydride (NaBH 4 ), the lactone function of the molecule is converted into a lactol (hemiacetal) known as dihydroartemisinin in which the peroxide bridge is intact. When dihydroartemisinin is crystalline, it is in the ~ form, and when in solution, it is a mixture of a and ~ epimers.

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Dihydroartemisinin can be converted into either ethers or esters (Luo and Shen 1987). Reduction of artemisinin with NaBH 4 , using boron trifluoride as a catalyst and dry tetrahydrofuran as the solvent, resulted in 71 % deoxoartemisinin. This compound is ca. 8 times more active against chloroquine-resistant malaria in vitro than artemisinin (Jung et al. 1990b). C. Isolation and Synthesis The first published laboratory procedure for isolation of artemisinin has been described by Klayman et al. (1984). Air-dried leaves were extracted with petroleum ether (bp 30 to 60°C), which was subsequently removed in vacuo. The residue was redissolved in chloroform to which acetonitrile was added to precipitate inert plant components such as waxes. The concentrated extract was then chromatographed on a column of silica gel. Fractions with a high artemisinin content crystallized readily; recrystallization was achieved with cyclohexane or 50% ethanol. Complete chemical (de novo) synthesis of artemisinin was achieved by Schmidt and Hofheinz (1983), Xu et a1. (1986), Ravindranathan et a1. (1990), and Avery et al. (1992). Each procedure for the chemical synthesis of artemisinin requires a final photooxidative step. Low yield, complexity, and high cost of the de novo synthesis suggests that isolation from the plant is the optimum system. However, there have been great advances in the synthetic chemistry of artemisinin and related compounds, which have enabled the radiolabeling of compounds to be used in studies on pharmacokinetic, metabolism, mode of action, and toxicity. Synthesis of greatly modified structures has provided a better understanding of structure-activity relationships and led to the synthesis of analogs with considerably increased antimalarial activity. Although artemisinin is the starting material for the synthesis of other more soluble and stable compounds, the most abundant sesquiterpene in A. annua is artemisinic acid (arteannuic acid, qinghao acid), which occurs at specific content 8- to 10-fold higher than artemisinin (Roth and Acton 1987; Jung et a1. 1990a), followed by arteannuin B (Klayman 1993). The usual extraction method for artemisinin neglects artemisinic acid, but recently Vonwiller et al. (1993) devised an efficient method to extract both artemisinic acid and artemisinin from the same material. Artemisinic acid can then be converted to artemisinin (Xu et al. 1983), which greatly increases the yield of artemisinin.

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D. Detection and Quantification 1. Sample Preparation. Shoots of A. annua should be air dried before sample preparation and analysis. A low-temperature (ca. 40°C), air-flow oven can be used for drying, if a large biomass is involved, and if the humidity is too high for air drying. Fresh samples should not be used for extraction and analysis of artemisinin because artemisinin content detected in fresh samples may be lower than dried samples. This appears to be an artifact of the extraction because, although artemisinin is poorly soluble in water (Lin et al. 1989), part may be lost in the warm (45°C) aqueous phase under the petroleum ether fraction. Extraction of fresh samples might be acceptable for rapid screening of clones by enzyme immunoassays if the samples are dissolved in DMSO, methanol, or other water-miscible solvent. High-performance chromatography with electrochemical detection (HPLC-EC) was able to detect artemisinin in dried samples of 10 mg but samples smaller than 500 mg are less reliable (Ferreira et al. 1992). 2. Chemical Detection. Artemisinin has been detected and quantified by many different methods in the last 20 years. These include thin-layer chromatography (TLC) (Tu et al. 1982; Klayman et al. 1984; Luo and Shen 1987; Roth and Acton 1989; Tawfiq et al. 1989; Pras et al. 1991), high-performance liquid chromatography with UV detection (HPLC-UV) (Zhao and Zeng 1985; Acton and Klayman 1985; Liersh et al. 1986; Singh et al. 1988; Pras et al. 1991), high-performance liquid chromatography with electrochemical detection (HPLCEC) (Acton et al. 1985; Zhou et al. 1988; Charles et al. 1990; Melendez et al. 1991; Ferreira et al. 1994), gas chromatography (GC) (Fulzele et al. 1991; Sipahimalani et al. 1991; Woerdenbag et al. 1993a), GC combined with mass spectrometry (GC/MS) (Banthorpe and Brown 1989; Woerdenbag et al. 1991; Woerdenbag et al. 1993b) or MS/MS (Ranasinghe et al. 1993), radioimmunoassay (RIA) (Song et al. 1985; Zhao et al. 1986), and enzyme-immunoassay (ELISA) (Jaziri et al. 1993; Ferreira and Janick 1995b, 1996a). TLC is neither sensitive nor precise enough to quantify artemisinin in crude plant extracts without interference from other compounds, although some authors (Tu et al. 1982; Klayman et al. 1984; Luo and Shen 1987; Roth and Acton 1989; Tawfiq et al. 1989; Pras et al. 1991) have quantified artemisinin by this method. To be analyzed by HPLCUV, artemisinin needs to be derivatized due to its lack of chromophores. This process may also derivatize other compounds present in the crude extract and mask the results. Artemisinin can be ana-

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lyzed and quantified by GC alone or GC/MS through its degradation products since these methods usually use oven temperatures beyond artemisinin stability (ca. 150°C). Arteannuin B, when present in the plant extracts, generates a peak that appears with the same retention time as one of the artemisinin degradation peaks (Sipahimalani et al. 1991; Ferreira et al. 1994). HPLC-EC is a sensitive way of detecting and quantifying artemisinin in crude plant extracts without molecular breakdown, or interference from other related compounds such as artemisitene, and does not require previous derivatization or sample purification. Compounds without a peroxide bridge (such as arteannuin Band artemisinic acid) or with only one oxygen in the bridge (deoxyartemisinin) are undetected by this method. Enhanced selective and sensitive detection and quantification of peroxides, using high-performance liquid chromatography combined with electrochemical detection, at a negative potential, was initially demonstrated with benzoyl peroxide by Funk et al. (1980). Acton et al. (1985) first described the use of this method for detection (3 ng readily detected) and quantification of artemisinin from crude plant extracts. An improved HPLC-EC method, originally based on Acton's method, has been described by Ferreira et al. (1994). Absence of the degassing step was considered to be the key step in improving the efficiency of the HPLC-EC method since it hastened the analysis, eliminated baseline drifting, and simplified the analysis in that the system no longer required forcing the solution into the 10-~L loop under gas pressure. Absence of sample degassing did not cause any oxygen peak to show up in the chromatogram. In addition, the heating of the mobile phase for recycling was reduced from 2 h (Acton et al. 1985) to 15 min, allowing a second cycle of analysis/day using the same mobile phase. The detector setting at-1.0 V, with the mobile phase being constituted of 450/0 acetonitrile:55% 0.1 N amonium acetate, and a flow rate of 1.5 mL/min, brought the artemisinin retention time down to ca. 7 min. This improved HPLC-EC method effectively separated and quantified dihydroartemisinin (mobile phase changed to 35% acetonitrile:65% 0.1 N amonium acetate), artemisitene, artemisinin, and dihydroartemisinin carboxymethylester. This HPLC-EC method was compared to a GC method by Ferreira et al. (1994). The GC analysis degraded artemisinin to three other compounds, which generated peaks named A (identified as artemisinin G by EI mass spectrometry), B, and Z. Although peak A was the largest peak, it coincided with the peak generated by arteannuin B and thus could not be trusted for artemisinin quantifi-

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cation, which was possible only through peak B. Although Woerdenbag et al. (1991, 1993a) reported the separation of artemisinin from arteannuin B through GC/MS or CG alone, without degradation of artemisinin, HPLC-EC was found to be ca. 10 times more sensitive than GC and provided a better separation of dihydroartemisinin, artemisitene, and artemisinin (Ferreira et al. 1994). 3. Immunoassays. Radioimmunoassays (RIAs) and enzyme-linked immunosorbant assays (ELISAs) are more sensitive than the chemical methods discussed above, although they are more laborious, since generation of polyclonal or monoclonal antibodies is required. A radiommunoassay for artemisinin has been described by Song et al. (1985) and Zhao et al. (1986) but great importance has been given to enzyme immunoassays because they do not involve the problems of legal use and disposal, instability, high prices, or health hazards associated with radioactive labels. Because artemisinin is a very small molecule and thus nonimmunogenic, it needs to be linked to a carrier protein which will function as the primary immunogen. To be attached to the protein, artemisinin must first be derivatized to a compound with a reactive group that will act as a chemical handle. The first step in the derivatization of artemisinin is its reduction to dihydroartemisinin by sodium borohydride, which does not destroy the peroxide group. Dihydroartemisinin can then be derivatized to dihydroartemisinin carboxymethylester, which is then hydrolyzed to dihydroartemisinin carboxymethylether (a carboxylic acid). This compound, which has a reactive carboxyl group, is then linked to free amino groups of lysine residues in the carrier protein (Song et al. 1985; Zhao et al. 1986; Ferreira and Janick 1996a). Dihydroartemisinin can also react with succinic anhydride to generate 10succinyldihydroartemisinin, which has the desired reactive carboxyl group (Jaziri et al. 1993) and can be linked to a protein using the mixed anhydride reaction (Song et al. 1985; Zhao et al. 1986) or by a reaction using a water-soluble carbodiimide (Staros et al. 1986). Zhao et al. (1986) used tritiated dihydroartemisinin to determine that an average molar ratio of 15:1 artemisinin to protein carrier had been achieved, and successfully generated polyclonal antibodies against artemisinin. One of the polyclonal antibodies obtained by Ferreira and Janick (1996a) produced a near linear standard curve that enabled the quantification of artemisinin from crude extracts of A. annua. The ELISA correlated highly with HPLC-EC in discriminating samples of tissue-cultured clones but overestimated artemisinin by 3- to 10-fold.

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This overestimation was due to cross-reactivity of polyclonal antibodies with artemisinin-related compounds, a problem also encountered by Jaziri et a1. (1993). Cross-reactivity with compounds produced by root extracts from A. annua, diluted 10 to 20x, suggested that structural similarity, as well as the peroxide bridge, is important for antibody recognition. Cross-reactivity with root extracts was circumvented by diluting samples 100 to 500x. Development of ELISAs using monoclonal antibodies might eliminate the cross-reactivity problem but may bring other problems such as false negative reactivity of antibodies caused by destruction or obstruction of the epitope involved in antibody recognition. E. Biosynthesis Although the complete biosynthetic pathway for artemisinin and some of its precursors has not been established, some biotransformation steps have been elucidated, in vitro and in vivo. Farnesyl pyrophosphate (FPP) is considered to be the precursor for sesquiterpenes (Akhila et a1. 1987). These authors proposed a complete biosynthetic pathway for artemisinin, starting from isopentenyl pyrophosphate (IPP). A four-step pathway for artemisinic acid proposed by Akhila et al (1990) is based on feeding the plant with [14Cf3H] mevalonic acid and starts from the cyclization of cis-FPP. Akhila's studies did not acknowledge artemisinic acid as a precursor for artemisinin but others (EI-Feraly et a1. 1986; Jung et a1. 1990a; Roth and Acton 1987,1989; Kim and Kim 1992; Sangwan et a1. 1993) consider artemisinic acid to be a possible biogenetic precursor for both arteannuin Band artemisinin, sequentially or independently. Arteannuin B occurs naturally in A. annua and has been considered as another precursor for artemisinin (Nair et a1. 1985; Roth and Acton 1989). Arteannuin B was converted into artemisinin in cellfree extracts of A. annua leaves (Nair et a1. 1985). EI-Feraly et a1. (1986) demonstrated that artemisinic (arteannuic) acid could be converted to arteannuin B, in vitro, by singlet oxygen (102) through dyesensitized photooxygenation. Artemisinic acid was converted into artemisinin (Roth and Acton 1989) and other artemisinin derivatives and correlated compounds such as artemisitene and arteannuin B. However, Nair et a1. (1986) suggested that artemisinin and arteannuin B are produced independently from artemisinic acid. Kim and Kim (1992) reported the transformation of dihydroartemisinic acid into artemisinin by A. annua tumor cell-free extracts but not by leaves or callus cell-free extracts. Tumor cell-free

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extracts, however, failed to transform artemisinic acid into dihydroartemisinic acid, both of which are found in crude plant extracts. Sangwan et al. (1993) reported in vivo and in vitro transformation of artemisinic acid to arteannuin Band artemisinin. Using an in vitro system, artemisinic acid was transformed into arteannuin B (1.58%) and artemisinin (3.59%). Horseradish peroxidase in the presence of HzO z further enhanced the yields to 4.68 and 7.19%, respectively. Peroxidases are known to catalyze a variety of reactions in the metabolism of natural products, such as oxidation of substrates with HzO z and introduction of oxygen into a substrate (Barz and Koster 1981). Although in vitro and in vivo studies suggest that artemisininic acid may serve as a biogenetic precursor for the synthesis of arteannuin Band artemisinin, the intermediate products or enzymes after FPP and before artemisinic acid, arteannuin B, and artemisinin, have not been isolated in vivo.

F. Mode of Action 1. Plant Growth Regulator. Artemisinin and other sesquiterpene lactones from the genus Artemisia have been shown to regulate plant growth (Duke et al. 1988). Arbusculin-A and other sesquiterpene lactones isolated from A. tridentata var. vaseyana and other species of Artemisia inhibited lateral root growth but stimulated respiration in Cucumis sativus (McCahon et al. 1973). Artemisinin inhibited germination of lettuce (Lactuca sativa) and A. annua and root and shoot growth of lettuce, redroot pigweed (Amaranthus retroflexus) , pitted morning glory (Ipomoea lacunosa), A. annua, and common purslane (Portulaca oleracea) at 33 IJ.M, although this concentration did not affect velvetleaf (Abutilon theophrasti) or grain sorghum (Sorghum bicolor) (Duke et al. 1987). Artemisinin was not as effective as 2,4-D, but was more effective than glyphosate, when tested as an herbicide in the mung bean (Vigna radiata) bioassay at 5, 10, and 20 IJ.M (Chen and Polatnick 1991). However, its herbicidal mode of action has not been elucidated. 2. Antimalarial. Based on studies in monkeys, artemisinin has

proven effective in impairing the life cycle, and thus transmission, of Plasmodium cynomolgi B (simian malaria) within 18-24 h even in small (5 mg/kg) single intramuscular doses (Dutta et al. 1989). Pharmacological studies and clinical observations in every type of malaria infection indicate that artemisinin has direct parasiticidal

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action on Plasmodium in the erythrocytic stage but is ineffective in the exoerythrocytic (liver) stage. In cases of uncomplicated falciparum malaria, artemisinin-derived drugs, such as artemether and artesunate (Fig. 6.1), require a 5-day treatment to avoid recrudescence. Under these conditions, artesunate or artemether, in a total dose of 600 mg given over 5 days, produced cure rates of over 90%. However, the cure rates depend on the severity of the diseasethe more severe, the lower the cure rate (Looareesuwan 1994). The same dose over the same period, produced a cure rate of only 76% in severe malaria, when intramuscular artemether was administered, and addition of chloroquine or sulfadoxine-pyrimethamine resulted in no improvement in these severe cases. However, the addition of mefloquine (25 mg/kg at the end of the course of artesunate) or doxycycline produced higher cure rates when combined with artesunate at 600 mg total dose over a 5-day period (Bunnag et a1. 1992). For the treatment of severe and complicated malaria, various formulations of artemisinin, artemether, and artesunate have been used. In patients who are comatose or vomiting, the administration of intramuscular artemether, at a dose of 3.2 mg/kg on the first day followed by 1.6 mg/kg per day until oral therapy can be given, has been effective (WHO 1994). All the artemisinin-related drugs (e.g., artesenuate, and artemether) when injected intramuscularly or intravenously, act faster than most antimalarial drugs and are well tolerated, without evident toxicity (White 1994). Studies carried out by the Chinese Qinghaosu Antimalaria Coordinating Research Group, reported by Dutta et a1. (1989), indicated that artemisinin is relatively nontoxic, with a LD 50 in mouse of 5.1 g/kg orally and 2.8 g/kg intramuscularly. Studies on the initial mechanism of action have established that artemisinin causes structural changes in the erythrocite stage of the parasite, that affect the membranes surrounding the food vacuole, the nucleus, the mitochondria, endoplasmic reticulum, and nucleoplasm. Such changes lead to the formation of autophagic vacuoles and the loss of cytoplasm, which kill the parasites (Anon. 1979; Maeno et a1. 1993). It is well known that the peroxide function in artemisinin and related compounds is vital for antimalarial activity, and results of biological activity studies in vitro indicate that the carbonyl function is not necessary for antimalarial activity (Jung et a1. 1990b). The biochemical action of artemisinin depends on two sequential steps (Meshnick 1994): (1) activation, which comprises an iron-mediated cleavage of the endoperoxide bridge generating an unstable organic free radical and/or other electrophilic species; and (2) alky-

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lation, which involves the formation of covalent adducts between the drug and proteins synthesized by the Plasmodium. Since iron readily catalyzes the cleavage of artemisinin endoperoxide bridge, the same reaction is thought to occur in vivo, catalyzed by the heme group of hemoglobin. Plasmodium multiplication occurs inside the erythrocytes and is dependent on heme groups of hemoglobin. If these heme groups form complexes with the peroxide group of artemisinin, they would be unavailable for the Plasmodium, thus impairing its cycle.

Activation. This iron-mediated decomposition mechanism of artemisinin has been elucidated by Posner and Oh (1992); data indicate that iron activates artemisinin into a free radical. However, since there are various iron pools in the infected red blood cell, it is not known for sure which ones are responsible for artemisinin activation (Meshnick 1994). The observation that chloroquine, which binds heme (Chou et a1. 1980), antagonizes the antimalarial activity of artemisinin against P. falciparum (Stahel et a1. 1988) suggests that the free heme pool might be important. This hypothesis is reinforced by the fact that iron chelators, which bind free iron, have been observed to antagonize the effect of artemisinin (Kamchonwongpaisan et a1. 1992; Meshnick et a1. 1993). Alkylation. After artemisinin-related drugs are converted to a reactive free radical, they can covalently bind to proteins. Radiolabeled artemisinin was shown to bind covalently mostly with free amino groups in human serum albumin (Yang et a1. 1993). Radiolabeled drug is also taken up by isolated red cell membranes, where it forms covalent bonds with various membrane proteins. In contrast, when artemisinin is incubated with intact erythrocytes, there is no uptake or protein alkylation (Asawamahasakda et a1. cited by Meshnick [1994]). However, the alkylation of heme appears to have little biological significance since, when infected red blood cells are exposed to high levels of artemisinin, there is no diminution in haemozoin content (Chang et a1.; Asawamahasakda et a1. cited by Meshnick [1994]).

A more recent series of water-soluble artemisinin derivatives has been developed in which the linkage of the water-solubilizing function to dihydroartemisinin was mediated by an ether, rather than an ester, functional group. The most active compound of this series is sodium artelinate, which, although less potent than sodium artesunate, compares favorably with this drug both in vitro against

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P. falciparum and in vivo against P. berghei and is more stable than

artesunate, remaining available in solution longer. Sodium artesunate, in alkaline solution, hydrolyzes into the insoluble dihydroartemisinin in a few hours, while sodium artelinate is detected with minor decomposition after 3 months (Lin et a1. 1987). Arteether, artemether, sodium artesunate, artelininic acid (Fig. 6.1), and sodium artelinate are more stable and effective against malaria than artemisinin and dihydroartemisinin (dihydroqinghaosu), but the latter two compounds were less cytotoxic when tested in vitro (Woerdenbag et a1. 1993c). However, in whole animal systems, dihydroartemisinin is by far the most potent and the most toxic compound, and although artemisinin is the least effective, it is also the easiest compound to manufacture. Artemisinin has been of great use in Vietnam, where the technology to derivatize it has not yet been available (D. Davidson, personal communication). G. Drug Development Artemisinin is the main active principle of A. annua effective against malaria. The peroxide moiety of the molecule appears to be responsible for the antimalarial activity, although other compounds produced by the plant, without the peroxide bridge, might have helped activate the antimalarial activity of plant extracts originally used by the Chinese. The first clinical studies conducted in China in 1972 showed excellent activity against malaria caused by both P. falciparum and P. vivax. Artemisinin was shown to clear parasitemia faster than chloroquine besides being effective against chloroquineresistant Plasmodium and against potentially fatal cases of cerebral malaria. Artemisinin formulations in China were suspended in oil or water for intramuscular injection, or were prepared as tablets and suppositories. However, due to its poor solubility in water or oil, researchers from the Walter Reed Army Institute have tried to develop semisynthetic derivatives of artemisinin with better solubility, such as arteether, artemether (both soluble in oil), and sodium artesunate and sodium artelinate (both water soluble). However, both artemether and artesunate are susceptible to breakdown by humidity, light, or acidic conditions. At room temperature, an aqueous solution of sodium artesunate at pH 7-8 hydrolyzes within hours to dihydroartemisinin. Sodium artesunate, a water-soluble salt derivative, is available commercially in China and Vietnam. It is 5-fold more effective than artemisinin, is well tolerated by test animals, and is

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far less harmful to the heart than chloroquine. The prescribed dosage is 60 mg (or 1.2 mg/kg) for adults, administered intravenously or intramuscularly and repeated 4, 24, and 48 h after the initial dose. Sodium artelinate was developed by Lin et al. (1987) and is more potent and stable than artemisinin, remaining available, undegraded, longer in the bloodstream, and providing a better control against Plasmodium berghei and P. falciparum. Sodium artesunate and sodium artelinate have special application for treatment of the potentially fatal cerebral malaria (Klayman 1993). Though still under clinical investigation, sodium artesenuate is available and routinely used as an injectable treatment for malaria. Artemether in oil solution, ready for intramuscular injection, is available commercially in the People's Republic of China and is now registered in countries in Africa, Asia, and Latin America under the name Paluther, but it is still awaiting approval in others (Rhone-Poulenc Rorer Inc., personal communication; Roche and Helenport 1994). Other artemisinin-related drugs including arteether and artelinic acid are potential candidates for commercial drug development. Artelinic acid is a second generation, water soluble derivative with improved stability in solution. It has been effective in rodent malaria when administered orally, parenterally, or transdermally. An intramuscular formulation of arteether, an ethyl ether of artemisinin, is now undergoing phase-I clinical trials and the synthetic analog yinghaosu is in phase-II trials. Other related compounds such as the trioxane and dioxane artemisinin derivatives are at earlier stages of development (WHO 1994). 1. Drug Availability. At the present, a number of formulations of artemisinin and its derivatives are being marketed. The most widely available preparations are (WHO 1994):

Oral Artemisinin tablets 250mg Artemisinin capsules 250 mg Artemether capsules 40mg Artesunic acid (artesunate) tablets 50 mg 60 mg Dihydroartemisinin tablets Intramuscular Artemether oily solution 80 mg/L mL ampoule Artesunate (anhydrous powder) 60 mg/L mL ampoule (reconstituted in 0.6 mL of 5% (w/v) sodium bicarbonate and diluted in 5.4 mL of dextrose solution or dextrose in saline just before use, because of the instability of the acid)

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Intravenous Artesunic acid identical to the formulation for intramuscular administration Suppositories Artemisinin 100 mg In China, artemisinin is no longer used clinically, but dihydroartemisinin in tablet form has been approved for marketing in 1993 (WHO 1994). Vietnam produces artemisinin tablets, capsules, and suppositories as well as artesuna~e tablets and capsules, which are authorized for domestic use, as are artesunate intravenous preparations imported from China (WHO 1994). Raw artemisinin is purchased from Vietnam for derivation and drug formulation by a Belgian pharmaceutical enterprise. In Myanmar, Thailand, and Brazil, artesunate tablets and intravenous formulations, as well as injectable artemether preparations, are imported. In a few African countries, the parenteral formulation of artemether has been registered (e.g, Paluther), but by June 1995 it was not yet authorized for use by the malaria programs by most Health Ministries. Although about 1000 cases of malaria are registered per year in the United States, no artemisinin-derived drug is approved by the Food and Drug Administration. Most of the information on the use of these derivatives relates to adults, but there are sufficient data to conclude that children also tolerate the drugs well and that their therapeutic response resembles that of adults having similar levels of immunity (Hien et al. 1991; White et al. 1992; Taylor et al. 1993). In an ongoing trial in Vietnam, 90 children have been treated in a three-way comparison of intramuscular artesunate, intravenous quinine, and artemisinin suppositories. Interim analysis indicates that the two artemisinin compounds are equivalent and more effective than intravenous quinine as an antimalarial (WHO 1994). 2. The Future of Artemisinin as an Antimalarial. Effective control of malaria is complicated by the fact that the mosquito vector develops resistance to available insecticides and Plasmodium falciparum develops resistance to the currently used antimalarial drugs. The best solution for malaria would be an effective, low-cost vaccine. This, however, has not yet been achieved and cannot be expected in the foreseeable future because of the different stages of the life cycle of Plasmodium in humans and the mutation frequency of this protozoa. Recently, the Pan American Health Organization reported a plasmid DNA vaccine, raised by producing antibodies and lymphocytes,

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with a 68% effectiveness in mice. Another vaccine (SPf66) was developed in Colombia under the supervision of M. Patarroyo and collaborators (Valero et al. 1993) and has been shown to induce a substantial antibody response with three doses. It is well tolerated and safe for children but its safety and immunogenicity against malaria remain to be determined in a phase III trial in Kilombero, Africa, where malaria is highly endemic (Teuscher et al. 1994). At present, short-term solutions to malaria involve: (1) providing early diagnosis and prompt treatment; (2) planning and implementing selective and sustainable preventive measures, including vector control; (3) encouraging early detection and prevention of epidemics; (4) strenthening of local capacities in basic and applied research to permit and promote regular assessment of a country's malaria situation, in particular the ecological, social, and economic determinants of the disease. The development of drug-resistant strains of Plasmodium has greatly complicated drug therapy. Artemisinin-derived drugs, used in association with other antimalarials, and strategies to control transmission of Plasmodium may be the only effective means of malaria control. Artemisinin-derived drugs are now available commercially in a few countries but they are restricted because of high cost; e.g., oral artesunate currently costs about $5-6 per treatment, with about 5 doses, as compared to $1.85 for mefloquine and 7ft for drugs such as chloroquine and amodiaquine. Injectable artesunate and artemether cost even more, compared to quinine injections at less than $2.00 per treatment. The fear that the Plasmodium will develop resistance to artemisinin has led to the suggestion (WHO/ MAL 1994) that these drugs be restricted to those areas where multidrug-resistantPlasmodium is endemic in order to avoid evolution of new artemisinin-resistant strains. V. CONCLUSIONS

Artemisia annua is an example of an ancient medicinal plant that has steadfastly remained a traditional Chinese herb, and whose virtues are only now being recognized. Although prized in the United States for the dried flowers and use in the craft trade, the plant has recently received enormous attention as the only practical source of artemisinin that is effective against drug-resistant strains of Plasmodium falciparum, the pathogen responsible for the most severe form of malaria. As malaria has increased, so too has medicinal and agricultural research on A. annua and artemisinin. The discovery of the

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plant's antimalarial activity and the isolation and identification of the main bioactive compound, artemisinin, is among the most exciting and successful breakthroughs in medicinal plant drug development in the past two decades. China remains the world's major supplier of artemisinin and, at present, most is extracted from natural stands, although some lines are now being cultivated as a crop in Szechwan Province. Unfortunately, crop information and crop statistics have been unavailable, because the Chinese consider this proprietary information. The potential market for artemisinin and related derivatives has prompted the establishment of many research programs to develop A. annua as a commercial crop around the world including Australia, Brazil, India, The Netherlands, Switzerland, the United States, and Vietnam, which, in 1995, exported artemisinin at the price of $300-400/ kg. These crop studies are in their early stages, and their success is closely linked with the potential of industrial commercialization. Agricultural field production of A. annua rather than in vitro culture appears to be the most practical method to produce artemisinin. The low yield of artemisinin may be overcome by the breeding of high-yielding, adapted clones. Early success toward this goal has been reported in Switzerland, but little is known concerning the genetics or breeding behavior of this species, and research in this area is needed. In addition to artemisinin, other closely related sesquiterpenes, such as artemisinic acid, may also be of clinical interest in the development of antimalarials. Harvesting the plant for both artemisinin and artemisinic acid, which occurs in levels 8 to 10 times higher than artemisinin, is feasible. Artemisinin is associated with the glandular trichomes in leaves and flowers, with the highest concentrations being associated with the inflorescence. Because A. annua is a short-day plant with a critical photoperiod of about 13.5 h, and unless day neutral genotypes are discovered, the likely areas of production will be temperate areas in which the daylength is long enough to achieve sufficient biomass development before flowering. Although it is clear that artemisinin is sequestered in glandular trichomes, it is not certain if actual synthesis takes place in these glands. Immunolocalization studies, now made possible through the development of antibodies against artemisinin, could resolve this question. In vitro studies suggest that roots may playa role in production of artemisinin and this warrants further study. Agronomic research carried out to date indicates that A. annua can be easily grown in temperate climates. High biomass yields have

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resulted from relatively high plant densities (5 to 11 plants/m 2 ) and the plant is responsive to nitrogen fertilization. The optimum time of harvest must take into consideration the maximum yields of artemisinin per unit area, balanced against extraction costs. At the present time, harvest close to anthesis, which coincides with the maximum content per plants, appears to be optimum. The development of efficient production systems will require effective strategies in weed control, mechanical sowing and harvesting, and postharvest handling. The low content of artemisinin, even in the best lines, suggest that processing facilities should be close to production areas. Despite the positive attributes of A. annua, the pollen, as in many other species of the Asteraceae, causes allergic rhinitis (hay fever), and is of concern. As A. annua generates greater research based on artemisinin production, we predict that wider medical and agricultural uses will be identified. For example, the bioactivity of the volatile and nonvolatile oils of this plant also are of interest. Artemisinin and 1,8-cineole have been identified as possible allelopathic agents exhibiting activity against the seed germination of other weed species. Although A. annua is presently marketed as a limited-volume essential oil, this could change with a significant increase in field production for artemisinin. Large quantities of such essential oil could stimulate new uses in beverages and fragrances, and generate new markets. The development of a processing system whereby both the artemisinic acid and the essential oil could be extracted from the same harvested plant material would increase the economic viability of production. Progress toward approval and commercialization of artemisinin and its antimalarial derivatives as pharmaceuticals has been slow. Although artemisinin has been substituted for dihydroartemisinin in China, more potent and stable artemisinin-derived antimalarials are still undergoing clinical trials. Artemether is now approved and available in Africa as Paluther (Rhone-Poulenc-Rorer) and is awaiting approval in other countries. Although over 1000 cases of malaria per year are reported in the United States, none of the artemisinin derivatives have been approved for human use by the Food and Drug Administration, despite their low toxicity. In some countries, these derivatives reach the population, at inflated prices, through the black market. This constitutes a serious hazard to the control of malaria, since the indiscriminate use of artemisinin-related drugs raises the risk of creating zones of artemisinin-resistant Plasmodium in areas where quinine-derived drugs are still effective. The emergence and

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spread of drug resistance by Plasmodium is a serious threat to effective treatment, particularly in Southeast Asia and Latin America. Complete or partial resistance to chloroquine has now spread to all endemic areas, including Africa, although the drug is still useful in some areas. Malaria control was once based on the control of the mosquito with DDT, but this approach is no longer practical, safe, or cost-effective. The global malaria control strategy adopted in 1992 emphasized the need for early diagnosis, appropriate treatment with antimalarial drugs, and selective use of preventive measures, including elimination of potential sites for mosquito breeding and use of mosquito nets. The improvement and approval of artemisinin-derived drugs and the possible release of a malaria vaccine are the current hope for the control of this scourge of humankind. LITERATURE CITED Acton, N., and D. L. Klayman. 1985. Artemisitene, a new sesquiterpene lactone endoperoxide from Artemisia annua. Planta Med. 51:441-442. Acton, N., D. L. Klayman, and I. J. Rollman. 1985. Reductive electrochemical HPLC assay for artemisinin (qinghaosu). Planta Med. 51:445-446. Ahmad, A., and L. M. Misra. 1994. Terpenoids from Artemisia annua and constituents of its essential oil. Phytochemistry 37:183-186. Akhila, A., R. S. Thakur, and S. P. Popli. 1987. Biosynthesis of artemisinin in Artemisia annua. Phytochemistry 26:1927-1930. Akhila, A., K. Rani, and R. S. Thakur. 1990. Biosynthesis of artemisinic acid in Artemisia annua. Phytochemistry 29:2129-2132. Anon. 1979. Qinghaosu Antimalarial Coordinating Research Group. Antimalarial studies on qinghaosu. Chin. Med. J. 92:811-816. Anon. 1982. China Cooperative Group on Qinghaosu and Its Derivatives as Antimalarials. Chemical studies on qinghaosu (Artemisinin). J. Trad. Chin. Med. 2:3-8. Arora, N., and S. V. Gangal. 1991. Liposomes are vehicle for allergen presentation in the immunotherapy of allergic diseases. Allergy 46:386-392. Ascensao, 1., and M. S. S. Pais. 1987. Glandular trichomes of Artemisia campestris (ssp Maritima): ontogeny and histochemistry of the secretory product. Bot. Gaz. 148:221-227. Avery, M. A., W. K. M. Chong, and C. Jennings-White. 1992. Stereoselective total synthesis of (+)-artemisinin, the antimalarial constituent of Artemisia annua L. J. Am. Chern. Soc. 114:974-979. Bailey, L. H. 1951. Manual of cultivated plants. Macmillan, New York. Bailey, L. H., and E. Z. Bailey. 1976. Hortus third. Macmillan, New York. Baker, J. (ed). 1994. Artemisinin: proceedings of a meeting convened by the Wellcome Trust, 25-27, April 1993. Trans. R. Soc. Trap. Med. Hyg. 88, Suppl. 1, London,

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Banthorpe, D. V., and G. D. Brown. 1989. Two unexpected coumarin derivatives from tissue cultures of Compositae species. Phytochemistry 28:3003-3007. Barz, W., and J. Koster. 1981. Turnover and degradation of secondary (natural) products. p. 35-84. In: E. E. Conn (ed.), The biochemistry of plants. Academic, New York. Beetle, A. A., and A. Young. 1965. A third subspecies in the Artemisia tridentata complex. Rhodora 67:405-406. Bennett, M. D., J. B. Smith, and J. S. Heslop-Harrison. 1982. Nuclear DNA amounts in angiosperms. Proc. R. Soc. London Ser. B 216:179-199. Besser, W. S. J. G. 1843. Monographiae Artemisiarum, Section 1 (Dracunculi). Memoires, L'Academie Imperiale des Sciences de Saint Petersbourg. Bostock, J., and H. T. Riley (Transl.). 1855-1857. The natural history of Pliny (6 vols.). Henry G. Bohn, London (see Vol. 5, Book XXV, Chapter 36, p. 106-107). Brown, G. D. 1993a. Annulide, a sesquiterpene lactone from Artemisia annua. Phytochemistry 32:391-393. Brown, G. D. 1993b. Production of anti-malarial and anti-migraine drugs in tissue culture of Artemisia annua and Tanacetum parthenium. Acta Hort. 330:269-276. Bryson, C. T., and E. M. Croom Jr. 1991. Herbicide inputs for a new agronomic crop, annual wormwood (Artemisia annual. Weed Technol. 5:117-124. Bunnag, D., J. Karbwang, and T. Harinasuta. 1992. Artemether in the treatment of multiple drug resistant falciparum malaria. Southeast Asian J. Trop. Med. Pub. Health. 23:762-768. (Cited by Looareesuwan [1994]). Cappelletti, E. M., R. Caniato, and G. Appendino. 1986. Localization of cytotoxic hydroper-oxyeudesmanolides in Artemisia umbelliformis. Biochem. System. Ecol. 14:183-190. Charles, D. J., J. E. Simon, K. V. Wood, and P. Heinstein. 1990. Germplasm variation in artemisinin content of Artemisia annua using an alternative method of artemisinin analysis from crude plant extracts. J. Nat. Prod. 53:157-160. Charles, D. J., E. Cebert, and J. E. Simon. 1991. Characterization of the essential oil of Artemisia annua L. J. Ess. Oil Res. 3:33-39. Charles, D. J., J. E. Simon, C. C. Shock, E. B. G. Feibert, and R. M. Smith. 1993. Effect of water stress and post-harvest handling on artemisinin content in the leaves of Artemisia annua L. p. 640-642. In: J. Janick and J. E. Simon (eds.), New crops. Wiley, New York. Chen, P. K., and M. Polatnick. 1991. Comparative study on artemisinin, 2,4-D, and glyphosate. J. Agr. Food Chern. 39:991-994. Chou, A., R. Chevli, and C. D. Fitch. 1980. Ferriprotoporphyrin IX fulfills the criteria for identification as the chloroquine receptor of malaria parasites. Biochemistry 19:1543-1549. Degener, O. 1939. Flora Hawaiiensis, Vol. 6, New York. De Kruif, P. 1939. Ross vs. Grassi, Malaria. p. 290-323. In: H. G. Grover (ed.), Microbe hunters. Harcourt, Brace, New York. Delabays, N., C. Blank, and G. Collet. 1992. The culture and the selection of Artemisia annua L. for the production of artemisinin. Fed. Agr. Res. Sta. Changins. Rev. Suisse Vitic. Arboric. Hort. 24:245-251. Delabays, N., A. Benakis, and G. Collet. 1993. Selection and breeding for high artemisinin (qinghaosu) yielding strains of Artemisia annua. Acta Hort. 330:203207.

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Woerdenbag, H. J., T. A. Moskal, N. Pras, T. M. Malingre, F. S. El-Feraly, H. M. Kampinga, and A. W. T. Konings. 1993c. Cytotoxicity of artemisinin-related endoperoxides to ehrlich ascites tumor cells. J. Nat. Prod. 56:849-856. Woerdenbag, H J., N. Pras, N. G. Chan, B. T. Bang, R. Bos, W. van Uden, P. Van Y., N. V. Boi, S. Batterman, and C.B. Lugt. 1994a. Artemisinin, related sesquiterpenes, and essential oil in Artemisia annua during a vegetation period in Vietnam. Planta Med. 60:272-275. Woerdenbag, H. J., N. Pras, W. Van Uden, T. E. Wallaart, A. C. Beekman, and C. B. Lugt. 1994b. Progress in the research of artemisinin-related antimalarials: an update. Pharm. World Sci. 16:169-180. Xu, X. X., J. Zhu, D. Z. Huang, and W. S. Zhou. 1983. The stereocontrolled synthesis of qinghaosu and deoxyqinghaosu from artennuic acid. Huaxue Xuebao 42:940. (Cited by Luo and Shen [1987].) Xu, X. X., J. Zhu, D. Z. Huang, W. S. Zhou. 1986. Total synthesis of arteannuin and deoxyarteannuin. Tetrahedron 42:819-828. Yang, Y. Z., W. Asawamahasakda, and S. R. Meshnick. 1993. Alkylation of human albumin by the antimalarial artemisinin. Biochem. Pharmacology 46:336-339. Yeou-ruenn, 1. 1994. The genera Artemisia 1. and Seriphidium (Bess.) Poljak. in the world. Compo Newslett. 25:39-45. Zaman, S. S., and R. P. Sharma. 1991. Some aspects of the chemistry and biological activity of artemisinin and related antimalarials. Heterocycles 32:1593-1638. Zhao, K. C., C. X. Liu, X. T. Liang, Y. Mingguang, and Z. Y. Song. 1986. Development of radioimmunoassay for determination of artesunate, a derivative of the antimalarial qinghaosu. Proc. Chinese Acad. Med. Sci., Pekin Union Medical College 1:213-217. Zhao, S. S., and M. Y. Zeng. 1985. Spektrometrische hochdruck-flussigkeitschromatographische (HPLC) untersuchungen zur analytic von qinghaosu. Planta Med. 51:233-237. Zhou, W. S., and X. X. Xu. 1989. The structure, reactions and syntheses of arteannuin (qinghaosu) and related compounds. In: Atta-Ur-Rahman (ed.). Studies in natural products chemistry. Stereoselective Synth. 3B:495-527. Zhou, Z., Y. Huang, G. Xie, X. Sun, Y. Wang, L. Fu, H. Jian, X. Guo, and G. Li. 1988. HPLC with polarographic detection of artemisinin and its derivatives and aplication of the method to the pharmacokinetic study of artemether. J. Liq. Chromatogr. 1:1117-1137.

7 Opium Poppy (Papaver somniferum): Botany and Horticulture Peter Tetenyi * Research Institute for Medicinal Plants, Budakalasz Hungary I. II.

III.

IV.

Introduction Botany A. Origin and Distribution 1. History 2. Spreading and Diversity B. Taxonomy 1. Species Level 2. Infraspecific Systematics Horticulture A. Cultivation Practices 1. Choice and Preparation of the Soil 2. Choice of Cultivar and Preparation of Seed Material 3. Operations During the First Developmental Period 4. Operations During the Second Developmental Period 5. Ripening and Harvest B. Utilization 1. Medicinal 2. Culinary (Oil and Spice) 3. Ornamental and Other Uses Summary and Prospects Literature Cited

I. INTRODUCTION

The word poppy has been used for many species of the Papaveraceae, but is only a part of the full name of the opium or oil poppy (Papaver *1 thank the International Narcotics Control Board for providing the 1995 Report on Narcotic Drugs World Requirement and Statistics.

Horticultural Reviews, Volume 19, Edited by Jules Janick ISBN 0-471-16529-8 © 1997 John Wiley & Sons, Inc.

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somniferum 1.), one of the most useful plant species. Opium poppy belongs to the section Papaver of the tribe Papavereae, and of subfamily Papaveroideae. Plants of the family are characterized by the watery or milky latex of vessels in all parts except seeds. Of about 240 species of the family, 170 belong to Papaveroideae, and of these 80 are members of the genus Papaver. Of four Papaveraceae subfamilies, Platystemonoideae and Eschscholzioideae are restricted in their natural distribution to the New World, and each have 3 genera. Chelidonioideae has 6 New World and 2 Old World genera, while Papaveroideae has 5 and 2, respectively. Only species belonging to Stylopborum (Chelidonoideae) and Papaver are present both in America and Eurasia. At the tribal level, Fedde (1909) separated Romneyeae (two genera), because of water-like colorless juice, from Papavereae with milky latex. In the restricted Papaver tribe (seven genera), Roemeria and Meconopsis of Eurasia and Stylomecon from America seem the nearest chemotaxinomical relatives of the genus Papaver (Tetenyi 1993). Species of the genus Papaver are classified according to life cycle and connected characters in 9 sections. Perennial sections are considered phylogenetically older, while biennial and annual sections are younger. Opium poppy and 3 other species (~ glaucum, ~ gracile, P. decaisnei ) form the most developed, annual Papaver section. The bush poppy (Dendromecon virginiana) belongs to the subfamily Eschscholzioideae, and the horned or sea poppy (Glaucium) to the Chelidonioideae. Mastilija poppy (Romneya coulteri) is a member of the tribe Romneyeae. In this review the name opium poppy will be used to distinguish the species Papaver somniferum from all other taxa of the family. The two infraspecific taxa of opium poppy (subsp. somniferum and subsp. setigerum [DC] Arcangeli) represent one of the most ancient of crops. According to Bakels (1982), sites with opium poppy were observed between the Rhine and the Maas dating back to the earliest Neolithic Ages (at the fifth millenium B.C.). Seeds were first used as food, butthe sleep-inducing effect of the capsules and of the latex were recognized by the ancient Greeks in the middle of the second millenium B.C. Ornamental use of the plant occurred, presumably at the end of the first millenium AD, both in European cloister gardens and in China (Vesselovskaya 1975). The present cultivation of the plant is quite widespread in the temperate and subtropical zones of the northern and southern hemispheres, thus, considerably exceeding its natural habitat around the Mediterranean Sea. The cultivated area extends to Ethiopia,

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Scandinavia, Thailand, Tasmania, and Argentina. This wide horticultural distribution is due to the high accommodative capability of the species. Genetically based hybrid vigor helps it to survive different climatic and edaphic conditions, even up to altitudes of 2300 2400 m in subtropical Asia. At present, world opium poppy cultivation covers 270,000 to 300,000 ha. The five largest producers are India, Burma (Myanmar), Afghanistan, Turkey, and the former USSR, representing two-thirds of all of the cultivated fields (White and Raymer 1985). The official statistical report of the International Narcotic Control Board (INCB) for 1989-1993 stated 37,000 to 56,000 ha cultivated for opium production, based on official notices of states and territories. Obviously, most cultivation of opium poppy is illicit. The agricultural importance of opium poppy is based on pharmaceutical products such as morphine and codeine, culinary uses of the seed for bakery products and for oil, and as an ornamental. Opium poppy is one of the most precious domesticated plant species. The economic importance and the biological specificity of opium poppy have been the subject of a number of treatises. Previous reviews in this century include Fedde (1909) on systematics, Basilevskaya (1941) on distribution and microclassification, Hegnauer (1969, 1989) on chemotaxonomy, Duke (1973) on utilization, Krikorian and Ledbetter (1975) on worldwide opium production, Kadereit (1986b,1988) on systematics, and Kapoor (1995) on botany, chemistry, and pharmacology. In this review, the present scientific knowledge concerning Papaver somniferum is summarized with emphasis on botany and horticulture.

II. BOTANY

A. Origin and Distribution 1. History. The phylogenetic origin of the opium poppy can be studied by various approaches. Evolutional differentiation in the genus Papaver can be interpreted as an adaptation of the ancient perennial poppy taxa to more stressful conditions of the late Tertiary Age, which resulted in a shortening of the life cycle and the appearance of anthocyanate pigmentation. Thus, biennial or annual poppies with anthocyanated anthers and filaments are thought to be phylogenetically younger than perennial ones with yellow (nudicaulin containing) anthers. Papaver rupifragum taxa belonging to the ancestral

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P. TETENYI

Pilosa section of the genus, but with a shortened, 2- to 3-year life span, are descendants of this perennial section (Gunther 1975). Perennial or biennial taxa of the P. rhoeas group (P. rapiferum and P. rumelicum), however, should be considered as evolutionary older than the annual species of Rhoeadium section. Moreover, some plants belonging to P. rhoeas (corn poppy) have yellow filaments in contrast to the more widespread violet-black ones (Kadereit 1989), preserving another atavistic character. A third character, decrease of the nuclear DNA content (average of 5.7 pg for Pilosa section as compared to 4.7 pg, for P. rupifragum), provides additional evidence for phylogenetic advancement according to Srivastava and Lavania (1991). This diminution of DNA was observed also for Rhoeadium and Papaver sections. Based on this evidence, the present taxa of Papaver section including opium poppy are genealogical descendants from the ancestral Pilosa and of the younger Rhoeadium (TStenyi 1993) (Fig. 7.1). How did the genesis of opium poppy occur? Some botanists (DeCandolle 1883; Fedde 1909; S06 1968, Hammer and Fritsch 1977) assumed that opium poppy originated from P. setigerum. This opinion was based on morphological and chemical characteristics, as well as on the easy and fertile crossability of the two taxa. Another hypothesis is that P. glaucum is the ancestor of P. somniferum (Rothmaler 1949) on the basis of shape and surface of stigmatic lobes. Reckin (1970, 1973) assumed a parallel evolution for P. somniferum and of P. aculeatum (Horrida section), an annual endemic to South Africa, because both chromosome number and morphology are very similar. An alternative hypothesis assumes a triploid hybrid origin for P. somniferum (2n = 22), which is supported by morphological evidence derived from interspecific crosses (Kadereit 1986a, 1987). The cross of P. glaucum (2n = 14) with P. gracile (2n = 14) produced progeny with capsules similar to P. somniferum ssp. setigerum, but leaves and petals similar to ssp. somniferum. Divergence of the same type was also present in hybrids of P. glaucum x ssp. somniferum, which formed more or less normally flowered hybrids, while P. glaucum x ssp. setigerum produced hybrids with distorted flowers. P. gracile could be crossed with ssp. setigerum, but not with ssp. somniferum. The hypothesis that P. somniferum originated from spontaneous crosses of P. gracile with P. glaucum in their ancient points of contact between Anatolia and the Syrian desert, however, was not confirmed by karyologic observations. Nevertheless, the triploid origin of P. somniferum was supported by cytogenetic studies. Thus, F 1 hybrid of ssp. setigerum (2n = 44) with ssp. somniferum (2n = 22) are 2n = 33, and when these were selfed, the F 2 descendants were

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\ I

Sections 1. Meconella 2. Meconidium 3. Californicum 4. Pilosa 5. Horrida 6. Macrantha 7. Argemonidium 8. Rhoeadium 9. Papaver

Symbols of alkaloids inside (wood) of cut Phtalide

IIIJII] Morphinoide

m

Promorphinane

~ Latericine

Symbols of filaments' colors margin (bark) of cut

D

[±] Isothebaine Retroprotoberi ne 01sopavine

Pale violet or whitish Light red

•

Dark violet Yellow

Fig. 7.1. Chemotaxonomic classification of the genus Papaver (Tetenyi 1993).

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mostly 2n = 28, regaining their original basic number of the genus Papaver (x = 7). Moreover, it seemed to be characteristic that P. somniferum as well as its hybrids with congeneric species formed 3 to 4 chromosome bivalents at meiosis with a secondary association. This phenomenon confirms the participation of 2 homologous and 1 homoeologous genome in the origin of P. somniferum (Kadereit 1991a). In the tribe Papavereae, species with similar chromosome number occur. Papaver aculeatum has the same chromosome number (2n = 22) as opium poppy, and Roemeria hybrida, Meconopsis bella, and M. cambrica have the same divergence from the tribe's basic number of x = 7. Kadereit (1986a, 1987) concluded that triploidy has not been an evolutionary dead end, always causing sterility, but might be perpetuated by bivalent formation, which produced fertile and meiotically stable taxa. Papaver glaucum and P. gracile are not identical genetically with P. somniferum, but these three species seem to be the result of a convergent evolution (Kadereit and Sytsma 1992). Sequence analysis of laticifer-specific polypeptides supports the triploid-hybrid origin of the opium poppy (Nessler 1994). Despite the above considerations, a common ancestor for P. somiferum, P. glaucum, and P. gracile cannot be excluded, given the postulated origin in the Near Eastern Center including the NuboSindian and the Irano-Anatolian flora provinces (Zohary 1973). Mansfeld (1962) and Simmonds (1976) both assumed that opium poppy probably originated in Asia Minor. This approach was supported by the presence of both ssp. setigerum and P. gracile in Cyprus and in Asia Minor. Cyprus separated from mainland Asia at the end of the Neogenic Age, about 4 million years ago. Thus, opium poppy must have evolved earlier, presumably during the Miocene Ages (Kadereit 1988). 2. Spreading and Diversity. The history of opium poppy as an early companion to humans is established by archeological excavations of the last 100 years. These findings were evaluated by Schultze-Motel (1979), and arranged according to sites and epochs by Frenzel (1992), as shown in Fig. 7.2. The most ancient prehistoric remains were excavated between the Rhine and Maas rivers (Netherlands), at Granada (Spain), Marseille (France), Milan (Italy), and in Switzerland and Poland from the earliest Neolithic period (before 4000 B.C.) as well as from the Middle and the Late Neolithic (before 2500 B.C.) Ages. Seeds found in these places differed from present ssp. setigerum and from ssp. somniferum var. nigrum. The capsule excavated in

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(D)

Fig. 7.2. Sites with P. somniferum in Europe (Frenzel (1992): (A) earliest (before 4000 B.C.) Neolithic; (B) middle and late (before 2500 B.C.) Neolithic times; (C) early and middle Bronze Age; (D) late Bronze Age; (E) older pre-Roman Iron Ages; (F) time of Roman Emperors.

Robenhausen (Switzerland) differed from ssp. setigerum by its globular shape, which had an inverse oval form. Capsules found in the Granada province were poricidal as in ssp. setigerum and similar to present self-propagating taxa of ssp. somniferum, while the shape of their capsules was similar to those of the present ssp. somniferum. No poppy findings were discovered from the Near East excavations of the epoch from 7500 B.C. to A.D. 1000 and no mention on poppy can be found in cuneiform Sumeric, Accad, or Assyric writ-

380

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ings (Krikorian 1975). Excavations of Neolithic sites in north and northwest China did not uncover evidence of opium poppy, and it was missing from the broad lists of cultivated plants of China during the historical period of the second millennium B.C. (Vesselovskaya 1975). It is certain, however, that opium poppy was known in the kingdom of Mycenae (second millennium B.C.), and to the Greeks of Peloponnese. An excavated terracotta female figurine from the sixteenth to twelfth century B.C. has closed eyes and holds poppy capsules on her head (Renfrew 1973), indicating that the Greeks of antiquity knew of the sleep-inducing effect of the plant. It could also be deduced from Homer's Illiad and Odessy that opium poppy was cultivated at the Aegean seaside of Asia Minor in the 12th century B.C. Excavations from Lydia and Phrygia (now West Anatolia) dated from the eight to sixth century B.C have confirmed this. Based on these observations Vesselovskaya (1975) suggested that the domestication process of opium poppy occurred around the Aegean Sea, where the first differentiation took place for the two utilizations of the plant: seeds for food or latex for sleep induction. In her view, this ancestral diversification was the origin of two existing infraspecific taxa of the opium poppy, that of the proles oleiferum Basil (syn. convar somniferum Vess.) and that of the proles opiiferum Basil. (Proles and convar are both terms used in the classification of infraspecific entities at the level just beneath subspecies. Proles have been used for wild and subspontaneous taxa, and convar is used for cultivated taxa [Mansfeld 1953].) Both proles of opium poppy can escape easily from cultivation and have small capsules, <2 cm in diameter, but differ in the number of flowers: 20 to 60 for opiiferum vs. 6 to 20 for oleiferum, confirming the effect of a primary, unconscious selection. Their ancestors have spread from the Aegean Center to the northwest (proles oleiferum) and to the southeast (proles opiiferum), as shown in Fig. 7.3. The ancient commercial links between the Greek Islands and Egypt since the beginning of the second millennium B.C. led to the the first introduction of opium poppy to Africa. Merrillees (1988/89) has hypothesized that the plant was used in the time of the eighteenth dynasty (1550 to 1400 B.C.), on the basis of excavations uncovering that the so-called base-ring juglets produced in Cyprus, which were exported to Egypt and other Mediterranean countries. However, Bisset et al. (1994) concluded that these can be no longer accepted as evidence for the presence of opium poppy in Egypt during this period. It is certain, however, that the Greek colonization of the Nile delta (ninth to sixth century B.C.) as well as the Macedonian reign (fourth

Fig. 7.3. Spontaneous presence of P. somniferum ssp. setigerum (Kadereit 1991b), and supposed Gene Center 1 for ssp. somniferum with two ancestral taxa belonging to descendant proles (Vesselovskaya 1975).

w

ex>

I-'

382

P. TETENYI

century B.C.) introduced the cultivation and the utilization of poppy to the Egyptians. The eastern spread of opium poppy and the culture of the plant was surely the consequence of Alexander's conquests (fourth century B.C.) up to India and Central Asia. Selection for larger but poricide capsules, since the main use was for the latex, occurred unconsciously, resulting in a still-existing, descendant taxon cultivated in this area (convar turcicum). The northern spread of opium poppy went through a selection adaptation process at the more humid and cooler regions of Europe. The Greek colonizers at the northern border of the Mediterranean and the Black Sea, as well as peasants of the Roman Empire, gradually improved their plant cultures. Opium poppy, like many other annuals of the genus, dispersed ripe seeds with various level of dormancy. Most seeds germinated immediately after the end of the summer dryness, just at the autumnal rains, but some remained dormant in the soil during winter, and some for 1 or more years. This phenomenon had been utilized by peasants to broaden the cultivation area to the north of Europe, as they selected these dormant variants for alternate, autumn-spring sowing, and later solely for spring sowings. This selection for a shorter life cycle was associated with the reduction of the number of flowers and, therefore, with the larger and indehiscent capsules. Selection for other characters occurred in Christian monasteries (founded in the sixth to ninth centuries). In cloister gardens, poppy was cultivated for food, but also as an ornamental. Ornamental poppies preserved poricide capsules, but were selected for peony-like (semifilled) or carnation-like (filled) variants, and for various colors of petals and petal blotches. Opium poppy was transferred along the Silk Road and arrived in South China during the Roman Ages. This connection persisted during the Byzantine Ages. The rise of Islam (eighth to eleventh century) brought important changes for the propagation of opium poppy, especially for medicinal use of opium. Arabian merchants passed this knowledge to India (ninth century) and to China, and cultivation broadened to Southeast Asia. The first Chinese description on the sleep-inducing effect of the milky juice was mentioned by SuTche (eleventh century). The present convar persicum was formed during this period. European expansion also spread poppy cultivation eastward. Opium poppy was introduced to Russia and to the forest steppe of western and southern parts of Siberia in the seventeenth century.

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Considering the northern and eastern development of this spring group of poppies, Vesselovskaya (1975) named the present descendants as taxa of the convar eurasiaticum. The northern spread of opium poppy cultivation was joined to the southern one roughly at about the start of the eighteenth century. The so-called Baluchi opium caravans carried the trade from the Persian Gulf to Meshed (northern Iran) and to Herat. Turkestans operated caravans from here to Faizabad (Afghanistan) and Yarkand (Sinkiang, Turkestan) during many centuries. Propagation of the two poppy taxa (convar eurasiaticum and turcicum) was accompanied by spontaneous hybridization, which resulted in a second gene center at the western slopes of the Dsungarian mountains. The progenitor of the present convar tianschanicum with large poricidal capsules and the convar songaricum with indehiscent, ribbed capsules originated in this area. Selections from the Dsungarian mountains were introduced into northwestern China, which has more rigorous climatic conditions than the south of the country, and led to a variant of opium poppy with unbranched axis bearing huge capsules (>12-cm diameter) suitable for the production of opium. Descendants of this selection form the present convar chinense . The spreading of opium poppy in the Old World and the origin of infraspecific taxa is shown in Fig. 7.4. The last steps of this develop-

Fig. 7.4. Supposed spreading of ssp. somniferum with Gene Center 2, and the diverse present descendant convars (Vesselovskaya 1975).

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ment were carried out by immigrants from Europe to North and South America and to Oceania (New Zealand and Australia). In addition to development during cultivation, opium poppy can also escape from the culture as a result of seed shatter and revert to a wild form. Three examples are the appearance of poppies in Newfoundland (Krikorian and Ledbetter 1975) and Kamchatka (Shaulskaya 1993) and the northern advance of land races belonging to the convar songaricum from the Balkans into Slovakia and Poland (Hammer 1981). B. Taxonomy 1. Species Level. Linnaeus (1753) provided the Latin binomial Pa-

paver somniferum to opium poppy and described the distinct characteristics differentiating it from other poppies of the genus, e.g., from the field poppy (P. rhoeas) or from the Welsh poppy (P. cambricum, later becoming the synonym of Meconopsis cambrica). Miller (1768) recognized 2 species: the ~ somniferum witp. dark colored seeds and flowers, and ~ album with white seeds and flowers. His opinion was accepted by Gmelin (1806), who added the character of poricide capsule to define ~ somniferum and the indehiscent capsule to the ~ officinale (syn. of ~ album). Hussenot (1835) distinguished ~ hortense with red to dark flowers, dark seeds, and poricide capsules; ~ apodocarpon with red flowers, dark seeds, and indehiscent capsules; ~ stipitatum with pink flowers, dark seeds, and indehiscent capsules; and ~ album with white flowers, seeds, and indehiscent capsules. In contrast, opium poppy was considered to be a single species by Hayne (1819), DeCandolle (1821), and Alefeld (1866).

Another problem emerged, when A. P. DeCandolle, in 1815 recognized the spontaneous opium poppy taxon as an autonomous species and named it ~ setigerum. His opinion was unaccepted by most other botanists (e.g., Elkan 1839; Alefeld 1866), who judged the taxon as a varietas or a subspecies of the opium poppy, which has been native in the West Mediterranean region, and as a weed elsewhere (see Fig. 7.3). However, Fedde (1909) retained ~ setigerum as an independent species in his monumental work on the Papaveraceae. When chromosome differences were detected (2n = 44 for setigerum and 2n = 22 for somniferum) , the controversy renewed. Schultze-Motel (1979) and Hammer (1981) after finding a setigerum variant with 2n = 22 concluded that this was proof of the unity of the ~ somniferum species. La Valva et al. (1985), however, refused the unification of the two taxa when collecting setigerum plants from

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DeCandolle's original collection locations, because of discriminative morphological and chemical characters on both taxa. Kadereit (1986b, 1991b) included setigerum as a subspecies of opium poppy principally on the theoretical basis that wild relatives of cultivated taxa had to be classified in the same species according to the International Code of Botanical Nomenclature. His later restriction analysis of chloroplast DNA confirmed this conclusion (Kadereit and Sytsma 1992). No differences were found between the two taxa in 273 sites, although the setigerum plant material was either from a Czech botanical garden, or from an unknown origin (P. Tetenyi, unpublished). It seems unlikely that a taxon with a higher chromosome number (tetraploid for setigerum) could be the progenitor of diploid opium poppy. Therefore, it would be better to follow the view of Sinskaya (1969) that both taxa originated from an ancient, undifferentiated extinct taxon, until new scientific evidence provides a better understanding of their relationship. The last taxonomic problem of the opium poppy on the species level appeared in the classification of Rothmaler (1949). He judged that all taxa of the cultivated poppy having flat lobes and round (entire), marginal, stigmatic disks belong to P. glaucum even if they produce opium. This rearrangement, however, was not accepted, because it broadened the notion of P. glaucum. This species was defined as a little capsule taxon living in the eastern part of the Mesopotamian and in the western part of Irano-Arabian flora provinces. It has undeniable morphologically similar characteristics to the opium poppy, maybe due to a convergent evolution (Kadereit and Sytsma 1992) or to the triploid origin of the opium poppy. Nevertheless, its basic chromosome number x = 7 differs from the opium poppy (x = 11). The lack of morphinoids in P. glaucum is a decisive argument against the transfer of any taxon from P. somniferum to it. Another chemical character, the accumulation of dihydroroemerine by P. glaucum, which is not present in P. somniferum, demonstrates convincingly that P. glaucum has similar alkaloid synthesis route to the P. dubium group (Rhoeadium) and to the Pilosa section of the genus (Tetenyi 1993). In conclusion, the removal of opium-bearing cultivated poppies from P. somniferum has been unanimously rejected by botanists. 2. Infraspecific Systematics. The first distinction of the poppy species is due to Bauhin (1623), who separated P. vulgare with poricide capsules from P. hortense semine alba with indehiscent ones. However, it was Linnaeus (1753) who distinguished 5 variants of P. somniferum:

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P. TETENYI

• a: white or yellowish seeds, white, pale violet, grayish-purple

flower petals with white or red blotches

• p: dark seeds

• y:dark seeds and white laciniate flowers • 8: dark seeds and red laciniate flowers • e: all double flowers

Hayne (1819), like DeCandolle (1821), distinguished var. album and var. nigrum for the cultivated poppy. Alefeld (1866) attached P. somniferum poecilospermum as a third group, also including the var. setigerum, among other varieties. Boissier (1867) distinguished var. album in Persia, var. glabrum in Asia Minor and in Egypt, and var. setigerum in the Peloponnese and in Cyprus based on area differences and hairiness. Fedde (1909) utilized the approach of Boissier, taking into account characters combined for the distinction of varieties. Nevertheless, he did not systematize the cultivated infraspecific taxa, but gave a list of distinct morphological characters (A to G), and enumerated their variation, e.g.:

c. Capsulae stipite Capsulae non stipitatae var. genuinum, var. paeoniflorum, var. dinocarpum, var. apodocarpum, var. caesium, var. Haageanum, var. griseum, var. quassandrum, var. roseum, var. luteum Capsulae stipitatae var. officinale, var. Hussenoti, var. stipitatum, var. hortense The next step for an infraspecific system of cultivated poppies was based on extensive collection of more than 1600 samples from Europe and Asia by the All-Union Institute for Plant Cultivation (Leningrad) during the 1920s and 1930s. Eight subspecies were defined by Bazilevskaya (1931,1941) based on morphology and on geography, including varieties differentiated by flower color. Bazilevskaya classified P. somniferum taxa according to the stigmatic lobes' margin and shape (entirely round and flat, or serrulate-dentate and sulcate). She felt it necessary to introduce the rank of proles as groups of varieties between these two levels. Vesselovskaya (1975) accepted the proles within the subspecies turcicum, persicum, and subspontaneum, but she could not agree with the separation for more proles within ssp. eurasiaticum. A classification of three levels for cultivated poppies was evolved by Danert (1958), based on the collection of Gatersleben (Germany).

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OPIUM POppy (PAPAVER SOMNIFERUM)

387

He considered Bazilevskaya's distinction for lobes as the most important character for evaluating the rank of subspecies, and he proposed to combine it with seed color to distinguish among convars. The poricide or indehiscent capsule character was subordinated to the convar level. The deepest rank was given to the flower colors. His hierarchic system included 52 varieties divided into 2 subspecies (somniferum and songaricum, with 26 varieties each). Danert's system was modified by Hammer (1981), who rearranged the 52 varieties, and added setigerum as a third subspecies. He stressed the importance of the dehiscence or indehiscence of the capsule by giving the convar level for this quality (somniferum, alefeldii, rothmaleri, orientale, each with 13 varieties). He considered capsule character and seed color from dark to light. Danert's and Hammer's systems are easy to use because ornamental cultivars and subspontaneous taxa were included. The systems, however, did not consider geographical conditions and, thus, were rather artificial. The classification of Vesselovskaya (1975) seems to be more acceptable. Her system has three levels: the subspecies based on origin (geography), morphology (height and branching of axis, shape of capsule), and physiology (life cycle). Varieties, including cultivars, were arranged according to petal color and blotch pattern (Table 7.1). None of these classifications dealt with differences in alkaloid biosynthesis, despite the fact that it was obvious that the worldwide distribution of species causes a wide spectrum of alkaloids. Morphine and codeine as well as the alkaloids narcotine or papaverine could be the main constituent of the capsule (Tetenyi and Vagujfalvi 1965), as shown in Fig. 7.5. Additional evidence for diversity was the demethylation process found in some poppy cultivars (Nielsen et al. 1983). Steps of the alkaloid biosyntheses of benzylisoquinolines were elucidated by enzymatic research of Zenk and coworkers during the last decade (Stadler et al. 1987; Gerardy and Zenk 1993). The most impressive part, the biosynthesis of morphinoids, is presented in Fig. 7.6. The demethylation of thebaine, which is widespread in the genus Papaver, can take place two ways: either from thebaine to oripavine or by thebaine to codeine to morphine. The latter pathway occurs in P. gracile and P. somniferum, but very rarely by other species. The ratio of the three alkaloids are similar (homologous) for opium poppy and P. gracile, confirming the relationship of these two species (Tetenyi 1993). The oripavine-morphine demethylation pathway is

w

o:J o:J

Table 7.1.

Infraspecific system of P. somniferum based on petal and blotch color. Varietas based on petal + blotch color

Convarietas

opiiferum somniferum eurasiaticum songaricum tarbagataicum chinense tianschanicum turcicum drogist persicum indicum

White

niveum album songaricum tarbagataicum chinense tianschanicum turcicum drogist persicum indicum

Source. Vesselovskaya (1975).

White + violet

Pink or red + white

Pink or red + violet

pictiflorum papyrinum

sanguineum roseolum rhodellum

apia tum

albooculatum rubriflorum

striatum punctatum

virga tum

Violet + dark violet

opiiferum rubrum quassandum atroviolaceum genuino-violaceum stipitatum erubescens violaceum livens

rubellum

ianthinum violascens pullatum violaceo-maculatum violiflorum

Light violet + dark violet

violet + white

lilacinum somniferum subviolaceum eurasiaticum subgriseum lividum

7.

OPIUM POPPY (PAPAVER SOMNIFERUM)

389

Capsule alkaloids

40-20%

70-40%

Cultivars or strains

20-10%

10-1%

, . - - - - - - - - - - - - narcotine + - - - - T - 4 thebaine codeine~ thebaine - - narcotine "Hatvani" ' " narcotine - - thebaine - - - - - T-16 papaverine narcotine T-21 thebaine - - codeine 5 thebaine - - codeine 6

morphine

17::;;;;;;;;;;;;;;;;;;~=======thebaine

thebaine + papaverine narcotine ~ codeine - - thebaine ~ papaverine codeine + - - - thebaine narcotoline - - codeine - - thebaine - - - - papaverine \ codeine - - thebaine thebaine - - codeine + narcotine narcotine - - codeine - - - - codeine ~morPhine ~ narcotin,e - - thebaine """ papaverine narcotine papaverine - - morphine - - narcotine papaverine --morphine - - codeine - - narcotine + narcotol ine narcotine - - morphine - - codeine - - - - - - - - - - ,

4 3

T-8 T-28 45

T-2 10 T-24 174 T-25 T-27 T-l T-23

Fig. 7.5. Infraspecific chemical diversity of opium poppy (Tetenyi and Vagujfalvi 1965).

found in some cultivars of opium poppy that have two pathways to accumulate morphine. An infraspecific classification of opium poppy was established by Tetenyi (1963) based on the diversity of alkaloid syntheses and accumulation. Chemoconvars Morphinan and Isoquinoline as well as their chemoprovars were distinguished. This concept was accepted by Nyman and Hansson (1978), in evaluating their cultivar collection (Table 7.2). The correlation between morphological taxa and their chemical characteristics was high (only 0.1 0/0 of error). The classification was presented at the International Botanical Congress in Berlin 1987 (Tetenyi 1988). An improved infraspecific classification of opium poppy is based on chemotaxonomy, including the two demethylation pathways and the response to photoperiod (Tetenyi 1989).

P. TETENYI

390

o HO~ H morphine

COFACTORS a: NADH (or NADPH) b: NADPH

H3

~+~fyme ~30~

i \ a

H3

H~ o o morphinone

t

HO

codeine " \ b + enzyme

H3

HO~ H3

o 3-0-demethyl-neopinone

\-o

H~

H3

b

CH3~ o H3

CH3 oripa-..1ne

CH30

thebaine

salutaridinol

Fig. 7.6. Biosynthetic pathways of morphinoids (Kodaira and Spector 1988; Lotter et al. 1992). Arrangement is based on a multidimensional scaling model (Tetenyi 1989).

7.

OPIUM POppy (PAPAVER SOMNIFERUM)

Table 7.2.

391

Chemical diversity of European and Asiatic cultivars. Chemoprovarieties

Botanical Convarieties Varieties

IsoIsoMorphinan Isoquinoline quinoline Morphine quinoline Phtalide Benzyl Total

somniferum serenum Danert roseolum Vess. candidum Vess. nigrum Hayne oculatum Rothm. somniferum papyrinum Danert paeoniflorum Alef. haageanum Alef. alefeldii spilanthum Danert pallidum Rothm. quassandum Alef. sanguineum Vess. contrasticum Danert rothmaleri albescens Vess. livens Vess. apia tum Vess. rubrum Basil. orientale fulgidum Danert ocellatum Danert leucomelum Danert glaucescens (Rothm.) Danert rotundilobum Danert Total

11

3 1

1 1

1 1

1 1

20 2

1

1 1

2 2 2

1 1 1 1

1 1 1 1

3

1

5

2

2

4

1

1 1

1 1 1 1

2

3

2

1 1 1

1 1 1

20

2

1 1 1 1 1

1 1 15

35 3

1

35

3

1 73

Source. From the collection of Nyman and Hansson (1978), rearranged Tetenyi (1988).

III. HORTICULTURE

A. Cultivation Practices Small-scale cultivation data are reviewed from Bartos et al. (1960), subtropical-tropical cultivation data from Krikorian and Ledbetter (1975), and large-scale cultivation data from Catizone et al. (1986) and F6ldesi (1992). 1. Choice and Preparation of the Soil. Fertile soils with high organic matter and crumbly structure are well suited to opium poppy.

392

P. TETENYI

Sandy or loamy black soils are the best, but loam, sandy loam, and brown sandy grounds are also suitable. Soil pH value should be between 6 and 8. Saline soils may increase morphine content (Jordanovska and Spasenoski 1990). Wet clay and alkaline soils are unsuitable, because their surfaces crack and seed emergence may be low. Soils with subsoil water, moorlands, or marshlands must be avoided. Fine sandy soils are dangerous for young plants because early spring winds cause abrasions. Windy areas must be avoided because plants with heavy capsules easily topple. In subtropical zones, mild, northward slopes at low altitudes and southward ones at high altitudes are preferred. Opium poppy requires little or no rain during the later periods of development, especially at harvest. Rotation is necessary to avoid soil exhaustion and the multiplication of soil disease and pests' biological enemies. Opium poppy grows well after almost any plant, especially legumes. Cereals (but not maize) fit in rotation because early harvest leaves time for land preparation before sowing. Poppy should not return to the same area for 5 to 6 years. Because poppy seeds are so small, good seed bed preparation is necessary to permit rapid germination. Manure (20 t/ha) can be plowed down. Opium poppy removes 20 to 65 kg P, 60 to 115 kg K, and 60 to 80 kg N per hectare. Boron is an important micro element for poppy. 2. Choice of Cultivar and Preparation of Seed Material. The goals and the zone of opium poppy cultivation determine the choice of a suitable cultivar or land race. In 1993, the Institute of Cultivar Testing in Hungary prepared guidelines for opium poppy's cultivars by the request of the International Union for the Protection of New Varieties of Plants (UPOV). A total of 59 characteristics were considered, including morphological, physiological and chemical qualities in 6 stages of the life cycle of poppy, from seedling to the mature phase of dry capsules. The tested cultivars were grouped according to flower color, shape of stigmatic disk, seed color, and capsule alkaloid constituents. Table 7.3 serves as a basis for the choice of a suitable cultivar for any purpose (Tetenyi 1995). Breeders of many countries are producing new cultivars of poppy continuously, while institutes preserve and multiply established ones. In addition, many land races are available that are adapted to local conditions. Thus, the spectrum for the choice of an appropriate cultivar is very large. High seed quality is required (98% purity, with no tolerance for henbane Hyoscyamus niger L.), 90% germination, and trueness to

7.

OPIUM POppy (PAPAVER SOMNIFERUM)

393

Table 7.3. Diversity of poppy cultivars characteristics fundamental for infraspecific systematization.

Character Habit of axis Branching Height Flower Category Color Petal blotch Seed Color Oil content Capsule Shape Stigmatic disk Surface Dehiscence Morphine level Oripavine Vegetation cycle Course Rate Ripening Distribution Height of axis Surface of capsule Genealogy Axis branching Dehiscence

Range Branched monopodially, 8-60 flowers Short, 40-90 cm

Tall, 160-200 cm

Simple-single, 4 petals White or pink Absent

Filled with petaloids Red or violet White or violet

Dark/brown,blue, or gray Low, 35-40%

Light/white,yellowish, or pink High, 50-55%

Globose-ovoid Flat-dish-like Smooth Poricidal Low «0.1%) Absent

Flattened-cylindrical Overlapping-pyramidical Ribbed Indehiscent High (>1.8%) Present

Overwintering, 8-10 months Early, 84-110 days All at once

Rapid. 3-5 months Late, 115-136 days Sluggish

Southern-autumnal Small Smooth Ancestral Gene Center 1 Multibranched Poricidal

Unbranched, 1-2 flowers

Northern-vernal Tall Ribbed Descendant Gene Center 2 Less or unbranched Indehiscent

Source: Tetenyi 1995.

type. However, many farmers use their own seed. Selfing predominates in opium poppy, and the species is mostly autogamous. Nevertheless, considerable outcrossing can occur if foreign pollen is available during flowering (Patra et al. 1992). Usually 2.5 to 8.0 kg seed/ha are required. The mass of 1000 seed varies from 0.2 to 0.7 g according to the cultivar. It is important to disinfect the seed material with authorized chemicals against fungal pathogens such as Helminthosporium papaveris Saw, Peronaspora arborescens (Berk.) DeBary, and Xanthomonas campestris pv.

394

P. TETENYI

papavericola (Bryan & M. E. Whorter) Dye. Because of small seed size, sand or ash is often added in Turkey and in some other countries, but the difference in specific gravity between seeds and the inert constituent causes serious difficulties. A German method using cooked seeds in the mixture eliminates the specific gravity difference. A Hungarian technique using irradiated seed will be discussed in the following section. 3. Operations During the First Developmental Period. Soil disinfection to prevent such pests as grubs (Ceutorrhynchus macula-alba Herbst., Melolontha melolontha L.), wireworms (Stenocarus fuliginosus Marsham), and weevils can be achieved with Basudin (diazinon) or Furadan (carbofurane). Control of weeds can be achieved with a number of herbicides including Dicuran (chlortoluron), Mesotox (nitrophen), or Reglone (diquat-dibromide). Seeds should be sown not deeper than 1 cm in wet soil and to a maximum of 2 cm in dry soils. Distance between rows extends from 20-25 cm in gardens to 30-50 cm in large fields. The crucial step is to press the wet soil by rolling the seeds immediately after sowing. The ancient method of sowing on snow was perfect for germination in northern countries, but it is risky because of later freezes. Germination does not require high temperatures, and begins at 3°C, but 710°C is required for sprouting. The speed of germination increases until 26°C. Temperatures above this are harmful to the seedlings. Seed emergence is critical and high germination is necessary to break the soil crust. If populations are too dense, manual thinning, which is laborious, will be required. Mechanical thinning, by "bouqueting" (disking the rows crosswise with or without use of herbicide) is only a partial solution. The Research Institute for Medicinal Plant in Budakalasz (Hungary) has patented and published a method (Tetenyi and Foldesi 1970) that has been put into practice to help seed emergence and to obtain uniform plant spacing. This method is based on a special seed mixture composed of 20% viable seeds (about 800-900 thousand intact seeds/hal and 80% irradiated seeds. All seeds germinate jointly and break the surface, but irradiated seedlings cease growth, while unirradiated seedlings develop without the need of thinning. Germination and seed emergence usually takes 12-14 days. When the rows become apparent at the 2- to 4-leaf stage, hand or mechanical cultivation is carried out to improve aeration and to eliminate weeds. The first thinning can be carried out at this time and postemergence herbicides (e.g, Asulox, asulam) can be applied.

7.

OPIUM POppy (PAPAVER SOMNIFERUM)

395

Opium poppy has no side branches at the basis of the axis and Gunther (1975) distinguished it from other annual taxa of the Rhoeadium or of the Papaver section by the lack of the typical rosette structure. Mika (1955) linked the rosette state of opium poppy to the transformation of the apical meristem to the dome-shaped and partly to semiconical stages. When 2 to 6 true leaves are present at the first phase of the rosette stage, the plant does not appear to grow, but at this stage assimilates are transported to the roots, which develop quickly into the deeper layers of the soil. Opium poppy is a long-day plant, which remains vegetative under short days «15 h) and can produce 100 internodes under continual short days and appropriate temperatures. Flowering can be induced by 10 long days after 2 weeks from germination, but there is a significantly lower production of dry matter and of internode number compared with later stage inductions (Mika 1955). Gentner et al. (1975) found that cultivars differed in respect to the critical photoperiod; one Afghan cultivar could be induced to flower in a 12-h photoperiod. During the vegetative stage, a second thinning can be carried out, a second herbicide spray is advisable, fertilizer can be added, and irrigation is critical. In India, plants are irrigated every 10-14 days. 4. Operations During the Second Developmental Period. The end

of the vegetative stage is evoked by the conical transformation of the apex, and is followed by shoot elongation and by the presence of at least one sepaline division (Mika 1955). Shoot height reaches 1.0 to 1.2 m as compared to a maximum of 25 em high in the rosette stage. Gentner et al. (1975) demonstrated that elongation could be achieved by two days of 24-h photoperiod without evoking flowering. During elongation, aphids (Myzus persicae Sulz. and Aphis papaveris Scop.) can cause extensive damage and can spread downy mildew (Peronospora arborescens (Berk.) De Bary) and powdery mildew (Erysiphe polygoni Cando ex Saint-Amans). Insecticide sprays or dusts are beneficial at this stage; afterward, only insecticides noninjurious to bees can be used. In small gardens the first attacked plants or leaves are often removed and destroyed. Plants may elongate precociously in hot, dry weather; therefore, irrigation is advisable. However, in the highlands of Afghanistan there is no need of irrigation, because the cool nights cause heavy dew. The first flower opens on the main axis of the plant, and side branch flowers follow sequentially. Flowering lasts 10 to 14 days. Bright, sunny, dry days favor fertilization. Cool, rainy, windy weather hinders the continuity of flowering and seed setting.

396

P. TETENYI

The so-called opium-ripe stage occurs about 10-14 days after petals dehiscence, when the first lancing of the capsules is carried out. The incising is a delicate operation carried out with a sharp tool. The cut must not go through the wall of the capsule. Deeper wounds can ruin conditions in the inner part of the capsule, stop seed development, and facilitate entry of insect pests. An appropriate cut permits the release of latex, an alkaloid-rich, yellowish-white or light pink colored, milky juice, which forms in drops on the surface of the capsule. The exudate spills out from the special inner laticiferous branching network of phloem. Alkaloids of this juice are produced in cells around the laticifers and are sequestered into the vacuoles of the laticiferous cells (Roberts et al. 1991). The drops become brownish on the outer surface of the capsule as the air starts an enzymatic reaction, and the loss of water transforms the liquid to an elastic structure in one day. This material can be collected by scraping it off and molding it to the desired form to produce the so-called raw opium. Successive lancing can be followed 3 to 5 times in this stage. The yield of opium ranges from 27 to 35 kg/ ha in India and 61 to 83 kg/ha in China. (ICNB 1995). When it is extracted, the alkaloid content is 6.2 to 10.1 %, mostly morphine. Opium contains 75% proteins, sugars, various ferments, waxes, resins, pectins, mucilage, and rubber material (Karsten et al. 1962). Ramenathan (1981) stimulated the yield and morphine content of opium by the application of liquid ethephon. Opium yield can be increased by heterosis breeding (Singh and Khanna 1991a). Since opium has been misused, there has been a world effort to minimize illegal opium production by limiting the actual extent of opium poppy cultivation. The UN Commission on Narcotic Drugs determines the cultivated surface that must be controlled by the states, according to the UN Conference Protocol agreement of 1953 and the Single Convention on Narcotic Drugs in 1961. Some countries have the right to produce opium for their inner needs, e.g., Japan and Russia, while legal export is allowed for other states. Since the acceptation of this protocol, Yugoslavia (Macedonia), Greece, Turkey, and Iran have stopped opium production. India produced 350-490 t of opium yearly from 1989 to 1993 (INCB 1995). This legal cultivation was concentrated into three small regions of India: in the valley of the Ganges around Benares, in Madhya Pradesh, and in Rajasthan with about 12,000 to 15,000 ha (Fig. 7.7). China, the second country in legal production, collected only 15-20 t. Major opium importers were the United States and Japan. Illegal world production is estimated to be the same quantity as that of India, and comes from the Golden Triangle of Burma

7.

OPIUM POppy (PAPAVER SOMNIFERUM)

397

CHINA

PAKISTAN

BURMA

BAY Of BENGAL ARABIAN

SEA

I Z 3 " , •

BOMBAY NEEMUCH DELHI AGRA GWALIOR VARANASI (BENARES I 7 GtiAZIf>UR 8 Plt.TNA 9 CALCUTTA

Fig. 7.7. Cultivated areas for opium poppy in India (Krikorian and Ledbetter 1975).

(Myanmar)-Thailand-Laos. The area of Burma is estimated at 60,000 ha, but has poor farming practices. Smuggling routes of heroin (transformed from the morphine alkaloid of opium) were first started from the Golden Crescent: Pakistan and Afghanistan (White and Raymer 1985). The illegal cultures of poppy in Mexico were destroyed by defoliants applied from helicopters. 5. Ripening and Harvest. The last stage of poppy's ontogeny is the period of ripening, which ends about 6 weeks after flowering. Leaves wither from base to apex along the axis and on the side branches. Capsules lose water continuously and their vivid green color becomes

398

P. TETENYI

yellowish. During this stage there is a decreased resistance to disease pathogens (e.g., Entyloma fuscum Schroet, Peronospora arborescens (Berk.) DeBary) and pests. If application of insecticides has been omitted, the damage to capsules becomes visible by the burrows indicating a mass of internal grubs (Ceutorrhynchus maculaalba Herbst, Dasyneura papaveris Winn.) and by secondary attack of saprophytic fungi. The last step of ripening is capsule drying and initiation of seed dormancy. The physiological and metabolic changes of this maturity stage have been described by Tookey et al. (1976). Capsule ripening is connected with an accelerated demethylation process of thebaine to morphine. Hastened ripening due to unfavorable climatic conditions can partly hinder this process, resulting in higher level of thebaine and codeine. Ripe seeds become separated from the partition walls of the capsule, and can disperse through the pores of dehiscent (shattering) cultivars. Most ornamental opium poppies belong to this group. Indehiscent cultivars respond to unfavorable conditions during ripening by half-open pores (Danert 1958) (Fig. 7.8). Harvest of poppy is optimal when capsules are dry, straw-yellow (brown color indicates adverse weather or pest attacks), and fragile. Shaking ripe seeds produces a rattling sound. In small-scale cultivation harvest is achieved by cutting stems at 20 to 25 cm from the capsules. A deeper cut above the ground is necessary when additional drying is required due to rainy weather, and 10 to 15 plants are stocked cone-shaped. The stalk after drying can be used for fuel. In large-scale cultivation harvest is achieved by specially adapted combines, which thresh poppy heads within 10 to 20 cm of stem. Seeds and capsule pieces must be separated quickly to avoid rancidity caused by green leaves or stems. Seeds and capsules can be dried by aeration, but under normal conditions there is no need for artificial drying. Seeds with 9% moisture and crushed capsules with 12% moisture can be preserved in sacks. Yields vary from 0.8 to 2 t/ha for seed and 0.6 to 2 t/ha for dry capsule. Heritability of seed yield is high (74%) and could be increased by heterosis breeding (Singh and Khanna 1991a,b). Because capsules contain 0.4 to 1.5% morphine, yield can vary from 5 to 30 kg/ha, depending on the cultivar. Yield depends on plant density and the number of capsules per plant. In temperate climates the optimum density is 450,000 plant/ha with 1-2 capsules per plant, while in southern countries, such as Turkey, small farmers are satisfied on smaller grounds with 25,000 plants/ha, with 5 to 8 capsules/ plants.

7.

OPIUM POppy (PAPAVER SOMNIFERUM)

399

III

5 mil Fig. 7.8. Dehiscent (I), semidehiscent (II), and indehiscent (III) capsules of opium poppy (Danert 1958).

Opium poppy may be intercropped. In Laos, poppy was traditionally cultivated with 30 different plant species The best companion crops for poppy are leafy vegetables, which can be harvested before shoot elongation, or root vegetables, which develop after the har-

400

P. TETENYI

vest. Double cultivation in humid temperate climates (e.g., in Poland) is achieved by a common sowing of poppy at spring with biennial caraway (Carum carvi L.).

B. Utilization 1. Medicinal. The ancient Greeks discovered the narcotic effect of opium poppy latex, which was attributed to the god Hypnos, who personified sleep and dreams. He was portrayed as having a poppy plant in his hands and a horn used for sprinkling of dreams. Dried capsules were used for colic hypertension and were extracted with water to prepare tinctures for medicinal use. Poppy "tea" has long been a part of folk medicine. The Ayurvedic treatises, classic Hindu works on materia medica in the eighth century, considered poppy seeds an aphrodisiac, while the capsules were used to alleviate cough and to induce delirium. Avicenna (986-1037) wrote about the dazing effect of opium, and Sydenham (1624-1689) declared that "Sine opio nollem esse medicus" ("I would not be a doctor without opium"). This opinion was accepted by many doctors, until the nineteenth century, when morphinomania (morphine abuse) became recognized. Duke (1973) gave a detailed overview of the utilization of poppy worldwide. Leaves have been rubbed on the body as an anodyne (India), and decoction of the stem has been used for stomachache and diarrhea (Okinawa, Japan). Flowers have been active against some human microorganisms (Bulgaria) and dried capsules have been used for coughs (Europe). Opium has had many traditional uses in medicine: smoked or eaten for malaria, applied externally for headaches (Southeast Asia), and used in formulations as analgesics, coagulants, and hypnotic agents, and for constipation (India, Pakistan). Opium was given to war elephants to calm them in the Mogul Empire. Headspace olfactoric evaluation of medicinal opium disclosed pyrazines of special interest (Buchbauer et al. 1994). Pharmaceutical opium powder is adjusted to 10% morphine. The effects are due to a combination of different alkaloids with either synergistic or antagonistic:: effects. The use is limited for grave pains with spasms. Tincture of opium is permitted as an antidiarrhoeic, when other medicines are unsuccessful (Braun and Frohne 1987). Seeds of poppy contain alkaloids on the outer surface only, when capsules have been injuried during cultivation, or as traces in the endosperm. Therefore, sedative effects were mostly due to unripe seeds or to the capsule powder being mixed by chance during treat-

7.

OPIUM POPPY (PAPAVER SOMNIFERUM)

401

ing or storing. Thus, seed washing is indispensable before the utilization for food. Opium poppy seeds significantly inhibited neoplasia as a valuable anti carcinogenic agent (Aruna and Sivaramakrishnan 1992). Seed oils have a confirmed vitamin F effect in skin treatments (Duke 1973). A poppy capsule contains more than 50 alkaloids. The presence of morphinoids, phtalide-isoquinoline alkaloids (narceine, narcotine, narcotoline), and of papaverine is characteristic (Preininger 1986). However, La Valva et a1. (1985) could find no morphinane alkaloids in plants of ssp. setigerum, where papaverine dominates. Morphine is a powerful analgesic, narcotic, and stimulant and serves also as raw material for synthesizing other antitussive (pholcodine) or veterinary (etorphine) drugs. Morphine is unique in treating acute pain, such as that from kidney stones or pancreatitis. Codeine is a valuable suppressant of cough, but its quantity and ratio are small when opium or poppy capsule is processed. To cover this need, about 7080% of morphine used to be methylated for codeine. Narcotine and papaverine are spasmolytics. For the extraction of all these poppy alkaloids, about 31,000 t straw (capsule with short stem), 300 t concentrate of straw, and 900 t opium was used in 1993 (INCB 1995). These alkaloids are poisons, thus, everyone must be utilized exclusively according to medical prescriptions. There have been many attempts to select for high opium content, the main value of the capsule. The maintenance of individual alkaloids in opium has been studied by Khanna and Shukla (1991), and Lal and Sharma (1991). Capsule morphine content of hybrids were close to parental means (Morice and Louarn 1971). Interspecific hybridization of poppy strains with three species of the Papaver section Macrantba indicated the presence of both parental spectra in F 1 hybrids (Pyysalo et a1. 1988). A cross of opium poppy with P. pseudoorientale (Lorincz and Tetenyi 1966) produced one new cultivar after 8 years of selection with more alkaloids and higher ratio of codeine and thebaine, but the proportion of morphine did not significantly increase. This cultivar, 'Kek Duna' ('Blue Danube'), was accepted by the Hungarian Testing authorities and is now considered the standard for any new cultivar produced by breeding in Hungary. A breakthrough was realized in Tasmania by a triple crossing of existing poppy cultivars (Nielsen et a1. 1983). The researchers involved one cultivar from India, characterized by the ability to demethylate oripavine to morphine. The combination resulted in hybrids having both demethylation pathways. The new selections

402

P. TETENYI

produced morphine content as high as 2.3%, with an average at 1.3 to 1.6% as compared with the 0.6 to 0.8% average value in earlier cultivars. 2. Culinary (Oil and Spice). Ancient Egyptians ground poppy seeds between stones and used this material as a condiment mixed into or sprinkled on the surface of batter. According to an ancient Chinese opinion, ground poppy seeds lubricate the mouth, harmonize the lungs, and nourish the stomach. At present, seed color preference varies: Turks prefer brown, central Europeans blue, and northern Europeans white seeds. The flavor of opium poppy seed is based partly on the oil and protein constituents. However, poppy seeds, even in very small quantities, liberate their special, pleasant, crunchy, nut-like aroma when eaten unbroken in the mouth. Although consumed as a condiment, poppy seed is very nutritious, containing 35 to 55% oil, 25 to 35% proteins, and an abundance of vitamins E, F, panthotenic acid, biotin, niacin, thiamine, and minerals (Ca, K, Mg, Fe) (Nergiz and Otles 1994). Because its high oil content poppy seed is very caloric (100 g

= 2250 kJ)

Leaves from thinned plants, although bitter, are eaten in salads or potherbs as spring greens in Southeast Asia. This bitter taste is due to the presence of compounds in the latex that cause different physiological reactions in the human body (e.g., caffeine and humulon). Seeds, which contain no alkaloids, may also liberate tasty compounds. Are poppy seeds a spice or a condiment? Tucker (1986) included poppy as a spice based on the legal definition by the American trade and tariff regulations. Previously, the concept of spice was restricted to products imported from the tropics. The notion of condiment refers to a pungent, prepared mixture of seasonings, sometimes in liquid form (Collins 1980). Poppy seeds are surely neither pungent nor piquant seasonings. Two liquid uses are known: One consists of whole poppy seeds mixed in beverages, producing an addictive effect by free serine (Duke 1973). The other one is the preparation in salad dressings, which are especially delicious with fruit salads (Collins 1980).

Whole poppy seeds are used for glazing cakes, croissants, and milk loaves. Scrambled eggs and preserved fruits have a special flavor when they contain poppy seeds. Yet much of the breeding and selection efforts are aimed at appearance, particularly the color of the seed coat (Schijfsma et al. 1960). Continuous selection is needed to

7.

OPIUM POppy (PAPAVER SOMNIFERUM)

403

ensure attractive eye appeal. Minor differences of the seed (bluish gray vs. sky blue) are important distinctions between cultivars. High seed ridges improve appearance and are appreciated by the baking industry. Ground poppy seeds are utilized for porridge, sometimes with honey, for dusting pasta or pie crust, and for sprinkling over fruit salads when mixed with sugar. Ground seeds can be used as the main constituent of pastry fillings and in various cheese products (Collins 1980). Finely ground poppy seed material can be used for the preparation of ice creams and candies. The seed oil is used for cooking, but it must be preserved under favorable conditions to prevent rancidity since it is composed from many unsaturated lipids. The oil contains 50 to 65% linoleic, 30% oleic, and 6 to 9% palmitic acid (Karsten et al. 1962; Nergiz and Otles 1994). There is a large variation in oil content of seeds. Oil content ranges from 37 to 54%, with the higher values found in lightcolored cultivars (Vesselovskaya 1975), but taxa belonging to Asiatic subspecies have less oil content than the European ones, in spite of their white colored seeds. In Czecho-Slovakian trials there was a slight correlation between seed oil content and morphine level of capsules (r = 0.21 ±0.12). Heritability of this character is high (Singh and Khanna 1991b). Poppy seed oil is one of the best constituents for paints, varnishes, and soaps. At one time poppy seed oil was videly used by artists, but it is relatively expensive. Poppy products are also used as animal feed. Oil cakes contain 88 feed units per 100 kg and are utilized for feeding dairy cows, sheep, and horses. Poppy straw, however, can induce cerebro-spinal excitations in cattle (Duke 1973). Poppy seed is valuable as bird feed. 3. Ornamental and Other Uses. The decoration value of poppy flowers had been lauded by the poets of China for a thousand years. The ancient Chinese also plugged citrus fruits with opium and hung them in rooms where they released a special perfumed atmosphere. Poppy, as carnation or peony, was selected in cloister-gardens of Europe and their ornamental use spread to peasant gardens. Bauhin and later Linnaeus based their infraspecific classification of poppy on flower differences. Selection was directed from dark-violet petals (as in ssp. setigerum) to red and pink and white with different blotch patterns from almost black-violet to white. The blotch can be confined to the base of the petal, present between veins, or marginal. Cultivars also exist with speckled petals. Petal shape also varies from the entire

404

P. TETENYI

undulate to serrated, incised, fringed, and laciniated forms. Many ornamental poppies have double flowers due to the transformation of anthers, partly (flare semi-plena, peony-like ) or totally (flare plena, carnation-like), to petaloids. The genetics of petal color and pattern has been studied by B5hm (1986), who established a physiological relationship between anther color and blotches. He established the role of anthocyanates in the color tint of petals by including species of the Papaver section Macrantha in crosses. Poppy capsules also have decorative value. They can be used in their natural state (straw-yellow or violet) and can be also gilded or painted and utilized in dried arrangements. The huge capsules of ssp. chinense, with a diameter of 12-18 cm, are often used as a solitary decoration. Heritability of capsule size is very high (h 2 = 84 % ) (Singh and Khanna 1991b). IV. SUMMARY AND PROSPECTS

Opium poppy is one of the earliest domesticated plant species and its botanical and historical origins, although partially revealed from a number of studies, still remain obscure. Future research, especially with molecular techniques, will undoubtedly cast more light on the genesis and development of this cultivated species. The dispersal of opium poppy extends from its primary gene center around the Aegean Sea to the far reaches of the globe, including Tasmania, Japan, Newfoundland, and Argentina. The present biodiversity of this species is due to its high genetic plasticity which has adapted to various environmental conditions and cultivation systems, as well as to continuous selection and breeding to accommodate the different goals of users. The taxon has become differentiated karyologically, morphologically, physiologically, and biosynthetically, and these factors must be integrated to produce a consistent infraspecific taxonomy. The present cultural practices for opium poppy vary tremendously, from primitive manual systems to highly mechanical operations. Similarly, germplasm varies from traditional land races to highly adapted, productive cultivars. This results in tremendous differences in yield and quality. Economic profitability of the crops ultimately depends on high rates of seed or opium production based on adapted cultivars and efficient production practices. Alkaloids produced by opium poppy are of special importance for a broad spectrum of medical uses, from the alleviation of acute

7.

OPIUM POppy (PAPAVER SOMNIFERUM)

405

pain to cough suppression. It has been possible to select poppy for high alkaloid production and altered ratio of specific alkaloids in the dry capsule. This trend can be expected to continue. The illicit production of opium may be reduced in the future as more and more governments acknowledge the dangers of misuse to society. International cooperation is needed to end this damaging activity, but alternative options must be made available to small growers that presently produce opium destined for illegal use. The use of poppy seed and seed oil for culinary purposes has increased potential. Improvements are required in characters such as seed color, seed ridges, and the ratio of oil constituents. The ornamental uses of poppy make them a valuable horticultural crop. Improvement in these areas will require increasing the pool of genetic information of Papaver. The huge biodiversity of opium poppy represents a tremendous resource for humankind, and it is essential that this biodiviersity be preserved for the interest of future generations. LITERATURE CITED Alefeld, F. 1866. p. 227-229. In: Landwirtschaftliche Flora Mitteleuropas. WiegandtHempel, Berlin. Aruna, K., and V. M. Shivaramakrishnan. 1992. Anticarcinogenic effects of some Indian plants. Food Chem.Tox. 30:953-956. Bakels, C. C. 1982. Der Mohn, die Linearbandkeramik. Archeologisches Korrespondenzblatt 12:11-13. Bartos, J., S. M6rasz, S. Sarkany, J. Vuk, and K. Zsoar. 1960. A mak es termesztese (Poppy and its cultivation). Mg. Kiad6 (Agron. Publ.). Budapest. Bauhin, G. 1623. p.170. In: Pinax Theatri Botanici. L. Regis Basileae. Bazilevskaya, N. A. 1931. Botanical groups of poppy. Trudy Prikl. Bot. Gen. Selekc. 25:185-196. Bazilevskaya, N. A. 1941. Sem.Papaveraceae Juss. Makovie (Poppies). Kulturn. (Cultiv.) Flora USSR. Leningrad. 7:1-39. Bisset, N. G., J. G. Bruhn, S. Curto, B. Holmstedt, U. Nyman, and M. H. Zenk. 1994. J. Ethnopharm. 41:99-114. B6hm, H. 1986. Basalflecke und Fleckenlosigkeit. Z. Naturforsch. 41:158-163. Boissier, E. 1867. Flora orientalis. H. Georg, Basel. 1:105-118. Braun, H., and D. Frohne. 1987. p. 173-176. In: Heilpflanzen Lexikon. G. Fischer, Stuttgart. Buchbauer, G., A. Nikiforov, and B. Remberg. 1994. Planta Med. 60:181-183. Catizone, P., M. Marotti, G. Toderi, and P. Tetenyi. 1986. p. 245-252. In: Coltivazione delle Pianti Medicinali. Patron Ed. Bologna. Collins, M. 1980. Spices of the world. p. 7,47-48,142,277,318-319,334,347-352. In: Cookbook McCormick. McGraw-Hill, New York. Danert, S. 1958. Zur Systematik von P.somniferum. Kulturpflanze 6:61-88. DeCandolle, A. 1883. Origine des plantes cultivees. Baillere, Paris.

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DeCandolle, A. P. 1819. Flore fran«;;aise. Paris. 5:585. DeCandolle, A. P. 1821. Regni vegetabilis systema naturale. Paris. 2:81-82. Duke, J. A. 1973. Utilization of Papaver. Econ. Bot. 27:390-400. Elkan, L. 1839. p. 30. In: Tentamen monographiae generis Papaver. E. J. Dalkowski Konigsberg, Berlin. Fedde, F. 1909. Papaveraceae-Papaveroideae. p. 1-430. In: A. Engler (ed.), Das Pflanzenreich, Vol. 40. Engelmann. Leipzig. Foldesi, D. 1992. Poppy. p. 119-128. In: L. Hornok (ed.), Cultivation and processing of medicinal plants. Wiley, Chichester, and Mg. Kiad6 (Agron.), Budapest. Frenzel, R. 1992. The history of flora and vegetation during the quaternary. Progr. Bot. 53:370, 378. Gentner, W. A., R B. Taylorson, and H. A. Borthwick. 1975. Responses of poppy to photoperiod. Bull. Narc. 27:23-31. Gerardy, R, and M. H. Zenk. 1993. Formation of salutaridine. Phytochemistry 32:7986. Gmelin, G. G. 1806. Flora Badensis alsatica Carlsruhense. II:479. Gunther, K. F. 1975. Beitraege zur Morphologie der Papaveraceae. Flora. 164:393436. Hammer, K. 1981. Probleme der Klassifikation von P.somniferum. Kulturpflanze 29:287-296. Hammer, K., and R Fritsch. 1977. Zur Frage der Ursprungsart des Kulturmohns. Kulturpflanze 25:113-124. Hayne, F. G. 1819. Getreue Darstellung der in der Arzneykunde gebrauchlichen Gewaechse Berlin. 6:40. Hegnauer, R. 1969. Chemotaxonomie der Pflanzen. Birkhauser Basel, Stuttgart. 5: 264-293. Hegnauer, R 1989 Chemotaxonomie der Pflanzen Birkhauser Basel, Stuttgart. 9:187208. Hussenot, F. 1835. p. 39. In: Chardons Nanciens. Nancy. International Narcotics Control Board. 1995. p. 208. In: Report on narcotic drugs: world requirement and statistics for 1993. UN, Vienna. Jordanovska, V., and M. Spasenoski. 1990. Effect of salinity on the production of morphine. Acta BioI. Med. Exp. 15:69-74. Kadereit, J. W. 1986a. P. somniferum a triploid hybrid? Bot. Jahrb. Syst. 106:221244. Kadereit, J. W. 1986b. A revision of the Papaver section Papaver. Bot.Jahrb.Syst. 108:1-16. Kadereit, J. W. 1987. Experimental evidence on the affinities of P.somniferum. Plant Syst.Evol. 156:189-195. Kadereit, J. W. 1988. Sectional affinities and geographical distribution in the genus Papaver. Beitr.Biol.Pflanzen. 63:139-156. Kadereit, J. W. 1989. A revision of the Papaver section Rhoeadium. Notes R. Bot. Garden Edinburgh. 45:225-286. Kadereit, J. W. 1991a. A note on the genomic consequences in triploids. Plant Syst. Evol. 175:93-99. Kadereit, J. W. 1991b. Papaveraceae-Papaveroideae, Vol. 1. p. 297-302. In: T.G. Tutin, N. A. Burges, A. O. Chater, J. R. Edmondson, W. H. Heyward, D. M. Valentine, S. M. Walters, and D. A. Webb (eds.), Flora Europaea, 2d ed. Univ. Press. Cambridge, UK.

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407

Kadereit, J. W., and K. 1. Sytsma. 1992. Disassembling Papaver: a restriction site analysis of chloroplast DNA. Nord.J.Bot. 12:205-217. Kapoor, L. D. 1995. Opium poppy: botany, chemistry, and pharmacology. Haworth, Binghamton, NY. Karsten, G., U. Weber, and E. Stahl. 1962. p. 555-558. In: Lehrbuch der Pharmakognosie. Fischer Stuttgart. Khanna K. R, and Sudhir Shukla. 1991. Studies on the inheritance of papaverine. Herba Hung. 30:7-10. Kodaira, H., and S. Spector. 1988. Transformation of morphinane alkaloids. Proc. NatI. Acad. Sci. (USA) 85:1267. Krikorian, A. D. 1975. Were the opium poppy known in the ancient Near East? J. Hist. BioI. 8:95-114. Krikorian, A. D., and M. D. Ledbetter. 1975. Some observations on P. somniferum cultivated for its latex. Bot. Rev. 41:30-103. Lal, R K., and J. R Sharma. 1991. Genetics of alkaloids in Papaver somniferum.Planta Med. 57:271-274. La Valva, V., S. Sabato, and S. G. Gigliano. 1985. Morphology and alkaloid-chemistry of P. setigerum. Taxon 34:191-196. Linnaeus, C. 1753. Species plantarum. Stockholm. 1:506-508. Lorincz, C., and P. Tetenyi. 1966. Otdalennaya gibridizacia (Distant hybridization) P. somniferum and P. orientale. Herba Hung. 5:97-106. Lotter, H., J. Gollwitzer, and M. H. Zenk. 1992. Revision of the configuration at C-7 of salutaridinol-I. Tetrahedron Lett. 33:2433-2346. Mansfeld, R. 1953. Zur allgemeine Systematik der Kulturpflanze. Kulturpflanze 1:138-155. Mansfeld, R. 1962. p. 659. VorHiufiges Verzeichnis kultivierte Pflanzenarten. Kulturpflanze Beiheft 2. Merrillees, R S. 1988/89. Highs and lows in the Holy Land: Opium in biblical times. Eretz Israel 20:148-153. Mika, E. S. 1955. Studies on the growths and development of opium poppy. Bot. Gaz. 116:323-339. Miller, P. 1768. p. 9. In: The gardener's and florist's dictionary, 8th ed. Morice, J., and J. Louarn. 1971. Etude de la teneur en morphine chez l'oeillette (P. somniferum). Ann. Amelior. Plantes 21:465-484 Nergiz, C., and S. Otles. 1994. The proximate composition and minor constituents of poppy seeds. J. Sci. Food Agr. 66:117-120. Nessler, C. 1. 1994. Sequence analysis of two new members of the latex protein supports the triploid-hybrid origin of the opium poppy. Gene 139:207-209. Nielsen, B., J. Roe, and E. Brochmann-Hanssen. 1983. A new opium alkaloid. Plant Med. 48:205-206. Nyman, U., and B. Hansson. 1978. Morphine content in P. somniferum. Hereditas 88:17-26. Patra, N. K., R S. Ram, S. P. Chauchan, and A. K. Singh. 1992. Quantitative studies on mating system of opium poppy. Theor. AppI. Genet. 84:299-303. Preininger, V. 1986. Chemotaxonomy ofthe Papaveraceae alkaloids. p.1-64. In: The alkaloids, Vol. 29. Academic, New York. Pyysalo, H., C-J. Widen, C.A. Salemink, E. Lewing, A. Rousi, and A. Ojala. 1988. Interspecific hybridization in Papaver 2. Ann. Bot. Fenn. 25:1-10. Ramenathan, V. S. 1981. Effect of ethrel oil in opium poppy. J. Agr. Res. 15:223-226.

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Reckin, J. 1970. A contribution to the cytology ofP. aculeatum, P. gracile. Caryologia. 23:461-464. Reckin, J. 1973. Proposals for the revision of the section Mecones.Caryologia 26:245251. Renfrew, J. M. 1973. p. 248. In: Paleobotany: the prehistoric food plants of the Near East and Europe. Methuen, London. Roberts, M. F., B. C. Homeyer, and T. D. T. Pham.1991.Further studies of sequestration of alkaloids in P. somniferum. Z.Naturforsch. 46:377-388. Rothmaler, W. 1949. Notulae Systematicae, 4: Papaveres. p. 42-44. In: Index Seminum Gaterslebense. Schijfsma, L., M. Hoesbergen, and F. E. Nijdam.1960. A study on the colour of the seed of poppy. Euphytica 9:127-140. Schultze-Motel, J. 1979. Die urgeschichtlichen Reste des Schlafmohns. Kulturpflanze 27:207-215. Shaulskaya, N. A. 1993. Vascular plant species, new and rare for the South Kamchatka state reserv. Bot.Zh. 78:133-139. Simmonds, Q. W. 1976. p. 339. In: Evolution of crop plants. Longman, London. Singh, S. P., and R K. Khanna. 1991a. Heterozis effect on opium poppy. Ind.J. Agr. 259-263. Singh, S. P., and R. K.Khanna. 1991b. Genetic variability in opium poppy. Dev. J.Agr. Res. 6:88-92. Sinskaya, E. N. 1969. p. 179. In: Historical geography ofthe flora of cultivated plants. Kolos, Leningrad. S06, R 1968. A magyar fl6ra es vegetaci6 kezik6nyve (The manual of the Hungarian flora and vegetation). Akad.Kiad6 (Acad. Publ.). Budapest. 3:267. Srivastava, S., and U. C. Lavania.1991. Evolutionary DNA variaton in Papaver. Genome 34:763-768. Stadler, R, T. M. Kutchan, S. Loeffler, N. Nagakura, B. Cassels, and M. H. Zenk. 1987. Revision of the early steps of the reticuline biosynthesis. Tetrahedron Lett. 28:1251-1254. Tetenyi, P. 1963. Infraspecific chemical taxa of medicinal plants. Thesis, Hungarian Acad. Sci., Budapest. Tetenyi, P. 1988. Chemical characters as tools for the classification of cultivated plants. BioI. Zblatt.107:391-394. TStenyi, P. 1989. Morphinoids du genre Papaver. Acte du colloque a Montreal. Chicoutimi. p. 64-69. Tetenyi, P. 1993. Chemotaxonomy of the genus Papaver. Acta Hart. 344:154-165. Tetenyi, P. 1995. Biodiversity of P.somniferum. Acta Hart. 390:191-201. Tetenyi, P., and D. F6ldesi. 1970. Experiments with unthinned poppy cultures grown from irradiated seed mixtures. Acta Agr. Acad. Sci. Hungary. 19:147-161. TStenyi, P., and D.Vagujfalvi. 1965. Veraenderungen d. Alkaloidgehalts von P. somniferum. Pharmazie 20:731-734. Tookey, H. L., G. F. Spencer, M. D. Grove, and W. F. Kwalek. 1976. Codeine and morphine in P. omniferum. Planta Med. 30:340-347. Tucker, A. 0.1986. Botanical nomenclature of culinary herbs. p. 33~80. In: S. Cracker, and J. Simon (eds.), Herbs, spices,and medicinal plants: recent advances in botany, horticulture and pharmacology, Vol. 1. Oryx, Phoenix, AZ. Vesselovskaya, M. A. 1975. The poppy, its variability, classification and evolution. Trudy Prikl. Bot. Gen. Selekc. 55:83-169. White, P. T., and S. Raymer. 1985. The poppy. Natl. Geograph. February:143-189. Zohary, H. 1973. Geobotanical foundations of the Middle East. Swets & Zeitlinger, Amsterdam.

Subject Index A

Apple, fruit splitting, 217-262 Artemisia, 319-371 Artemisinin, 346-359

c Cherry, origin, 263-317 D

Dedication, Campbell, C.W., xiii F

Flower: development (postpollination),1-58 Fruit splitting and cracking, 217-262 Fruit crops: apple fruit splitting and cracking, 217-262 cherry origin, 263-317 G

Glucosinolates, 99-215 I

In vitro, artemisia, 342-345

M Medicinal crops: artemisia, 319-371 poppy,373-408 Mushroom cultivation, 59-97

o Opium poppy, 373-408 Orchid, pollination regulation of flower development, 28-38 Ornamental plants: orchid pollination regulation, 28-38 poppy, 373-408 p

Physiology: flower development, 1-58 glucosinolates, 99-215 Pollination, flower regulation, 1-58

s Senescence, pollinationinduced, 4-25

v Vegetable crops, mushroom cultivation, 59-97 409

Cumulative Subject Index (Volumes 1-19) A

Abscisic acid: chilling injury 15 :78-79 cold hardiness, 11:65 dormancy, 7:275-277 genetic regulation, 16:9-14, 20-21 mechanical stress, 17:20 rose senescence, 9:66 stress, 4:249-250 Abscission: anatomy and histochemistry, 1:172-203 citrus, 15:145-182 flower and petals, 3:104-107 regulation, 7:415-416 rose, 9:63-64 Acclimatization: foliage plants, 6:119-154 herbaceous plants, 6:379-395 micropropagation, 9:278-281, 316-317 Actinidia,6:4-12 Adzuki bean, genetics, 2:373 Agaricus, 6:85-118 Agrobacterium tumefaciens, 3:34 Air pollution, 8:1-42 Almond: bloom delay, 15:100-101 in vitro culture, 9:313

A10casia, 8:46, 57. See also Aroids Alternate bearing: chemical thinning, 1:285-289 fruit crops, 4:128-173 pistachio, 3:387-388 Aluminum: deficiency and toxicity symptoms in fruits and nuts, 2:154 Ericaceae, 10:195-196 Amorphophallus, 8:46, 57. See also Aroids Anatomy and morphology: apple flower and fruit, 10:273-308 apple tree, 12:265-305 asparagus, 12:71 cassava, 13:106-112 citrus, abscission, 15:147156 embryogenesis, 1:4-21, 35-40 fig,12:420-424 fruit abscission, 1 :172-203 fruit storage, 1:314 ginseng, 9:198-201 grape flower, 13:315-337 grape seedlessness, 11:160164 heliconia, 14:5-13 kiwifruit, 6:13-50 orchid,5:281-283 411

412

CUMULATIVE SUBJECT INDEX

Anatomy and morphology (cant'd)

navel orange, 8:132-133 pecan flower, 8:217-255 petal senescence, 1:212-216 pollution injury, 8:15 Androgenesis, woody species, 10:171-173 Angiosperms, embryogenesis, 1:1-78 Anthurium, see Aroids, ornamental fertilization, 5 :334-335 Antitranspirants, 7:334 cold hardiness, 11:65 Apical meristem, cryopreservation, 6:357372 Apple: alternate bearing, 4:136-137 anatomy and morphology of flower and fruit, 10:273309 bitter pit, 11 :289-355 bioregulation, 10:309-401 bloom delay, 15:102-104 CA storage, 1:303-306 chemical thinning, 1:270-300 fertilization, 1:105 fire blight control, 1:423-474 flavor, 16:197-234 flower induction, 4:174-203 fruit cracking and splitting, 19:217-262 fruiting, 11:229-287 in vitro, 5:241-243; 9:319321 light, 2:240-248 maturity indices, 13:407-432 nitrogen metabolism, 4:204246 replant disease, 2:3

root distribution, 2:453-456 stock-scion relationships, 3:315-375 summer pruning, 9:351-375 tree morphology and anatomy, 12:265-305 vegetative growth, 11 :229287 watercare, 6:189-251 yield,1:397-424 Apricot: bloom delay, 15:101-102 CA storage, 1:309 Aroids: edible, 8:43-99; 12:166-170 ornamental, 10:1-33 Arsenic, deficiency and toxicity symptoms in fruits and nuts, 2:154 Artemisia, 19:319-371 Artemisinin, 19:346-359 Artichoke, CA storage, 1:349350 Asexual embryogenesis, 1:1-78; 2:268-310; 3:214-314; 7:163-168,171-173,176177,184,185-187,187188,189; 10:153-181; 14:258-259,337-339 Asparagus: CA storage, 1:350-351 fluid drilling of seed, 3:21 postharvest biology, 12:69155 Auxin: abscission, citrus, 15: 161, 168-176 bloom delay, 15:114-115 citrus abscission, 15:161, 168-176 dormancy, 7:273-274 flowering, 15:290-291, 315

CUMULATIVE SUBJECT INDEX

genetic regulation 16:5-6, 14, 21-22 geotropism, 15:246-267 mechanical stress, 17:18-19 petal senescence, 11:31 Avocado: flowering,8:257-289 fruit development, 10:230238 fruit ripening, 10:238-259 rootstocks, 17:381-429 Azalea, fertilization, 5:335-337 B

Babaco, in vitro culture, 7:178 Bacteria: diseases of fig, 12:447-451 ice nucleating, 7:210-212, 11:69-71 pathogens of bean, 3:28-58 tree short life, 2:46-47 wilt of bean, 3:46-47 Bacteriocides, fire blight, 1:450-459 Bacteriophage, fire blight controL 1:449-450 Banana: CA storage, 1:311-312 fertilization, 1:105 in vitro culture, 7:178-180 Bean: CA storage, 1:352-353 fluid drilling of seed, 3:21 resistance to bacterial pathogens, 3:28-58 Bedding plants, fertilization, 1:99-100; 5:337-341 Beet: CA storage, 1:353 fluid drilling of seed, 3:18-19 Begonia (Rieger), fertilization,

413

1:104 Biochemistry, petal senescence, 11:15-43 Biennial bearing, see Alternate bearing Bioregulation, see Growth substances apple and pear, 10:309-401 Bird damage, 6:277-278 Bitter pit in apple, 11 :289-355 Blackberry harvesting, 16:282298 Black currant, bloom delay, 15:104 Bloom delay, deciduous fruits, 15:97 Blueberry: developmental physiology, 13:339-405 harvesting,16:257-282 nutrition, 10:183-227 Botanic gardens, 15:1-62 Boron: deficiency and toxicity symptoms in fruits and nuts, 2:151-152 foliar application, 6:328 nutrition, 5:327-328 pine bark media, 9:119-122 Bramble, harvesting, 16:282298 Branching, lateral: apple, 10:328-330 pear, 10:328-330 Brassicaceae, in vitro, 5:232235 Breeding, see Genetics and breeding Broccoli, CA storage, 1:354-355 Brussels sprouts, CA storage, 1:355 Bulb crops, see Tulip

414

CUMULATIVE SUBJECT INDEX

Bulb crops (cont'd) genetics and breeding, 18:119-123 in vitro, 18:87-169

micropropagation, 18:89-113 root physiology, 14:57-88 virus elimination, 18:113123

kiwifruit partitioning, 12:318-324

metabolism, 7:69-108 partitioning, 7:69-108 petal senescence, 11:19-20 reserves in deciduous fruit trees, 10:403-430 Carbon dioxide, enrichment, 7:345-398, 544-545

Carnation, fertilization, 1 :100;

c

5:341-345

CA storage, see Controlledatmosphere storage Cabbage: CA storage, 1:355-359 fertilization, 1:117-118 Cactus: crops, 18:291-320 reproductive biology, 18:321346

Caladium, see Aroids, ornamental Calciole, nutrition, 10:183-227 Calcifuge, nutrition, 10:183-227 Calcium: bitter pit, 11:289-355 cell wall, 5:203-205 container growing, 9:84-85 deficiency and toxicity symptoms in fruits and nuts, 2:148-149 Ericaceae nutrition, 10:196197

foliar application, 6:328-329 fruit softening, 10:107-152 nutrition, 5:322-323 pine bark media, 9:116-117 tipburn, disorder, 4:50-57 Calmodulin, 10:132-134, 137138

Carbohydrate: fig, 12:436-437

Carrot: CA storage, 1:362-366 fluid drilling of seed, 3:13-14 Caryophyllaceae, in vitro, 5:237-239

Cassava, 12:158-166; 13:105129

Cauliflower, CA storage, 1:359362

Celeriac, CA storage, 1:366-367 Celery: CA storage, 1:366-367 fluid drilling of seed, 3:14 Cell culture, 3:214-314 woody legumes, 14:265-332 Cell membrane: calcium, 10:126-140 petal senescence, 11 :20-26 Cellular mechanisms, salt tolerance, 16:33-69 Cell wall: calcium, 10:109-122 hydrolases, 5:169-219 ice spread, 13:245-246 tomato, 13:70-71 Chelates, 9:169-171 Cherry: bloom delay, 15:105 CA storage, 1:308 origin, 19:263-317 Chestnut:

CUMULATIVE SUBJECT INDEX

blight, 8:281-336 in vitro culture, 9:311-312 Chicory, CA storage, 1:379 Chilling: injury, 4:260-261,15:63-95 pistachio, 3:388-389 Chlorine: deficiency and toxicity symptoms in fruits and nuts, 2:153 nutrition, 5:239 Chlorosis, iron deficiency induced, 9:133-186 Chrysanthemum fertilization, 1:100-101; 5:345-352 Citrus: abscission, 15:145-182 alternate bearing, 4:141-144 asexual embryogenesis, 7:163-168 CA storage, 1:312-313 chlorosis, 9:166-168 cold hardiness, 7:201-238 fertilization, 1:105 flowering, 12:349-408 honey bee pollination, 9:247248 in vitro culture, 7:161-170 navel orange, 8:129-179 nitrogen metabolism, 8:181 rootstock, 1 :23 7-269 Cloche (tunnel), 7:356-357 Coconut palm: asexual embryogenesis, 7:184 in vitro culture, 7:183-185 Cold hardiness, 2:33-34 apple and pear bioregulation, 10:374-375 citrus, 7:201-238 factors affecting, 11:55-56 herbaceous plants, 6:373-417 injury, 2:26-27

415

nutrition, 3:144-171 pruning, 8:356-357 Co10casia, 8:45, 55-56. See also Aroids Common blight of bean, 3:45-46 Compositae, in vitro, 5:235-237 Container production, nursery crops, 9:75-101 Controlled-atmosphere (CA) storage: asparagus, 12:76-77, 127-130 chilling injury, 15:74-77 flowers, 3:98; 10:52-55 fruit quality, 8:101-127 fruits, 1:301-336; 4:259-260 pathogens, 3:412-461 seeds, 2:134-135 tulip,5:105 vegetable quality, 8:101-127 vegetables, 1:337-394; 4:259260 Controlled environment agriculture, 7:534-545. See also Greenhouse and greenhouse crops; Hydroponic culture; Protected crops, carbon dioxide Copper: deficiency and toxicity symptoms in fruits and nuts, 2:153 foliar application, 6:329-330 nutrition, 5:326-327 pine bark media, 9:122-123

Corynebacterium flaccumfaciens, 3:33,46 Cowpea: genetics, 2:317-348 U.S. production, 12:197-222 Cranberry: fertilization, 1:106

416

Cranberry (cont'd) harvesting, 16:298-311 Cryphonectria parasitica, see Endothia parasitica Cryopreservation: apical meristems, 6:357-372 cold hardiness, 11:65-66 Crytosperma, 8:47, 58. See also Aroids Cucumber, CA storage, 1:367368 Currant, harvesting, 16:311-327 Cytokinin: cold hardiness, 11:65 dormancy, 7:272-273 floral promoter, 4:112-113 flowering, 15:294-295, 318 genetic regulation, 16:4-5, 14,22-23 grape root, 5:150, 153-156 lettuce tipburn, 4:57-58 petal senescence, 11:30-31 rose senescence, 9:66 D

Date palm: asexual embryogenesis, 7:185-187 in vitro culture, 7:185-187 Daylength, see Photoperiod Dedication: Bailey, L.H., 1:v-viii Beach, S.A., l:v-viii Bukovac, M.J., 6:x-xii Campbell, C.W., 19:xiii Cummins, J.N., 15:xii-xv Faust, Miklos, 5:vi-x Hackett, W.P., 12:x-xiii Halevy, A.H., 8:x-xii Hess, C.E., 13:x-xii Kader, A.A., 16:xii-xv

CUMULATIVE SUBJECT INDEX

Looney, N.E., 18:xiii Magness, J.R., 2:vi-viii Moore, J.N., 14:xii-xv Proebsting, Jr., E.L., 9:x-xiv Rick, Jr., C.M., 4:vi-ix Sansavini, S., 17:xii-xiv Smock, R.M., 7:x-xiii Weiser, C.J., 11:x-xiii Whitaker, T.W., 3:vi-x Wittwer, S.H., 10:x-xiii Deficiency symptoms, in fruit and nut crops, 2:145-154 Defoliation, apple and pear bioregulation, 10:326-328 'Delicious' apple, 1:397-424 Desiccation tolerance, 18:171213 Dieffenbachia, see Aroids, ornamental Dioscorea, see Yam Disease: and air pollution, 8:25 aroids, 8:67-69; 10:18; 12:168-169 bacterial, of bean, 3:28-58 cassava, 12:163-164 control by virus, 3:399-403 cantrolled-atmos p here storage, 3:412-461 cowpea, 12:210-213 fig, 12:447-479 flooding, 13:288-299 hydroponic crops, 7:530-534 lettuce, 2:187-197 mycorrhizal fungi, 3:182-185 ornamental aroids, 10:18 resistance, acquired, 18:247289 root, 5:29-31 stress, 4:261-262 sweet potato, 12:173-175 tulip, 5:63, 92

CUMULATIVE SUBJECT INDEX

turnip moasic virus, 14:199238 yam (Dioscorea), 12:181-183 Disorder, see Postharvest physiology bitterpit, 11:289-355 fig, 12:477-479 pear fruit, 11:357-411 watercore, 6:189-251; 11:385-387 Dormancy, 2:27-30 blueberry,13:362-370 release in fruit trees, 7:239300 tulip, 5:93 Drip irrigation, 4:1-48 Drought resistance, 4:250-251 cassava, 13:114-115 Dwarfing: apple, 3:315-375 apple mutants, 12:297-298 by virus, 3:404-405

E Easter lily, fertilization, 5:352355 Embryogenesis, see Asexual embryogenesis Endothia parasitica, 8:291-336 Energy efficiency, in greenhouses, 1:141-171; 9:1-52 Environment: air pollution, 8:20-22 controlled for agriculture, 7:534-545 controlled for energy efficiency, 1:141-171; 9:1-52 embryogenesis, 1:22, 43-44 fruit set, 1:411-412 ginseng,9:211-226 greenhouse management,

417

9:32-38 navel orange, 8:138-140 nutrient film technique, 5:13-26 Epipremnum, see Aroids, ornamental Erwinia: amylovora, 1:423-474 lathyri,3:34 Essential elements: foliar nutrition, 6:287-355 pine bark media, 9:103-131 plant nutrition 5:318-330 soil testing, 7:1-68 Ethylene: abscission, citrus, 15:158161,168-176 apple bioregulation, 10:366369 avocado, 10:239-241 bloom delay, 15:107-111 CA storage, 1:317-319, 348 chilling injury, 15 :80 citrus abscission, 15:158161,168-176 cut flower storage, 10:44-46 dormancy,7:277-279 flowering, 15:295-296, 319 flower longevity, 3:66-75 genetic regulation, 16:6-7, 14-15,19-20 kiwifruit respiration, 6:47-48 mechanical stress, 17:16-17 petal senescence, 11:16-19, 27-30 rose senescence, 9:65-66

F Feed crops, cactus, 18:298-300 Fertilization and fertilizer: anthurium, 5:334-335

CUMULATIVE SUBJECT INDEX

418

Fertilization and fertilizer

(cont'd)

azalea, 5:335-337 bedding plants, 5:337-341 blueberry,10:183-227 carnation, 5:341-345 chrysanthemum, 5:345-352 controlled release, 1:79-139; 5:347-348 Easter lily, 5:352-355 Ericaceae, 10:183-227 foliage plants, 5:367-380 foliar, 6:287-355 geranium, 5:355-357 greenhouse crops, 5:317-403 lettuce, 2:175 nitrogen, 2:401-404 orchid,5:357-358 poinsettia, 5:358-360 rose, 5:361-363 snapdragon, 5:363-364 soil testing, 7:1-68 trickle irrigation, 4:28-31 tulip, 5:364-366 Vaccinium, 10:183-227 Fig: industry, 12:409-490 ripening,4:258-259 Filbert, in vitro culture, 9:313314 Fire blight, 1:423-474 Flooding: fruit crops, 13:257-313 Floricultural crops, see indi-

vidual crops fertilization, 1:98-104 growth regulation, 7:399-481 heliconia, 14:1-55 postharvest physiology and senescence, 1:204-236; 3:59-143; 10:35-62; 11:1543

Florigen, 4:94-98 Flower and flowering: alternate bearing, 4:149 apple anatomy and morphology, 10:277-283 apple bioregulation, 10:344348 aroids, ornamental, 10:19-24 avocado, 8:257-289 blueberry development, 13:354-378 cactus, 18:325-335 citrus, 12:349-408 control, 4:159-160; 15:279334 development (postpollination),19:1-58 fig, 12:424-429 grape anatomy and morphology,13:354-378 honey bee pollination, 9:239243 induction, 4:174-203; 254256 initiation, 4:152-153 in vitro, 4:106-127 kiwifruit, 6:21-35; 12:316318 orchid,5:297-300 pear bioregulation, 10:344348 pecan, 8:217-255 perennial fruit crops, 12:223264 phase change, 7:109-155 photoperiod,4:66-105 pistachio, 3:378-387 postharvest physiology, 1:204-236; 3:59-143; 10:35-62; 11:15-43 postpollination development, 19:1-58

CUMULATIVE SUBJECT INDEX

protea leaf blackening, 17:173-201 pruning, 8:359-362 raspberry,11:187-188 regulation in floriculture, 7:416-424 rhododendron, 12:1-42 rose, 9:60-66 senescence, 1:204-236; 3:59143; 10:35-62; 11:15-43 18:1-85 sugars, 4:114 thin cell layer morphogenesis, 14:239-256 tulip, 5:57-59 water relations, 18:1-85 Fluid drilling, 3:1-58 Foliage plants: acclimatization, 6: 119-154 fertilization, 1:102-103; 5:367-380 Foliar nutrition, 6:287-355 Freeze protection, see Frost protection Frost: apple fruit set, 1:407-408 citrus, 7:201-238 protection, 11:45-109 Fruit: abscission, 1:172-203 citrus, 15:145-182 apple anatomy and morphology, 10:283-297 apple bioregulation, 10:348374 apple bitter pit, 11 :289-355 apple flavor, 16:197-234 apple maturity indices, 13:407-432 apple ripening and quality, 10:361-374 avocado development and

419

ripening, 10:229-271 bloom delay, 15:97-144 blueberry development, 13:378-390 cactus physiology, 18:335341 CA storage and quality, 8:101-127 chilling injury, 15:63-95 cracking, 19:217-262 diseases in CA storage, 3:412-461 drop, apple and pear, 10:359361 fig, 12:424-429 kiwifruit, 6:35-48; 12:316318 maturity indices, 13:407-432 navel orange, 8:129-179 nectarine, postharvest, 11:413-452 peach, postharvest, 11:413452 pear, bioregulation, 10:348374 pear, fruit disorders, 11:357411 pear maturity indices, 13:407-432 pear ripening and quality, 10:361-374 pistachio, 3:382-391 quality and pruning, 8:365367 ripening, 5:190-205 set, 1:397-424; 4:153-154 set in navel oranges, 8:140142 size and thinning, 1:293-294; 4:161 softening, 5:109-219,10:107152

420

Fruit (cont'd) splitting,19:217-262 strawberry growth and ripening, 17:267-297 thinning, apple and pear, 10:353-359 tomato parthenocarpy, 6:6584 tomato ripening, 13:67-103 Fruit crops: alternate bearing, 4:128-173 apple bitter pit, 11:289-355 apple flavor, 16:197-234 apple fruit splitting and cracking, 19:217-262 apple growth, 11:229-287 apple maturity indices, 13:407-432 avocado flowering, 8:257-289 avocado rootstocks, 17:381429 berry crop harvesting, 16:255-382 bloom delay, 15:97-144 blueberry developmental physiology,13:339-405 blueberry harvesting, 16:257282 blueberry nutrition, 10:183227 bramble harvesting, 16:282298 cactus, 18:302-309 carbohydrate reserves, 10:403-430 CA storage, 1:301-336 CA storage diseases, 3:412461 cherry origin, 19:263-317 chilling injury, 15:145-182 chlorosis, 9:161-165 citrus abscission, 15:145-182

CUMULATIVE SUBJECT INDEX

citrus cold hardiness, 7:201238 citrus flowering, 12:349-408 cranberry harvesting, 16:298311 currant harvesting, 16:311327 dormancy release, 7:239-300 Ericaceae nutrition, 10:183227 fertilization, 1:104-106 fig, industry, 12:409-490 fireblight, 11:423-474 flowering, 12:223-264 foliar nutrition, 6:287-355 frost control, 11 :45-109 grape flower anatomy and morphology, 13:315-337 grape harvesting, 16:327-348 grape nitrogen metabolism, 14:407-452 grape pruning, 16:235-254, 336-340 grape root, 5:127-168 grape seedlessness, 11:164176 grapevine pruning, 16:235254,336-340 honey bee pollination, 9:244250,254-256 jojoba, 17:233-266 in vitro culture, 7:157-200; 9:273-349 kiwifruit, 6:1-64; 12:307-347 longan, 16:143-196 lychee, 16:143-196 muscadine grape breeding, 14:357-405 navel orange, 8:129-179 nectarine postharvest, 11:413-452 nutritional ranges, 2:143-164

CUMULATIVE SUBJECT INDEX

orange, navel, 8:129-179 orchard floor management, 9:377-430 peach origin, 17:331-379 peach postharvest, 11:413452 pear fruit disorders, 11:357411 pear maturity indices, 13:407-432 pecan flowering, 8:217-255 photosynthesis, 11:111157 Phytophthora control, 17:299-330 pruning, 8:339-380 rambutan, 16:143-196 raspberry, 11:185-228 roots, 2:453-457 sapindaceous fruits, 16:143196 short life and replant problem, 2:1-116 strawberry fruit growth, 17:267-297 strawberry harvesting, 16:348-365 summer pruning, 9:351-375 Vaccinium nutrition, 10:183227 water status, 7:301-344 Fungi: fig,12:451-474 mushroom, 6:85-118 mycorrhiza, 3:172-213; 10:211-212 pathogens in postharvest storage, 3:412-461 truffle cultivation, 16:71107 Fungicide, and apple fruit set, 1:416

421

G

Garlic, CA storage, 1:375 Genetics and breeding: aroids (edible), 8:72-75; 12:169 aroids (ornamental), 10:1825 bean, bacterial resistance, 3:28-58 bloom delay in fruits, 15:98107 bulbs, flowering, 18:119-123 cassava, 12:164 chestnut blight resistance, 8:313-321 citrus cold hardiness, 7:221223 embryogenesis, 1:23 fig, 12:432-433 fire blight resistance, 1:435436 flowering, 15:287-290, 303305,306-309,314-315 flower longevity, 1:208-209 ginseng, 9:197-198 in vitro techniques, 9:318324; 18:119-123 lettuce, 2:185-187 muscadine grapes, 14:357405 mushroom, 6:100-111 navel orange, 8:150-156 nitrogen nutrition, 2:410-411 plant regeneration, 3:278-283 pollution insensitivity, 8:1819 potato tuberization, 14:121124 rhododendron, 12:54-59 sweet potato, 12:175 tomato parthenocarpy, 6:69-

422

Genetics and breeding (cont'd) 70 tomato ripening, 13:77-98 tree short life, 2:66-70 Vigna, 2:311-394 woody legume tissue and cell culture, 14:311-314 yam (Dioscorea), 12:183 Genetic variation: alternate bearing, 4:146-150 photoperiodic response, 4:82 pollution injury, 8:16-19 ternperature-photoperiod interaction, 17:73-123 Geophyte, see Bulb crops Geranium, fertilization, 5:355357 Germination, seed, 2:117-141, 173-174 Germplasm preservation: cryopreservation, 6:357-372 in vitro, 5:261-264; 9:324325 Gibberellin: abscission, citrus, 15:166167 bloom delay, 15:111-114 citrus, abscission, 15:166167 cold hardiness, 11:63 dormancy, 7:270-271 floral promoter, 4:114 flowering, 15:219-293, 315318 genetic regulation, 16:15 grape root, 5:150-151 mechanical stress, 17:19-20 Ginseng, 9:187-236 Girdling, 4:251-252 Glucosinolates, 19:99-215 Graft and grafting: incompatibility, 15:183-232

CUMULATIVE SUBJECT INDEX

phase change, 7:136-137, 141-142 rose, 9:56-57 Grape: CA storage, 1 :308 chlorosis, 9:165-166 flower anatomy and morphology, 13:315-337 harvesting,16:327-348 muscadine breeding, 14:357405 nitrogen metabolism, 14:407452 pollen morphology, 13:331332 pruning, 16:235-254, 336340 root, 5:127-168 seedlessness, 11 :159-18 7 sex determination, 13:329331 Gravitropism, 15:233-278 Greenhouse and greenhouse crops: carbon dioxide, 7:357-360, 544-545 energy efficiency, 1:141-171; 9:1-52 growth substances, 7:399-481 nutrition and fertilization, 5:317-403 pest management, 13:1-66 Growth regulators, see Growth substances Growth substances, 2:60-66. See also Abscisic acid; Auxin; Cytokinins; Ethylene; Gibberellins abscission, citrus, 15:157176 apple bioregulation, 10:309401

CUMULATIVE SUBJECT INDEX

apple dwarfing, 3:315-375 apple fruit set, 1:417 apple thinning, 1:270-300 aroids, ornamental, 10:14-18 avocado fruit development, 10:229-243 bloom delay, 15:107-119 CA storage in vegetables, 1:346-348 cell cultures, 3:214-314 chilling injury, 15:77-83 citrus abscission, 15 :15 7176 cold hardiness 7:223-225; 11:58-66 dormancy,7:270-279 embryogenesis, 1:41-43; 2:277-281 floriculture, 7:399-481 flower induction, 4:190-195 flowering, 15:290-296 flower storage, 10:46-51 genetic regulation, 16:1-32 ginseng, 9:226 grape seedlessness, 11:177180 in vitro flowering, 4:112-115 mechanical stress, 17:16-21 meristem and shoot-tip culture, 5:221-227 navel oranges, 8:146-147 pear bioregulation, 10:309401 petal senescence, 3:76-78 phase change, 7:137-138, 142-143 raspberry,11:196-197 regulation, 11:1-14 rose, 9:53-73 seedlessness in grape, 11:177-180 triazole, 10:63-105

423

H

Halo blight of beans, 3:44-45 Hardiness, 4:250-251 Harvest: flower stage, 1:211-212 index, 7:72-74 lettuce, 2:176-181 mechanical of berry crops, 16:255-382 Hazelnut, see Filbert Heliconia, 14:1-55 Herbaceous plants, subzero stress, 6:373-417 Herbicide-resistant crops, 15:371-412 Histochemistry: flower induction, 4:177179 fruit abscission, 1 :172-203 Histology, flower induction 4:179-184. See Anato~y and morphology Honey bee, 9:237-272 Horseradish, CA storage, 1:368 Hydrolases, 5:169-219 Hydroponic culture, 5:1-44; 7:483-558 Hypovirulence, in Endothia parasitica, 8:299-310 I

Ice, formation and spread in tissues, 13 :215-255 Ice-nucleating bacteria, 7:210212; 13:230-235 Industrial crops, cactus, 18:309-312 Insects and mites: aroids, 8:65-66 avocado pollination, 8:275277

424

CUMULATIVE SUBJECT INDEX

Insects and mites (cont'd) fig, 12:442-447 hydroponic crops, 7:530-534 integrated pest management, 13:1-66

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

aroids, ornamental, 10:13-14 artemisia, 19:342-345 bulbs, flowering, 18:87-169 cassava propagation, 13:121123

cellular salinity tolerance, 16:33-69

cold acclimation, 6:382 cryopreservation, 6:357-372 embryogenesis, 1:1-78; 2:268-310; 7:157-200; 10:153-181

environmental control, 17:123-170 flowering, 4:106-127 flowering bulbs, 18:87-169 pear propagation, 10:325-326 phase change, 7:144-145 propagation, 3:214-314; 5:221-277; 7:157-200; 9:57-58,273-349; 17:125172

thin cell layer morphogenesis, 14:239-264 woody legume culture, 14:265-332

Iron:

deficiency chlorosis, 9:133186

deficiency and toxicity symptoms in fruits and nuts, 2:150 Ericaceae nutrition, 10:193195

foliar application, 6:330 nutrition, 5:324-325 pine bark media, 9:123 Irrigation: drip or trickle, 4:1-48 frost control, 11:76-82 fruit trees, 7:331-332 grape root growth, 5:140-141 lettuce industry, 2 :175 navel orange, 8:161-162 root growth, 2:464-465

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

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

Lamps, for plant growth, 2:514531

Leaves: apple morphology, 12:283288

flower induction, 4:188-189

CUMULATIVE SUBJECT INDEX

Leek: CA storage, 1:375 fertilization, 1:118 Leguminosae, in vitro, 5:227229; 14:265-332 Lemon, rootstock, 1:244-246. See also Citrus Lettuce: CA storage, 1:369-371 fertilization, 1:118 fluid drilling of seed, 3:14-17 industry, 2:164-207 tipburn, 4:49-65 Light: fertilization, greenhouse crops, 5:330-331 flowering, 15:282-287, 310312 fruit set, 1:412-413 lamps, 2:514-531 nitrogen nutrition, 2:406-407 orchards, 2:208-267 ornamental aroids, 10:4-6 photoperiod,4:66-105 photosynthesis, 11:117-121 plant growth, 2:491-537 tolerance, 18:215-246 Longan, see Sapindaceous fruits Lychee, see Sapindaceous fruits M

Magnesium: container growing, 9:84-85 deficiency and toxicity symptoms in fruits and nuts, 2:148 Ericaceae nutrition, 10:196198 foliar application, 6:331 nutrition, 5:323 pine bark media, 9:117-119

425

Male sterility, temperaturephotoperiod induction, 17:103-106 Mandarin, rootstock, 1:250-252 Manganese: deficiency and toxicity symptoms in fruits and nuts, 2:150-151 Ericaceae nutrition, 10:189193 foliar application, 6:331 nutrition, 5:235-326 pine bark media, 9:123-124 Mango: alternate bearing, 4:145-146 asexual embryogenesis, 7:171-173 CA storage, 1:313 in vitro culture, 7:171-173 Mechanical harvest, berry crops, 16:255-382 Mechanical stress regulation, 17:1-42 Media: fertilization, greenhouse crops, 5:333 pine bark, 9:103-131 Medicinal crops: artemisia, 19:319-371 poppy, 19:373-408 Meristem culture, 5:221-277 Metabolism: flower, 1:219-223 nitrogen in citrus, 8:181-215 seed,2:117-141 Micronutrients: container growing, 9:85-87 pine bark media, 9:119-124 Micropropagation, see In vitro; propagation bulbs, flowering, 18:89-113 environmental control,

426

Micropropagation (cont'd) 17:125-172 nuts, 9:273-349 rose, 9:57-58 temperate fruits, 9:273-349 tropical fruits and palms, 7:157-200 Microtus, see Vole Moisture, and seed storage, 2:125-132 Molybdenum nutrition, 5:328329 Monocot, in vitro, 5:253-257 Monstera, see Aroids, ornamental Morphology: navel orange, 8:132-133 orchid, 5:283-286 pecan flowering, 8:217-243 Moth bean, genetics, 2:373-374 Mung bean, genetics, 2:348-364 Mushroom: CA storage, 1:371-372 cultivation, 19:59-97 spawn, 6:85-118 Muskmelon, fertilization, 1:118-119 Mycoplasma-like organisms, tree short life, 2:50-51 Mycorrhizae: container growing, 9:93 Ericaceae, 10:211-212 fungi,3:172-213 grape root, 5:145-146 N

Navel orange, 8:129-179 Nectarine: bloom delay, 15:105-106 CA storage, 1:309-310 postharvest physiology,

CUMULATIVE SUBJECT INDEX

11:413-452 Nematodes: aroids, 8:66 fig, 12:475-477 lettuce, 2:197-198 tree short life, 2:49-50 NFT (nutrient film technique), 5:1-44 Nitrogen: CA storage, 8:116-117 container growing, 9:80-82 deficiency and toxicity symptoms in fruits and nuts, 2:146 in embryogenesis, 2:273-275 Ericaceae nutrition, 10:198202 fixation in woody legumes, 14:322-323 foliar application, 6:332 metabolism in apple, 4:204246 metabolism in citrus, 8:181215 metabolism in grapevine, 14:407-452 nutrition, 2:395,423; 5:319320 pine bark media, 9:108-112 trickle irrigation, 4:29-30 Nursery crops: fertilization, 1:106-112 nutrition, 9:75-101 Nut crops: chestnut blight, 8:291-336 fertilization, 1:106 honey bee pollination, 9:250251 in vitro culture, 9:273-349 nutritional ranges, 2:143-164 pistachio culture, 3:376-396 Nutrient:

CUMULATIVE SUBJECT INDEX

concentration in fruit and nut crops, 2:154-162 film technique, 5:1-44 foliar-applied,6:287-355 media, for asexual embryogenesis, 2:273-281 media, for organogenesis, 3:214-314 plant and tissue analysis, 7:30-56 solutions, 7:524-530 uptake, in trickle irrigation, 4:30-31 Nutrition (human): aroids, 8:79-84 CA storage, 8:101-127 Nutrition (plant): air pollution, 8:22-23, 26 blueberry, 10:183-227 calcifuge, 10:183-227 cold hardiness, 3:144-171 container nursery crops, 9:75-101 embryogenesis, 1:40-41 Ericaceae, 10:183-227 fire blight, 1:438-441 foliar, 6:287-355 fruit and nut crops, 2:143164 ginseng, 9:209-211 greenhouse crops, 5:317-403 kiwifruit,12:325-332 mycorrhizal fungi, 3:185-191 navel orange, 8:162-166 nitrogen in apple, 4:204-246 nutrient film techniques, 5:18-21,31-53 ornamental aroids, 10:7-14 pine bark media, 9:103-131 raspberry, 11:194-195 slow-release fertilizers, 1:79139

427

o Oil palm: asexual embryogenesis, 7:187-188 in vitro culture, 7:187-188 Okra, CA storage, 1:372-373 Olive, alternate bearing, 4:140141 Onion: CA storage, 1:373-375 fluid drilling of seed, 3:17-18 Opium poppy, 19:373-408 Orange, see Citrus alternate bearing, 4:143-144 sour, rootstock, 1:242-244 sweet, rootstock, 1:252-253 trifoliate, rootstock, 1:247250 Orchard and orchard systems: floor management, 9:377-430 light, 2:208-267 root growth, 2:469-470 water, 7:301-344 Orchid: fertilization, 5:35 7-358 physiology,5:279-315 pollination regulation of flower development, 19:28-38 Organogenesis, 3:214-314. See also In vitro; Tissue, culture Ornamental plants: chlorosis, 9:168-169 fertilization, 1:98-104, 106116 flowering bulb roots, 14:5788 flowering bulbs in vitro, 18:87-169 foliage acclimatization, 6:119-154

CUMULATIVE SUBJECT INDEX

428

Ornamental plants (coni'd) heliconia, 14:1-55 orchid pollination regulation, 19:28-38

poppy, 19:373-408 protea leaf blackening, 17:173-201

rhododendron, 12:1-42 p

Paclobutrazol, see Triazole Papaya: asexual embryogenesis, 7:176-177

CA storage, 1:314 in vitro culture, 7:175-178 Parsley: CA storage, 1:375 drilling of seed, 3:13-14 Parsnip, fluid drilling of seed, 3:13-14

Parthenocarpy, tomato, 6:65-84 Passion fruit, in vitro culture, 7:180-181

Pathogen elimination, in vitro, 5:257-261

Peach: bloom delay, 15:105-106 CA storage, 1:309-310 origin, 17:333-379 postharvest physiology, 11:413-452

short life, 2:4 summer pruning, 9:351-375 Peach palm (Pejibaye): in vitro culture, 7:187-188 Pear: bioregulation, 10:309-401 bloom delay, 15:106-107 CA storage, 1:306-308 decline, 2:11

fire blight control, 1:423-474 fruit disorders, 11 :35 7-411 in vitro, 9:321 maturity indices, 13:407-432 root distribution, 2:456 short life, 2:6 Pecan: alternate bearing, 4:139-140 fertilization, 1 :106 flowering, 8:217-255 in vitro culture, 9: 314-315 Pejibaye, in vitro culture, 7:189 Pepper (Capsicum): CA storage, 1:375-376 fertilization, 1:119 fluid drilling in seed, 3:20 Persimmon: CA storage, 1:314 quality, 4:259 Pest control: aroids (edible), 12:168-169 aroids (ornamental), 10:18 cassava, 12:163-164 cowpea, 12:210-213 fig, 12:442-477 fire blight, 1:423-474 ginseng, 9:227-229 greenhouse management, 13:1-66

hydroponics, 7:530-534 sweet potato, 12:173-175 vertebrate, 6:253-285 yam (Dioscorea), 12:181-183 Petal senescence, 11 :15-43 pH: container growing, 9:87-88 fertilization greenhouse crops, 5:332-333 pine bark media, 9:114-117 soil testing, 7:8-12, 19-23 Phase change, 7:109-155 Phenology:

CUMULATIVE SUBJECT INDEX

apple, 11:231-237 raspberry, 11:186-190 Philodendron, see Aroids, ornamental Phosphonates, Phytophthora control,17:299-330 Phosphorus: container growing, 9:82-84 deficiency and toxicity symptoms in fruits and nuts, 2:146-147 nutrition, 5:320-321 pine bark media, 9:112-113 trickle irrigation, 4:30 Photoautotrophic micropropagation, 17:125172 Photoperiod, 4:66-105,116117; 17:73-123 flowering, 15:282-284, 310312 Photosynthesis: cassava, 13:112-114 efficiency, 7:71-72; 10:378 fruit crops, 11:111-157 ginseng, 9:223-226 light, 2:23 7-238 Physiology, see Postharvest physiology bitter pit, 11:289-355 blueberry development, 13:339-405 cactus reproductive biology, 18:321-346 calcium, 10:107-152 carbohydrate metabolism, 7:69-108 cassava, 13:105-129 citrus cold hardiness, 7:201238 conditioning 13:131-181 cut flower, 1:204-236; 3:59-

429

143; 10:35-62 desiccation tolerance, 18:171-213 disease resistance, 18:247289 dormancy, 7:239-300 embryogenesis, 1:21-23; 2:268-310 flower development, 19:1-58 flowering, 4:106-127 fruit ripening, 13:67-103 fruit softening, 10:107-152 ginseng, 9:211-213 glucosinolates, 19:99-215 heliconia, 14:5-13 juvenility, 7:109-155 light tolerance, 18:215-246 male sterility, 17:103-106 mechanical stress, 17:1-42 nitrogen metabolism in grapevine, 14:407-452 nutritional quality and CA storage, 8:118-120 orchid,5:279-315 petal senescence, 11:15-43 photoperiodism, 17:73-123 pollution injury, 8:12-16 polyamines, 14:333-356 potato tuberization, 14:89188 pruning, 8:339-380 raspberry, 11:190-199 regulation, 11:1-14 root pruning, 6:158-171 roots of flowering bulbs, 14:57-88 rose, 9:3-53 salinity hormone action, 16:1-32 salinity tolerance, 16:33-69 seed,2:117-141 seed priming, 16:109-141

430

Physiology (cont'd) subzero stress, 6:373-417 summer pruning, 9:351-375 thin cell layer morphogenesis' 14:239-264 tomato fruit ripening, 13:67103 tomato parthenocarpy, 6:7174 triazole, 10:63-105 tulip, 5:45-125 vernalization, 17:73-123 volatiles, 17:43-72 watercore, 6:189-251 water relations cut flowers, 18:1-85 Phytohormones, see Growth substances Phytophthora control, 17:299330 Phytotoxins, 2:53-56 Pigmentation: flower, 1:216-219 rose, 9:64-65 Pinching, by chemicals, 7:453461 Pineapple: CA storage, 1:314 in vitro culture, 7:181-182 Pine bark, potting media, 9:103-131 Pistachio: alternate bearing, 4:137-139 culture, 3:376-393 in vitro culture, 9:315 Plantain, in vitro culture, 7:178-180 Plant protection, short life, 2:79-84 Plum, CA storage, 1:309 Poinsettia, fertilization, 1:103104; 5:358-360

CUMULATIVE SUBJECT INDEX

Pollen, desiccation tolerance, 18:195 Pollination: apple, 1:402-404 avocado,8:272-283 cactus, 18:331-335 embryogenesis, 1:21-22 fig,12:426-429 flower regulation, 19:1-58 fruit crops, 12:223-264 fruit set, 4:153-154 ginseng, 9:201-202 grape, 13:331-332 heliconia, 14:13-15 honey bee, 9:237-272 kiwifruit, 6:32-35 navel orange, 8:145-146 orchid,5:300-302 petal senescence, 11 :33-35 protection, 7:463-464 rhododendron, 12:1-67 Pollution, 8:1-42 Polyamines, 14:333-356 chilling injury, 15 :80 Polygalacturonase, 13:67-103 Postharvest physiology: apple bitter pit, 11 :289-355 apple maturity indices, 13:407-432 aroids, 8:84-86 asparagus, 12:69-155 CA storage and quality, 8:101-127 cut flower, 1:204-236; 3:59143; 10:35-62 foliage plants, 6:119-154 fruit, 1:301-336 fruit softening, 10:107-152 lettuce, 2:181-185 low-temperature sweetening, 17:203-231 navel orange, 8:166-172

CUMULATIVE SUBJECT INDEX

nectarine, 11:413-452 pathogens, 3:412-461 peach,11:413-452 pear disorders, 11:357-411 pear maturity indices, 13:407-432 petal senescence, 11 :15-43 protea leaf blackening, 17:173-201 seed,2:117-141 tomato fruit ripening, 13:67103 vegetables, 1:337-394 watercore, 6:189-251; 11:385-387 Potassium: container growing, 9:84 deficiency and toxicity symptoms in fruits and nuts, 2:147-148 foliar application, 6:331-332 nutrition, 5:321-322 pine bark media, 9:113-114 trickle irrigation, 4:29 Potato: CA storage, 1:376-378 fertilization, 1:120-121 low temperature sweetening, 17:203-231 tuberization, 14:89-198 Propagation, see In vitro apple, 10:324-326; 12:288295 aroids, ornamental, 10:12-13 cassava, 13:120-123 floricultural crops, 7:461-462 ginseng, 9:206-209 orchid,5:291-297 pear, 10:324-326 rose, 9:54-58 tropical fruit, palms 7:157200

431

woody legumes in vitro, 14:265-332 Protea, leaf blackening, 17:173201 Protected crops, carbon dioxide, 7:345-398 Protoplast culture, woody species, 10:173-201 Pruning, 4:161; 8:339-380 apple, 9:351-375 apple training, 1:414 chemical, 7:453-461 cold hardiness, 11:56 fire blight, 1:441-442 grapevines, 16:235-254 light interception, 2:250-251 peach,9:351-375 phase change, 7:143-144 root, 6:155-188 Prunus, see Almond; Cherry; Nectarine; Peach; Plum in vitro, 5:243-244; 9:322 root distribution, 2:456

Pseudomonas: phaseolicola, 3:32-33, 39, 44-45

solanacearum, 3:33 syringae, 3:33, 40; 7:210-212 R

Rabbit,6:275-276 Radish, fertilization, 1:121 Rambutan, see Sapindaceous fruits Raspberry: harvesting, 16:282-298 productivity, 11:185-228 Rejuvenation: rose, 9:59-60 woody plants, 7:109-155 Replant problem, deciduous

CUMULATIVE SUBJECT INDEX

432

Replant problem, deciduous (cont'd)

fruit trees, 2:1-116 Respiration: asparagus postharvest, 12:7277 fruit in CA storage, 1:315316 kiwifruit,6:47-48 vegetables in CA storage, 1:341-346 Rhizobium, 3:34,41 Rhododendron, 12:1-67 Rice bean, genetics, 2:375-376 Root: apple, 12:269-272 cactus, 18:297-298 diseases, 5:29-31 environment, nutrient film technique, 5:13-26 Ericaceae, 10:202-209 grape, 5:127-168 kiwifruit,12:310-313 physiology of bulbs, 14:57-88 pruning, 6:155-188 raspberry, 11:190 rose, 9:57 tree crops, 2:424-490 Root and tuber crops: aroids, 8:43-99; 12:166-170 cassava, 12:158-166 low-temperature sweetening, 17:203-231 minor crops, 12:184-188 potato tuberization, 14:89188 sweet potato, 12:170-176 yam (Dioscorea), 12:177-184 Rootstocks: alternate bearing, 4:148 apple, 1:405-407; 12:295-297 avocado, 17:381-429

citrus, 1:237-269 cold hardiness, 11:57-58 fire blight, 1:432-435 light interception, 2:249-250 navel orange, 8:156-161 root systems, 2:471-474 stress, 4:253-254 tree short life, 2:70-75 Rosaceae, in vitro, 5:239-248 Rose: fertilization, 1:104; 5:361363 growth substances, 9:3-53 in vitro, 5:244-248

s Salinity: air pollution, 8:25-26 soils, 4:22-27 tolerance, 16:33-69 Sapindaceous fruits, 16:143196 Scoring, and fruit set, 1 :416417 Seed: abortion, 1 :293-294 apple anatomy and morphology, 10:285-286 conditioning,13:131-181 desiccation tolerance, 18:196-203 environmental influences on size and composition, 13:183-213 flower induction, 4:190-195 fluid drilling, 3:1-58 grape seedlessness, 11:159184 kiwifruit,6:48-50 lettuce, 2:166-174 priming, 16:109-141

CUMULATIVE SUBJECT INDEX

rose propagation, 9:54-55 vegetable, 3:1-58 viability and storage, 2:117141 Secondary metabolites, woody legumes, 14:314-322 Senescence: cut flower, 1:204-236; 3:59143; 10:35-62; 18:1-85 petal, 11:15-43 pollination-induced, 19:4-25 rose, 9:65-66 whole plant, 15:335-370 Sensory quality, CA storage, 8:101-127 Shoot-tip culture, 5:221-277. See also Micropropagation Short life problem, fruit crops, 2:1-116 Small fruit, CA storage, 1:308 Snapdragon fertilization, 5:363364 Sodium, deficiency and toxicity symptoms in fruits and nuts, 2:153-154 Soil: grape root growth, 5:141-144 management and root growth, 2:465-469 orchard floor management, 9:377-430 plant relations, trickle irrigation, 4:18-21 stress, 4:151-152 testing, 7:1-68; 9:88-90 Soilless culture, 5:1-44 Solanaceae, in vitro, 5:229-232 Somatic embryogenesis, see Asexual embryogenesis Spathiphyllum, see Aroids, ornamental Stem, apple morphology,

433

12:272-283 Storage, see Postharvest physiology; Controlled-atmosphere (CA) storage cut flower, 3:96-100; 10:3562 rose plants, 9:58-59 seed,2:117-141 Strawberry: fertilization, 1:106 fruit growth and ripening, 17:267-297 harvesting, 16:348-365 in vitro, 5:239-241 Stress: benefits of, 4:247-271 climatic, 4:150-151 flooding, 13:257-313 mechanical,17:1-42 petal, 11:32-33 plant, 2:34-37 protection, 7:463-466 subzero temperature, 6:373417 Sugar beet, fluid drilling of seed, 3:18-19 Sugar, see Carbohydrate allocation, 7:74-94 flowering, 4:114 Sulfur: deficiency and toxicity symptoms in fruits and nuts, 2:154 nutrition, 5:323-324 Sweet potato: culture, 12:170-176 fertilization, 1:121 Symptoms, deficiency and toxicity symptoms in fruits and nuts, 2:145-154 Syngonium, see Aroids, ornamental

CUMULATIVE SUBJECT INDEX

434

T Taro, see Aroids, edible Temperature: apple fruit set, 1 :408-411 bloom delay, 15:119-128 CA storage of vegetables, 1:340-341

chilling injury, 15:67-74 cut flower storage, 10:40-43 cryopreservation, 6:357-372 fertilization, greenhouse crops, 5:331-332 fire blight forecasting, 1:456459

flowering, 15:284-287,312313

interaction with photoperiod, 4:80-81

low temperature sweetening, 17:203-231

navel orange, 8:142 nutrient film technique, 5:21-24

photoperiod interaction, 17:73-123

photosynthesis, 11:121-124 plant growth, 2:36-37 seed storage, 2:132-133 subzero stress, 6:373-417 Thinning, apple, 1:270-300 Tipburn, in lettuce, 4:49-65 Tissue, see In vitro culture, 1:1-78; 2:268-310; 3:214-314; 4:106-127; 5:221-277; 6:357-372; 7:157-200; 8:75-78; 9:273349; 10:153-181 dwarfing, 3:347-348 nutrient analysis, 7:52-56; 9:90

Tomato: CA storage, 1:380-386

fertilization, 1:121-123 fluid drilling of seed, 3:19-20 fruit ripening, 13:67-103 galacturonase, 13:67-103 parthenocarpy, 6:65-84 Toxicity symptoms in fruit and nut crops, 2:145-154 Transport, cut flowers, 3:100104

Tree decline, 2:1-116 Triazole, 10:63-105 chilling injury, 15:79-80 Trickle irrigation, 4:1-48 Truffle cultivation, 16:71-107 Tuber, potato, 14:89-188 Tuber and root crops, see Root and tuber crops Tulip, see Bulb crops fertilization, 5 :364-366 in vitro, 18:144-145 physiology, 5 :45-125 Tunnel (cloche), 7:356-357 Turfgrass, fertilization, 1:112117

Turnip, fertilization, 1:123-124 Turnip Mosaic Virus, 14:199238

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

v Vaccinium, 10:185-187. See also Blueberry; Cranberry Vase solutions, 3:82-95; 10:4651

Vegetable crops: aroids, 8:43-99; 12:166-170 asparagus postharvest, 12:69-

CUMULATIVE SUBJECT INDEX

155 cactus, 18:300-302 cassava, 12:158-166; 13:105129 CA storage, 1:337-394 CA storage diseases, 3:412461 CA storage and quality, 8:101-127 chilling injury, 15:63-95 fertilization, 1:117-124 fluid drilling of seeds, 3:1-58 greenhouse pest management, 13:1-66 honey bee pollination, 9:251254 hydroponics, 7:483-558 low-temperature sweetening, 17:203-231 minor root and tubers, 12:184-188 mushroom cultivation, 19:59-97 mushroom spawn, 6:85-118 potato tuberization, 14:89188 seed conditioing, 13:131-181 seed priming, 16:109-141 sweet potato, 12:170-176 tomato fruit ripening, 13:67103 tomato parthenocarpy, 6:6584 truffle cultivation, 16:71-107 yam (Dioscorea), 12:177-184 Vegetative tissue, desiccation tolerance, 18:176-195 Vernalization, 4:117, 15 :284287; 17:73-123 Vertebrate pests, 6:253-285 Vigna, see Cowpea genetics, 2:311-394

435

U.S. production, 12:197-222 Virus: benefits in horticulture, 3:394-411 elimination, 7:157-200; 9:318; 18:113-123 fig, 12:474-475 tree short life, 2:50-51 turnip mosaic, 14:199-238 Volatiles, 17:43-72 Vole, 6:254-274

w Walnut, in vitro culture, 9:312 Water relations: cut flower, 3:61-66; 18:1-85 desiccation tolerance, 18:171-213 fertilization, greenhouse crops, 5:332 fruit trees, 7:301-344 kiwifruit,12:332-339 light in orchards, 2:248-249 photosynthesis, 11:124-131 trickle irrigation, 4:1-48 Watercore, 6:189-251 pear, 11:385-387 Watermelon, fertilization, 1:124 Weed control, ginseng, 9:228229 Weeds: lettuce research, 2:198 virus, 3:403 Woodchuck,6:276-277 Woody species, somatic embryogenesis, 10:153-181

x Xanthomonas phaseoli, 3:2932,41,45-46

CUMULATIVE SUBJECT INDEX

436

Xanthophyll cycle, 18:226239

Xanthosoma, 8:45-46, 56-57. See also Aroids y

Yam (Dioscorea), 12:177-184 Yield: determinants, 7:70-74; 97-99 limiting factors, 15 :413-45 2

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

Cumulative Contributor Index (Volumes 1-19) Adams III, W.W., 18:215 Aldwinckle, H.S., 1:423; 15:xiii Anderson, J.L., 15:97 Anderson, P.C., 13:257 Andrews, P.K., 15:183 Ashworth, E.N., 13:215, 255 Asokan, M.P., 8:43 Atkinson, D., 2:424 Aung, L.H., 5:45 Bailey, W.G., 9:187 Baird, L.A.M., 1:172 Banks, N.H., 19:217 Barden, J.A., 9:351 Barker, A.V., 2:411 Bass, L.N., 2:117 Becker, J.S., 18:247 Beer, S.V., 1:423 Bennett, A.B., 13:67 Benschop, M., 5:45 Ben-Ya'acov, A., 17:381 Benzioni, A., 17:233 Bewley, J.D., 18:171 Binzel, M.L., 16:33 Blanpied, G.D., 7:xi Bliss, EA., 16:xiii Borochov, A., 11:15 Bower, J.P., 10:229 Bradley, G.A., 14:xiii Brennan, R., 16:255 Broschat, T.K., 14:1 Brown, S. 15:xiii

Buban, T., 4:174 Bukovac, M.J., 11:1 Burke, M.J., 11:xiii Buwalda, J.G., 12:307 Byers, R.E., 6:253 Caldas, L.S., 2:568 Campbell, L.E., 2:524 Cantliffe, D.J., 16:109, 17:43 Carter, J.V., 3:144 Cathey, H.M., 2:524 Chambers, R.J., 13:1 Charron, C.S., 17:43 Chin, C.K., 5:221 Cohen, M., 3:394 Collier, G.F., 4:49 Collins, W.L., 7:483 Compton, M.E., 14:239 Conover, C.A., 5:317; 6:119 Coyne, D.P., 3:28 Crane, J.C., 3:376 Criley, R.A., 14:1 Crawly, W., 15:1 Cutting, J.G., 10:229 Daie, J., 7:69 Dale, A., 11:185; 16:255 Darnell, R.L., 13:339 Davenport, T.L., 8:257; 12:349 Davies, F.S., 8:129 Davies, P.]., 15:335 Davis, T.D., 10:63 437

438

DeGrandi-Hoffman, G., 9:237 De Hertogh, A.A., 5:45; 14:57; 18:87 Deikman, J., 16:1 DellaPenna, D., 13:67 Demmig-Adams, B., 18:215 Dennis, F.G., Jr., 1 :395 Doud, S.L., 2:1 Duke, S.D., 15:371 Dunavent, M.G., 9:103 Dyer, W.E., 15:371 Early, J.D., 13:339 Elfving, D.C., 4:1; 11:229 EI-Goorani, M.A., 3:412 Esan, E.B., 1:1 Evans, D.A., 3:214 Ewing, E.E., 14:89 Faust, M., 2:vii, 142; 4:174; 6:287; 14:333; 17:331; 19:263 Fenner, M., 13:183 Fenwick, G.R., 19:99 Ferguson, A.R., 6:1 Ferguson, LB., 11:289 Ferguson, L., 12:409 Ferree, D.C., 6:155 Ferreira, J.F.S., 19:319 Fery, R.L., 2:311; 12:157 Fischer, R.L., 13:67 Flick, C.E., 3:214 Flore, J.A., 11:111 Forshey, e.G., 11:229 Fujiwara, K., 17:125 Geisler, D., 6:155 Geneve, R.L., 14:265 George, W.L., Jr., 6:25 Gerrath, J.M., 13:315 Giovannetti, G., 16:71 Giovannoni, J.J., 13:67

CUMULATIVE CONTRIBUTOR INDEX

Glenn, G.M., 10:107 Goldschmidt, E.E., 4:128 Goldy, R.G., 14:357 Goren, R., 15:145 Goszczynska, D.M., 10:35 Grace, S.C., 18:215 Graves, C.J., 5:1 Gray, D., 3:1 Grierson, W., 4:247 Griffen, G.J., 8:291 Grodzinski, B., 7:345 Guest, D.L, 17:299 Guiltinan, M.J., 16:1 Hackett, W.P., 7:109 Halevy, A.H., 1:204; 3:59 Hammerschmidt, R., 18:247 Hanson, E.J., 16:255 Heaney, R.K., 19:99 Heath, R.R., 17:43 Helzer, N.L., 13:1 Hendrix, }.W., 3:172 Henny, R.J., 10:1 Hergert, G.B., 16:255 Hess, F.D., 15:371 Heywood, V., 15:1 Hogue, E.}., 9:377 Holt, }.S., 15:371 Huber, D.}., 5:169 Hutchinson, J.F., 9:273 Isenberg, F.M.R., 1;337 Iwakiri, B.T., 3:376 Jackson, J.E., 2:208 Janick, J., l:ix; 8:xi; 17:xiii; 19:319 Jensen, M.H., 7:483 Jeong, B.R., 17:125 Joiner, J.N., 5:317 Jones, H.G., 7:301 Jones, J.B., Jr., 7:1

CUMULATIVE CONTRIBUTOR INDEX

Jones, R.B., 17:173 Kagan-Zur, V., 16:71 Kang, S.-M., 4:204 Kato, T., 8:181 Kawa, 1.,14:57 Kawada, K., 4:247 Kelly, J.F., 10:ix Khan, A.A., 13:131 Kierman, J., 3:172 Kim, K.-W., 18:87 Kinet, J.-M., 15:279 King, G.A., 11:413 Kingston, C.M., 13:407-432 Kliewer, W.M., 14:407 Knight, R.J., 19:xiii Knox, R.B., 12:1 Kofranek, A.M., 8:xi Korcak, R.F., 9:133; 10:183 Kozai, T., 17:125 Krezdorn, A.H., 1:vii Lakso, A.N., 7:301; 11:111 Lamb, R.C., 15:xiii Lang, G.A., 13:339 Larsen, R.P., 9:xi Larson, R.A., 7:399 Ledbetter, C.A., 11:159 Li, P.H., 6:373 Lill, R.E., 11:413 Lipton, W.J., 12:69 Litz, R.E., 7:157 Lockard, R.G., 3:315 Loescher, W.H., 6:198 Lorenz, O.A., 1:79 Maraffa, S.B., 2:268 Marangoni, A.G., 17:203 Marini, R.P., 9:351 Marlow, G.C., 6:189 Maronek, D.M., 3:172 Martin, G.G., 13:339

439

Mayak, S., 1:204; 3:59 Maynard, D.N., 1:79 McConchie, R., 17:173 McNicol, R.J., 16:255 Merkle, S.A., 14:265 Michailides, T.J., 12:409 Michelson, E., 17:381 Mika, A., 8:339 Miller, S.S., 10:309 Mills, H.A., 9:103 Mitchell, C.A., 17:1 Mizrahi, Y., 18:291,321 Molnar, J.M., 9:1 Monk, G.J., 9:1 Moore, G.A., 7:157 Mor, Y., 9:53 Morris, J.R., 16:255 Mills, H.A., 2:411 Monselise, S.P., 4:128 Murashige, T., 1:1 Myers, P.N., 17:1 Nadeau, J.A., 19:1 Neilsen, G.H., 9:377 Nerd, A., 18:291, 321 Niemiera, A.X., 9:75 Nobel, P.S., 18:291 O'Donoghue, E.M., 11:413 Ogden, R.J., 9:103 O'Hair, S.K., 8:43; 12:157 Oliveira, C.M., 10:403 Oliver, M.J., 18:171 O'Neill, S.D., 19:1 Opara, L.U., 19:217 Ormrod, D.P., 8:1 Palser, B.F., 12:1 Parera, C.A., 16:109 Pegg, K.G., 17:299 Pellett, H.M., 3:144 Perkins-Veazil, P., 17:267

440

Ploetz, R.C., 13:257 Pokorny, F.A., 9:103 Poole, R.T., 5:317;6:119 Poovaiah, B.W., 10:107 Portas, C.A.M., 19:99 Porter, M.A., 7:345 Possingham, J.V., 16:235 Pratt, C., 10:273; 12:265 Preece, J.E., 14:265 Priestley, C.A., 10:403 Proctor, J.T.A., 9:187 Quamme, H., 18:xiii Raese, J.T., 11:357 Ramming, D.W., 11:159 Reddy, A.S.N., 10:107 Reid, M., 12:xiii, 17:123 Reuveni, M., 16:33 Richards, D., 5:127 Rieger, M., 11:45 Rosa, E.A.S., 19:99 Roth-Bejerano, N., 16:71 Roubelakis-Angelakis, K.A., 14:407 Rouse, J.L., 12:1 Royse, D.J., 19:59 Rudnicki, R.M., 10:35 Ryder, E.J., 2:164; 3:vii Sachs, R., 12:xiii Sakai, A., 6:357 Salisbury, F.B., 4:66; 15:233 San Antonio, J.P., 6:85 Sankhla, N., 10:63 Saure, M.C., 7:239 Schaffer, B., 13:257 Schneider, G.W., 3;315 Schuster, M.L., 3:28 Scorza, R., 4:106 Scott, J.W., 6:25 Sedgley, M., 12:223

CUMULATIVE CONTRIBUTOR INDEX

Seeley, S.S., 15:97 Serrano Marquez, C., 15:183 Sharp, W.R., 2:268; 3:214 Shattuck, V.I., 14:199 Shear, C.B., 2:142 Sheehan, T.J., 5:279 Shorey, H.H., 12:409 Simon, J.E., 19:319 Sklensky, D.E., 15:335 Smith, G.S., 12:307 Smock, R.M., 1:301 Sommer, N.F., 3:412 Sondahl, M.R., 2:268 Sopp, P.L, 13:1 Soule, J., 4:247 Sparks, D., 8:217 Splittstoesser, W.E., 6:25; 13:105 Srinivasan, C., 7:157 Stang, E.J., 16:255 Steffens, G.L., 10:63 Stevens, M.A., 4:vii Struik, P.C., 14:89 Studman, C.J., 19:217 Stutte, G.W., 13:339 Styer, D.J., 5;221 Sunderland, K.D., 13:1 Suninyi, D., 19:263 Swanson, B., 12:xiii Swietlik, D., 6:287 Syvertsen, J.P., 7:301 Tetenyi, P., 19:373 Tibbitts, T.W., 4:49 Timon, B., 17:331 Tindall, H.D., 16:143 Tisserat, B., 1:1 Titus, J.S., 4:204 Trigiano, R.N., 14:265 Tunya, G.O., 13:105 van Doorn, W.G., 17:173; 18:1

CUMULATIVE CONTRIBUTOR INDEX

Veilleux, R.E., 14:239 Wallace, A., 15:413 Wallace, D.H., 17:73 Wallace, G.A., 15:413 Wang, C.Y., 15:63 Wang, S.Y., 14:333 Wann, S.R., 10:153 Watkins, C.B., 11:289 Watson, G.W., 15:1 Webster, B.D., 1:172; 13:xi Weichmann, J., 8:101 Wetzstein, H.Y., 8:217 Whiley, A.W., 17:299 Whitaker, T. W., 2: 164 White, J.W., 1:141 Williams, E.G., 12:1 Williams, M.W., 1:270

441

Wismer, W.V., 17:203 Wittwer, S.H., 6:xi Woodson, W.R., 11:15 Wright, R.D., 9:75 Wutscher, H.K., 1:237 Yada, R. Y., 17:203 Yadava, D.L., 2:1 Yahia, E.M., 16:197 Yan, W., 17:73 Yarborough, D.E., 16:255 Yelenosky, G., 7:201 Zanini, E., 16:71 Zieslin, N., 9:53 Zimmerman, R.H., 5:vii; 9:273 Zucconi, F., 11:1