Horticultural Reviews, Volume 27

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HORTICULTURAL REVIEWS Volume 27

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Horticultural Reviews is sponsored by: American Society for Horticultural Science

Editorial Board, Volume 27 Nigel H. Banks Frederick T. Davies Susan Lurie

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HORTICULTURAL REVIEWS Volume 27

edited by

Jules Janick Purdue University

NEW YORK

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

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Copyright © 2001 by John Wiley & Sons, Inc. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except as permitted under Sections 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4744. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 605 Third Avenue, New York, NY 10158-0012, (212) 850-6011, fax (212) 850-6008, E-Mail: PERMREQ @ WILEY.COM. 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 professional services. If professional advice or other expert assistance is required, the services of a competent professional person should be sought. This title is also available in print as ISBN 0-471-38790-8. Some content that appears in the print version of this book may not be available in this electronic edition. For more information about Wiley products, visit our web site at www.Wiley.com

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Contents

Contributors

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Dedication: John V. Possingham

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Keith Boardman

1. The Molecular Biology of Flowering

1

Steve van Nocker I. II. III. IV. V. VI. VII.

Introduction Arabidopsis as a Model for Flowering-Time Studies Floral Inductive Pathways Role of Carbohydrates Control of Meristem Identity Competency Conclusion and Perspectives Literature Cited

2. Floral Homeotic Gene Regulation

1 2 3 20 21 25 28 30

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Robert G. Franks and Zhongchi Liu I. II. III. IV. V.

Introduction Conservation of the ABC Functions in Angiosperms Positive Regulators of Floral Organ Identity Genes Negative Regulators of Floral Organ Identity Genes Summary Literature Cited

42 51 56 63 69 71 v

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CONTENTS

3. Lingonberry: Botany and Horticulture

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Inger Hjalmarsson and Rodomiro Ortiz I. II. III. IV. V. VI.

Introduction History Botany Management of Natural Stands Horticulture Summary and Future Prospects Literature Cited

4. Caper Bush: Botany and Horticulture

80 81 87 93 99 111 114

125

Gabriel O. Sozzi I. II. III. IV. V. VI. VII. VIII.

Introduction Botany Ecophysiology Horticulture Postharvest Technology Composition and Utilization International Trade Concluding Remarks Literature Cited

126 132 137 140 156 159 170 172 173

5. Water Relations and Irrigation Scheduling in Grapevine

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M. H. Behboudian and Zora Singh I. II. III. IV. V. VI.

Introduction Phenology Aspects of Water Relations Irrigation of Vineyards Quality Attributes for Wine, Dried, Table, and Juice Grapes Future Prospects Literature Cited

6. Physiology and Biochemistry of Superficial Scald of Apples and Pears

190 191 193 207 215 218 219

227

Morris Ingle I. II.

Introduction Scald Symptoms and Cell Changes

228 228

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CONTENTS

III. IV. V. VI.

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Biochemistry of Scald Physiology of Scald A Model of Scald Development Prospects Literature Cited

7. Health Functional Phytochemicals of Fruit

229 245 253 259 262

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Wilhelmina Kalt I. II. III. IV. V. VI.

Introduction Citrus Grapes and Wine Vaccinium Other Fruits Conclusions Literature Cited

8. Producing Sods over Plastic in Soilless Media

270 282 291 298 303 307 308

317

Henry F. Decker I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

Introduction Producing Sods in Soilless Media Development of the Concept Producing Mature Sods over Plastic Producing Sods for Golf Greens Solving the Problem of a Stable Continuum Subsequent Proposals in the Genre Manufacturing Sods New Machinery Future Potential Summary Literature Cited

318 319 321 327 330 331 333 338 340 342 345 346

Subject Index

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Cumulative Subject Index

355

Cumulative Contributor Index

377

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Contributors M. H. Behboudian, Institute of Natural Resources, College of Sciences, Massey University, Private Bag 11222, Palmerston North, New Zealand Keith Boardman, 6 Somers Crescent, Canberra ACT 2603, Australia Henry F. Decker, Buckeye Bluegrass Farms, Inc., Box 176, Ostrander, OH 43061 Robert G. Franks, Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742 Inger Hjalmarsson, The Nordic Gene Bank, Smedjevägen 3, PO Box 41, S-230 53 Alnarp, Sweden Morris Ingle, Division of Plant and Soil Sciences, 1090 AG SCI BD, PO Box 6108, West Virginia University, Morgantown, WV 26506 Wilhelmina Kalt, Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, Kentville, Nova Scotia B4N 1J5, Canada Zhongchi Liu, Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742 Steve van Nocker, Department of Horticulture, Michigan State University, 390 Plant and Soil Science Building, East Lansing, MI 48824 Rodomiro Ortiz, IITA c/o Lambourn & Co., Carolyn House, 26 Dingwall Road, Croydon CR9 3EE, UK Zora Singh, Department of Horticulture, Muresk Institute of Agriculture, Curtin University of Technology, GPO Box U 1987, Perth, WA 6845, Australia Gabriel O. Sozzi, Departamento de Biología Aplicada y Alimentos, Facultad de Agronomía, Universidad de Buenos Aires, Avda. San Martín 4453, C 1417 DSE Buenos Aires, Argentina

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John V. Possingham

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Dedication: John V. Possingham This volume is dedicated to Dr. John Possingham, an outstanding scientist and administrator, in recognition of his outstanding contribution to Australian horticulture and for his international efforts in horticultural science and viticulture. John was born in 1929 and grew up on a small horticultural property in rural South Australia. He studied agriculture and plant physiology at the Universities of Adelaide in Australia and at Oxford in England. He initially worked in the field of plant nutrition at the Laboratories of the Commonwealth Scientific and Industrial Research Organisation (CSIRO) in Canberra establishing that manganese was essential for the photochemical reactions of higher plant chloroplasts. In 1962 he accepted responsibility for CSIRO’s program of research in horticulture that was mainly concerned with grapevines and centered at Merbein Victoria. He subsequently established horticultural laboratories at Adelaide, Darwin, and Brisbane that, together with an existing postharvest horticulture group at Sydney, enabled CSIRO’s Division of Horticulture to carry out a wide-ranging program of basic and applied horticultural research concentrating mainly on wine and raisin grape vines and on a range of selected tropical and subtropical fruit crops. At Merbein, Dr. Possingham established a major grapevine improvement program that covered virus-tested introductions from overseas and the breeding of new cultivars suited to the warm irrigated conditions of inland Australia. This program also included the development of grapevine rootstocks that were tolerant of Australian plant parasitic nematodes. New cultivars released from the grape breeding program and adopted by the viticultural industries include ‘Tarrango’ for light red wine; ‘Carina’, a black seedless raisin; and the table grape ‘Marroo Seedless’. A number of other potential cultivars are being evaluated by industry. Dr. Possingham also obtained major government funding and established a vineyard mechanization program based on importing prototype grape harvesting machines from the University of California, Davis and Cornell University. Vine training and management systems were developed for use in mechanically harvested vineyards. These included minimal pruning, which is now used extensively in warm to hot grape growing regions in both Australia and California. xi

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DEDICATION: JOHN V. POSSINGHAM

The work of the Division of Horticulture assisted the recent rapid expansion of the Australian wine industry and enabled it to become a major player in world markets. It provided the industry with high-quality vine planting material and developed training systems that enabled Australian wine-grape vineyards to become highly mechanized. Together with other members of the Division he was awarded both a CSIRO Medal for his contributions to vine improvement and an Ian McLennan Award from Industry for his foresight in developing a program of work on vineyard mechanization for Australia. Dr. Possingham was involved with other scientists in many aspects of the Division’s program including the minimal pruning of grapevines and the evaluation of introduced grape cultivars. He contributed to the understanding of how the waxy layers of raisin grapes control water loss and the elucidation of a major role of endotrophic mycorrhizae in the uptake of phosphorus by grapevines. Research concerned with grapevine physiology, biotechnology, and molecular biology was developed at the Division’s laboratory located on the Waite Institute Campus. Studies on grapevines included the features of and factors affecting flowering, fruit set, photosynthesis, response to salinity, and root/shoot hormonal interactions. Biotechnology investigations were aimed at better methods for grapevine propagation and systems for virus elimination. Recent work has been concerned with methods for the DNA finger-printing of grapevines and techniques for their genetic modification. Some genetically modified grapevines are currently undergoing field trials. Throughout his career, Dr. Possingham has maintained a personal research program concerned with factors involved with the division of higher plant chloroplasts. This work included both structural and biochemical studies and provided support for the long-held hypothesis that the plastids of higher plants arise from the division of pre-existing plastids and cannot be formed de novo. He showed that chloroplasts of spinach are highly polyploid and that division can be temporally separated from the synthesis of chloroplast located DNA. Using barley mutants he demonstrated that the polymerase used for c-DNA replication is nuclear coded and a component in the complex control exerted by the nucleus over plastic division. Dr. Possingham has contributed to international horticulture via a number of FAO/United Nations Development Program (UNDP) missions, but more importantly through his work with the International Society for Horticultural Science (ISHS). He established the Viticulture Section within ISHS and has been a Council member for Australia for a number of years. He is in his second term as a member of the ISHS Board and is currently the Society’s Vice President. In Australia he was directly

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involved in setting up the Australian Society for Horticultural Science and was its second President. He is a fellow of the Australian Institute of Agricultural Science and Technology, the Australian Academy of Technological Science and Engineering, the Russian Academy of Agricultural Sciences, and holds a DSc from the University of Oxford. In his “retirement” he grows wine grapes and makes wine on highly mechanized vineyards near Adelaide. John is known throughout Australia and the world for his generosity and warmth, for his dedication to high standards, and for his love of good fellowship and fine wines. Keith Boardman Formerly Chairman and Chief Executive CSIRO, Canberra, Australia

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1 The Molecular Biology of Flowering Steve van Nocker Department of Horticulture Michigan State University East Lansing, MI 48824

I. INTRODUCTION II. ARABIDOPSIS AS A MODEL FOR FLOWERING-TIME STUDIES III. FLORAL INDUCTIVE PATHWAYS A. Photoperiodic Induction 1. Light Effects on Flowering in Arabidopsis 2. The Endogenous Clock 3. Entrainment of the Clock by Light 4. Other Photoperiodic Pathway Genes B. Non-Photoperiodic Induction: The Autonomous Pathway C. Vernalization D. Induction by Gibberellins IV. ROLE OF CARBOHYDRATES V. CONTROL OF MERISTEM IDENTITY A. Meristem Identity Genes B. Integration of Flowering Pathways and Activation of Meristem Identity Genes VI. COMPETENCY VII. CONCLUSION AND PERSPECTIVES LITERATURE CITED

I. INTRODUCTION Of the myriads of developmental processes that define plant form and function, flowering is of exceptional interest to horticulturalists. The vast majority of horticulturally important crops are in some way dependent upon flowering, whether the flower is the primary goal of production, or is simply required for a crop to be produced. Much effort is currently being put into regulating the timing of flowering. In floriculture crops, the interest is in abbreviating or extending the vegetative Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 1

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phase in order to create an aesthetically pleasing balance between leaves and flowers, or to conveniently induce or repress flowering to take advantage of market potential. In ornamental foliage plants, and agronomically important plants that are grown for their leaf tissues (e.g., lettuce, spinach, and other greens), it is highly desirable to suppress flowering as long as possible. Also, in woody plants, there is a great deal of interest in finding means to abbreviate the vegetative phase, which in some species can last ten or more years and is probably the single most limiting factor for germplasm improvement through traditional breeding techniques. Most efforts at controlling flowering time have involved manipulation of environmental conditions or the application of synthetic growth regulators. However, these approaches can increase production costs and labor requirements. In addition, the use of many traditionally utilized chemical compounds is becoming restricted. Alternative approaches to manipulate flowering—including biotechnology—will require a better understanding of the associated molecular mechanisms. The physiology and phenomenology of the developmental transition from vegetative growth to reproductive growth—flowering—has been studied for many years, but only in approximately the last 10 years have the molecular mechanisms begun to be addressed. Flowering is ultimately determined by genes that govern the identity of the meristem, promoting or repressing floral fate versus shoot fate. When and how these genes are activated, in response to environmental cues and/or developmental progression, is a fascinating question. As might be expected from the incredible diversity of flowering strategies employed in nature, it is now becoming apparent that flowering at the molecular level involves an extraordinarily complex web of interactive pathways. Here we review the current knowledge about the genetics and molecular biology of flowering in Arabidopsis thaliana, the only plant in which these aspects of flowering have been extensively studied.

II. ARABIDOPSIS AS A MODEL FOR FLOWERING-TIME STUDIES Arabidopsis thaliana is an herbaceous weed of the mustard family with a natural distribution throughout the Northern Hemisphere (Meyerowitz 1989; Meinke et al. 1998). In addition to its many qualities that make it a superior model for plant biology in general (i.e., small size, rapid life cycle, and well-characterized genome), Arabidopsis is especially attractive as a subject for flowering-time studies because the timing of flow-

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ering can be strongly influenced by environmental conditions (e.g., light and temperature), thus permitting the molecular analyses of the associated input pathways. In this species, at least fifty genes have been identified that act directly or indirectly either to promote or to repress flowering (Levy and Dean 1998). Many of these genes have been identified through a traditional genetic approach. Delayed flowering can result from loss of function of genes that presumably act to promote flowering, whereas accelerated flowering can result from loss of function of flowering-repressor genes. Although several repressor genes have been identified, most mutagenic approaches have targeted genes that act to promote flowering (Redei 1962; Koornneef et al. 1991). This is in part because the genotypes commonly used in the laboratory flower soon after germination in photoperiodically inductive conditions (long-day photoperiods), and mutants that flower even earlier, are difficult to discern in large populations. Screens designed to find early-flowering mutants among late-flowering genetic backgrounds, or employing photoperiodically noninductive conditions, should result in the identification of additional repressor genes.

III. FLORAL INDUCTIVE PATHWAYS An interesting finding coming from genetic analyses is that no single mutation completely eliminates flowering. This was an early indication that flowering is promoted by at least two pathways that can operate in a parallel, or partially redundant, manner. That such redundancy should have evolved makes sense, given the crucial importance of flowering in maintaining the species. The promotive genes identified through genetic analyses have traditionally been assigned into distinct groups based on the sensitivity of the mutant phenotype to environmental conditions, and these groups have formerly been considered to define the pathways (Martinez-Zapater et al. 1994; Coupland 1995). Mutations in a subset of flowering-time genes predominately affect the photoperiodic control of flowering, such that the flowering habit of the corresponding mutant tends toward day-neutrality. Mutations in another subset of floweringtime genes result in delayed flowering without a significant loss of photoperiodic sensitivity—i.e., these mutants flower later than wild-type plants under both photoperiodically inductive and noninductive conditions. Because mechanisms for sensing daylength are evidently intact in the latter mutants, the corresponding genes are supposed to function in an environmentally “autonomous” pathway that acts in parallel with the “photoperiodic” pathway to eventually initiate flowering, even

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under unfavorable conditions (Martinez-Zapater et al. 1994; Coupland 1995; Amasino 1996). Another characteristic of mutants in the autonomous pathway is that they exhibit a significant vernalization response—i.e., the late-flowering phenotype can be fully “rescued” by a long-term cold treatment given to the imbibed seed or young plant. In contrast, cold is largely ineffective to accelerate flowering of the photoperiodic pathway mutants (Martinez-Zapater et al. 1994). Koornneef et al. (1991, 1998a) used double-mutant analysis to examine the epistatic relationships between the commonly studied floweringpromoting genes in an attempt to better define such pathways. The rationale for this type of approach is as follows: if two genes operate in a more-or-less linear pathway, then loss of both genes’ function should confer a phenotype that is similar to that of the single mutant (i.e., the double mutant should flower no later than either single mutant). However, if genes operate in parallel pathways, a significant enhancement of the late flowering might be conferred by combining the mutant alleles. A caveat to this type of genetic approach is that it is only valid when using complete loss-of-function alleles, as enhancement of the phenotype should be expected when partially functional alleles operating in the same pathway are combined. In general, the results of these experiments were inconsistent with the simple assignment of flowering-time genes to independent pathways. This suggests that there is significant interaction (“crosstalk”) between pathways. Another finding from these studies was that flowering was not prevented even when combining mutations in genes considered to act in the photoperiodic and autonomous pathway. Thus, the redundancy of flowering pathways is more extensive than was previously thought. A. Photoperiodic Induction 1. Light Effects on Flowering in Arabidopsis. As in many other plants, both light quality (wavelength) and photoperiod strongly influence flowering time in Arabidopsis. In general, flowering in this species is delayed by red light and accelerated by blue light (Brown and Klein 1971; Eskins 1992). The molecular biology of the major photoreceptors in plants, the red/blue-sensitive phytochromes and green/blue/UV-A-sensitive cryptochromes, has been extensively reviewed and will not be discussed here (Barnes et al. 1997; Cashmore 1998; Whitelam and Devlin 1998; Ahmad 1999; Cashmore et al. 1999; Deng and Quail 1999; Briggs and Huala 1999). Mutations that abrogate synthesis of the phytochrome chromophore and therefore result in an absence of functional phytochrome, or mutations that specifically result in loss of the major light-stable phy-

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tochrome, PHYB, confer early flowering, suggesting that the negative effect of red light is mediated by PHYB. Mutants lacking function of the CRYPTOCHROME1 (CRY1) gene exhibit delayed flowering that is evident in both long and short days (King and Bagnall 1996; Coupland 1997). This phenotype is especially striking when plants are grown under blue light (Bagnall et al. 1996), suggesting that CRY1 mediates blue-light promotion of flowering. In contrast, mutants lacking function of CRY2 (allelic to the previously described flowering time gene FHA) exhibit a much-reduced photoperiodic response, flowering much later than wild type in long days and slightly earlier than wild type in short days (Koornneef et al. 1991; Guo et al. 1998). In addition, constitutive expression of the CRY2 gene in transgenic plants accelerates flowering in short days, but not long days. Unlike in cry1 mutants, flowering in plants lacking CRY2 is accelerated by blue light (Guo et al. 1998). Given the delay in flowering in white light conferred by loss of CRY2 activity, one interpretation of this data is that CRY2 normally acts not as a direct positive regulator under blue, but as a negative regulator of the repression of flowering imposed by PHYB (Guo et al. 1998). 2. The Endogenous Clock. In plants, as in other organisms, one or more molecular mechanisms sustain oscillations with periods of approximately 24 h. The circadian rhythms generated by these endogenous “clocks” allow plants to anticipate daily variations in environmental conditions and thereby optimize their responses to them. One example is the family of LHC genes encoding light-harvesting chlorophyll a/b-binding (CAB) proteins, which are upregulated in a diurnal manner before the expected onset of illumination (Piechulla 1988; Nagy et al. 1988). A large body of physiological evidence implicates the clock in mediating the effects of photoperiod on flowering. Evidence is also accumulating that light quality as well influences flowering time by virtue of its effects on the clock. Thus, the clock has a central and very important role in flowering. How might a self-sustaining oscillatory mechanism in plants be composed at the molecular level? Some clues come from research on the Drosophila (fruit fly) clock mechanism that controls eclosion (emergence from the pupae) and locomotor activity. This clock is essentially comprised of an oscillatory mechanism set up through the interactions between two proteins, TIMELESS (TIM) and PERIOD (PER). Transcription of both the PER and TIM genes increases during the subjective day, from a minimum rate near the onset of illumination (referred to as Zeitgeiber time 0, or Zt0) and reaching a maximum rate at approximately Zt12 (Hardin et al. 1992). Maximal accumulation of PER and

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TIM mRNA is offset 2 to 4 hours, whereas maximal accumulation of the proteins is offset another 2 to 4 hours (So and Rosbash 1997). Thus, PER and TIM protein levels reach a maximum at Zt16-20, a point during which transcription of the genes is rapidly decreasing. In fact, transcriptional repression of the PER and TIM genes is a direct result of the increase in protein levels. Heterodimerization between PER and TIM allow the proteins to gain entrance into the nucleus, where they block the transcription of their own genes by the CLOCK and BMAL1 transcription factors (Vosshall et al. 1994; Gekakis et al. 1995; Darlington et al. 1998). The inhibition of transcription by PER/TIM allows the circadian cycle to begin anew. Constant turnover of the mRNAs and proteins is necessary for the oscillations to continue. The TIM protein is thought to be destabilized through phosphorylation by the product of the DOUBLETIME gene, which is structurally related to the kinase domain of human casein kinase Iε (Kloss et al. 1998; Price et al. 1998). In addition, in the absence of TIM, PER protein fails to accumulate, suggesting that TIM functions directly or indirectly to stabilize PER (Price et al. 1995). Although great progress has been made in understanding the basics of this Drosophila clock mechanism, how the clock operates in plants is mostly unknown. PER, TIM, and other components of the fly clock were discovered through traditional genetic analysis. Arabidopsis displays numerous visible phenotypes that cycle in a circadian manner [e.g., movements of cotyledons and primary leaves (Engelmann et al. 1992), alterations in the rate of hypocotyl elongation (Dowson-Day and Millar 1999), and changes in stomatal aperture (Somers et al. 1998b)], but in all cases these phenotypes are subtle and thus not useful for mutant screening. Millar et al. (1995) generated a synthetic circadian phenotype by expressing the firefly luciferase gene under the control of an LHC gene promoter in transgenic plants. Screens using this LHC:LUC genetic background yielded numerous mutants. The best-characterized, designated toc1-1, exhibits a slightly shorter period length of LHC mRNA expression in both constant light and constant darkness (Millar et al. 1995; Somers et al. 1998b). In addition, the mRNA expression of members of at least one other circadian-cycling nuclear gene family, GRP7/8 (see below), is altered in a similar manner (Kreps and Simon 1997). Although toc1-1 plants were originally reported to be phenotypically indistinguishable from wild-type plants, more careful observations revealed that toc1-1 plants were disrupted in multiple circadian cycling phenotypes. In addition, toc1-1 plants exhibited aberrant floral initiation, flowering earlier than wild-type plants under short photoperiods and later than wild-type plants under long photoperiods (Somers et al. 1998b). These findings suggest that the multiple circadian processes

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and the timing of flowering are controlled either by a single clock, or by multiple related clocks sharing the TOC1 component. The TOC1 gene was recently cloned and found to encode a protein with homology to the receiver domain of response regulators from two-component signal transduction systems (Strayer et al. 2000). A dominant mutation in the LATE ELONGATED HYPOCOTYL (LHY) gene leads to loss of rhythmic mRNA expression of clock-regulated genes and defects in multiple clock-influenced phenotypes, including flowering time and circadian leaf movements (Schaffer et al. 1998). In wild-type plants, LHY mRNA levels oscillate in a circadian manner, whereas LHY mRNA is expressed at a constitutive high level in the lhy mutant (Schaffer et al. 1998). In addition, in transgenic plants containing a singly copy of a lhy mutant allele, cycling of the endogenous wildtype LHY mRNA is suppressed. These findings indicate that LHY is part of a feedback circuit that regulates its own mRNA expression. The LHY gene product is a member of a large family of proteins structurally related to the vertebrate proto-oncogenic transcription factor c-Myb (Martin and Paz-Ares 1997). In Arabidopsis, this family also includes the product of the CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) gene, originally identified as a factor that bound a LHC promoter element essential for its regulation by light and the clock (Wang et al. 1997). Like LHY mRNA, CCA1 mRNA and protein also cycle with a circadian rhythm (Wang and Tobin 1998). Constitutive expression of CCA1 mRNA under control of the strong, viral CaMV 35S promoter (35S:CCA1) in transgenic Arabidopsis, like constitutive expression of LHY mRNA from the mutant lhy allele, leads to the disruption of the circadian mRNA expression patterns of various clock-regulated genes, including LHY, and such plants exhibit delayed flowering in long-day conditions (Wang and Tobin 1998). These findings suggest that both LHY and CCA1 are potential key components of a central clock mechanism. That the clock defect conferred by loss of CCA1 function is apparent even in the presence of LHY activity indicates that, despite their structural similarities and similar effects by constitutive expression, LHY and CCA1 do not have strictly redundant roles (Green and Tobin 1999). Like that of TIM, the activity of the CCA1 and LHY proteins may be negatively regulated by phosphorylation. Both proteins are substrates for the protein kinase CK2 in vitro (Sugano et al. 1998, 1999). Constitutive expression of CKB3 mRNA, encoding a regulatory subunit of CK2, in Arabidopsis mimics the effects of loss of CCA1 function, substantially shortening the rhythm periods of multiple clock-regulated genes. However, in contrast to the delay of flowering conferred by loss of CCA1, flowering is accelerated in 35S:CKB3 plants (Sugano et al. 1999).

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GIGANTEA (GI) is another member of the set of genes involved in the promotion of flowering by long days. In plants grown in a regular lightdark photoperiod, mRNA levels for GI oscillate in a diurnal pattern, and studies in which plants were kept in constant light or darkness indicate that GI is under clock control (Fowler et al. 1999; Park et al. 1999). In 35S:CCA1 or 35S:LHY plants, this rhythmic expression pattern is disrupted, indicating that GI is regulated by these genes (Fowler et al. 1999). However, disruption of GI function also affects expression of CCA1 and LHY mRNA. In some gi mutant backgrounds, the amplitude of CCA1 and LHY oscillations are diminished and periodicity becomes less obvious (Fowler et al. 1999; Park et al. 1999). The finding that GI may act both upstream and downstream of the clock genes CCA1 and LHY suggests that GI is intimately associated with the clock. Mutants lacking GI function also exhibit reduced seedling deetiolation under red light, suggesting that GI could be involved in PHYB signaling (Huq et al. 2000). How GI might carry out its function has not been determined. GI was recently cloned and encodes a large protein that is predicted by computer modeling to contain transmembrane domains (Fowler et al. 1999; Park et al. 1999). However, more recent evidence indicates that the GI protein is localized to the nucleus (Huq et al. 2000). The GI transcript was detected in both very young seedlings and mature plants, and is apparently not restricted to any specific tissue type (Fowler et al. 1999). Open reading frames have been identified from both rice and maize that would encode proteins with significant amino acid sequence identity to the GI protein (Fowler et al. 1999; J. Liu and S. van Nocker, unpublished data). Because structural homology is often associated with functional homology, it is possible that these monocot GI orthologs are also involved in flowering. The current efforts to better characterize the rice genome, and determine gene function in maize through reversed genetics approaches, will allow this idea to be tested (Goff 1999; Martienssen 1998; Walbot, 2000). Other potential clock genes include members of the GRP7/8 (also called CCR1/2) family. These genes encode small proteins containing an interesting bipartite structure (van Nocker and Vierstra 1993; Carpenter et al. 1994). The amino-terminal domain contains a specific RNA-binding consensus sequence termed the RRM motif (found also in the flowering-time genes FCA and FPA; below), whereas the carboxyl-terminal region is greatly enriched in glycine residues, a configuration seen in many plant cell wall proteins (Showalter 1993; Cassab 1998). These genes are expressed to high levels in meristematic tissues, and, in addition to being regulated in a circadian pattern, are upregulated by lowered temperatures

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(Heintzen et al. 1994; Kreps and Simon 1997). The protein products of these genes also oscillate with a circadian period, and are localized to the nucleus (Heintzen et al. 1994). Constitutive expression of the GRP7 gene in transgenic Arabidopsis suppresses the circadian oscillations of mRNAs for both the endogenous GRP7 gene and for GRP8, suggesting that the respective proteins are involved in a mutual, autoregulatory feedback loop. This effect of GRP7 on the oscillations of its own transcript is not mediated entirely through its promoter, suggesting that at least some regulation occurs at the posttranscriptional level (Staiger and Apel 1999). As previously mentioned, GRP7/8 expression is affected by impairment of TOC1 function. Interestingly, however, unlike in toc1 mutants, the rhythmic expression patterns of specific clock-regulated genes were not affected in 35S:GRP7 plants (Heintzen et al. 1997). This suggests both that the GRP7/8 clock acts downstream from TOC1, and that the output of the AtGRP7/8 oscillator is limited. The function of the so-called AtGRP7/8 “slave” oscillator is not known, as phenotypic abnormalities associated with constitutive expression of GRP7 have not been reported. In light of the intimate relationship between circadian rhythms and flowering, it is reasonable to hypothesize that these genes are somehow involved in the regulation of flowering time. On the other hand, genes encoding small proteins exhibiting the RRM motif/glycine-rich bipartite structure have also been identified in mammals, amphibians, ascidians, and cyanobacteria (Nishiyama et al. 1997; Danno et al. 1997; Uochi and Asashima 1998; Tanaka et al. 2000; Maruyama et al. 1999). At least a subset of these genes cycle in a diurnal manner and/or are inducible by lowered temperatures (Nishiyama et al. 1997, 1998; Sato and Maruyama 1997; Maruyama et al. 1999; Danno et al. 1997). Thus, these RRM-GRP proteins may carry out a function that is conserved among kingdoms. 3. Entrainment of the Clock by Light. A notable feature of clocks in all organisms yet studied is that the innate period is somewhat longer or shorter than 24 h. In order to cycle with a precise daily rhythm, the clock must be entrained, or synchronized, each day. In Drosophila, light serves to entrain the clock by initiating the phosphorylation and rapid degradation of the TIM protein (Hunter-Ensor et al. 1996; Myers et al. 1996; Zeng et al. 1996). This results in a phase delay in the evening, when TIM is being continually resynthesized, and a phase advance in the morning, when TIM is not effectively replaced. The light signals are perceived by cryptochrome, a protein that is similar in amino acid sequence to the CRY proteins in plants. Upon illumination, CRY undergoes a photochemical change that allows a physical interaction with the TIM protein and presumably initiates the degradation process (Ceriani et al. 1999).

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As in other organisms, under constant illumination, the period of the clock can be modulated by light in an intensity-dependent manner (Somers et al. 1998a,b). Although this phenomenon may not be physiologically relevant, it has proven useful for determining the potential role of specific genes in the light entrainment of the clock. Millar et al. (1995) found that both red and blue wavelengths were effective in shortening the period of LHC:LUC expression, suggesting the involvement of phytochromes and potentially also cryptochromes. By examining the period length of LHC:LUC expression in phyA, phyB, cry1, or cry2 mutant plants, Somers et al. (1998a) concluded that red light signals are transmitted to the clock by phytochromes A and B, with PHYA acting under low intensities and PHYB acting under high intensities, whereas blue light inputs are provided by PHYA and cryptochromes. Interestingly, loss of CRY2 shortened the period under low-fluence blue light, but had little effect under the higher fluences where photoperiodic timing of flowering is affected. Unlike in Drosophila, no direct interactions between photoreceptors and clock components have been demonstrated in plants, and clock function may depend on a mechanism to transmit information from photoreceptors to the clock. The EARLY FLOWERING 3 (ELF3) gene may play such a role. Plants lacking ELF3 activity display phenotypes that mimic phyB mutations (i.e., increased hypocotyl elongation in red light and petiole length). However, mutations in ELF3 and PHYB have additive effects when combined, suggesting that ELF3 is not simply a component of a PHYB signal transduction pathway (Reed et al. 2000). Unlike mutations in PHYB, which alter the periodicity of the clock (above), mutations in ELF3 abolish rhythmicity, and do so in a light-dependent manner (Hicks et al. 1996). The CCA1 gene is rapidly and transiently upregulated in response to light, specifically red light (Wang et al. 1997), suggesting that CCA1 could also be involved in transmitting signals to the clock from phytochrome. Whether or not the phosphorylation of CCA1 or LHY is associated with their turnover, as is the case with TIM, has not been reported. However, as in the Drosophila clock, protein degradation is an important process of the plant clock mechanism. Two homologous genes have been identified in Arabidopsis that might play a role in the turnover of clock components. These genes, FKF1 and ZEITLUPE (ZTL), encode proteins containing an F-box motif (Nelson et al. 2000; Somers et al. 2000). Where studied in other organisms, F-box-containing proteins act in the recognition of degradation substrates for the ubiquitin proteolytic pathway (Patton et al. 1998; Kornitzer and Ciechanover 2000). FKF1 transcripts oscillate in a circadian manner, whereas ZTL mRNA expression

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is apparently not under clock control. However, mutations in both genes confer a similar phenotype, with flowering delayed primarily under photoinductive conditions. In addition to the F-box, the FKF1/ZTL proteins contain a segment similar to the flavin-binding domain in the bluelight receptor NPH1 involved in phototropism. At least ztl mutants exhibit a period-lengthening phenotype that is strongly light-dependent, and at least the FKF1 promoter is selectively activated under white or blue light. Taken together, these observations indicate that these proteins may function as light-dependent clock regulators (Nelson et al. 2000; Somers et al. 2000). 4. Other Photoperiodic Pathway Genes. CONSTANS (CO), another of the promotive photoperiod pathway genes, was one of the first of the flowering-time genes to be cloned, and thus has been the most extensively studied (Putterill et al. 1995). The CO protein contains zincfinger-type DNA-binding domains common to the GATA1 family of transcription factors, and thus likely acts as a component of the transcriptional apparatus. Known mutations in CO are semidominant. Where a mutation results in complete loss of function of the gene, semidominance is an indicator that the respective gene product is limiting for the respective process (i.e., that relative levels of the gene product are important). Consistent with this, increasing CO activity, either constitutively through adding extra copies of the gene in transgenic plants (Putterill et al. 1995), or transiently by activating the protein in an inducible system (Simon et al. 1996), is sufficient to trigger flowering. In addition, consistent with its role in promoting flowering under photoinductive conditions, the mRNA levels of CO are elevated in long-day grown Arabidopsis plants relative to short-day grown plants (Putterill et al. 1995), and this regulation is accomplished at least in part by transcriptional upregulation of the gene (Suarez-Lopez et al. 1998). CO was found to be expressed in both leaf and stem tissue, but the very low abundance of the mRNA complicated a more thorough analysis of spatial expression patterns (Putterill et al. 1995). Its likely function as a transcription factor and regulation at the transcriptional level suggests that CO is an intermediate in a cascade of transcriptional events. What could be upstream regulators and downstream targets of CO? CRY2 and PHYB likely act upstream of CO as indirect positive and negative regulators, respectively, because mutations in CRY2 decrease CO mRNA expression (Guo et al. 1998), and the early-flowering seen in phyB mutants in short days is alleviated in a genetic background compromised for CO activity (Putterill et al. 1995). Onouchi et al. (1998) clarified the relationship between CO and several other photoperiodic

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pathway genes by examining the effect of 35S:CO expression in photoperiodic pathway-mutant backgrounds. The premise of this experiment was that if CO acted in a genetic pathway downstream from GENE X, then removal of GENE X function should have no effect on the phenotype conferred by constitutive expression of CO (i.e., constitutive expression of CO would be epistatic to loss of GENE X function). In contrast, if CO acted upstream of GENE X, then adding CO activity should make no difference to the phenotype conferred by loss of GENE X function. In this case, the 35S:CO transgene was completely epistatic to gi and lhy, suggesting that CO acts downstream from these two genes. In contrast, 35S:CO had only a small effect in genetic backgrounds in which expression of the FT or FWA flowering-time genes was disrupted (see below), suggesting that these two genes function downstream from CO. Extending this experimental approach, Onouchi et al. (2000) discovered a target of CO by searching for mutations that suppressed the earlyflowering phenotype conferred by constitutive CO expression. This gene, designated SUPPRESSOR OF CONSTITUTIVE EXPRESSION OF CO1 (SOC1), encodes a protein containing a domain designated the MADS box. This motif is present in other proteins known to bind DNA as homo- or heterodimeric complexes (Trobner et al. 1992; Riechmann and Meyerowitz 1997). SOC1 is expressed in the shoot and inflorescence apical meristem as well as the leaf primordia in response to inductive photoperiods (Samach et al. 2000). Consistent with the results of Onouchi et al. (1998), Samach et al. (2000) identified the FT gene (see below) as a very early downstream target of CO activity. In the approach used here, the CO coding sequence was translationally fused to the ligand-binding domain of the rat glucocorticoid receptor. This CO-GR fusion protein was expressed constitutively in transgenic plants, and could be directed to the nucleus and thus “activated” by application of the synthetic glucocorticoid hormone dexamethasone. In this case, FT transcript accumulation was seen within two hours of dexamethasone application. This experimental approach also resulted in the identification of two other early downstream targets of CO, AtP5CS2 involved in proline biosynthesis, and ACS10, encoding a potential 1-aminocyclopropane-1-carboxylic acid (ACC) synthase involved in the production of ethylene. AtP5CS2 is apparently essential for the elongation of the internodes that occurs upon flowering in Arabidopsis (bolting), as reduction in AtP5CS2 expression in transgenic plants eliminated this response (Nanjo et al. 1999). Although ethylene plays an obvious role in flowering in other species (e.g., Bromeliads), the precise role of ethylene in flowering in Arabidopsis is not known (Bernier et al. 1981). Mutants insensitive to ethylene exhibit slightly

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delayed flowering, but the molecular mechanism of this effect has not been explored (Guzman and Ecker 1990). CO exists as a member of a gene “family,” or group of genes that encode structurally related proteins (Ledger et al. 1996). It is possible that these CO-like (COL) genes also have a role in flowering, however Putterill et al. (1997) have noted that at least one of these genes, COL1, does not seem to be expressed at higher levels in inductive photoperiods, and to date has not been identified as important in flowering by traditional genetic analyses. In other species studied [i.e., apple (Hoon et al. 1999) and Brassica napus (Robert et al. 1998)], families of CO homologs also exist. It has been suggested that the function of CO in apple could be different from that in Arabidopsis based on the apparent abundance of mRNAs of two of the apple genes in the developing flower and fruit (Hoon et al. 1999), but such studies are complicated by the ambiguity of the evolutionary relationships between the identified apple genes and the Arabidopsis CO and COL genes, and the fact that the spatial pattern of CO expression in Arabidopsis has not been fully established. In Brassica napus, CO homologs are found at genomic positions corresponding to quantitative trait loci (QTL) affecting flowering time, and at least one of the Brassica CO homologs is functionally homologous to its Arabidopsis counterpart, because it is able to complement the flowering-time defect conferred by the co-2 mutation when expressed in transgenic Arabidopsis (Robert et al. 1998). A gene encoding a protein closely related to CO has recently been cloned from the short-day plant Pharbitis nil through an assay designed to identify genes that are upregulated in response to inductive photoperiods (i.e., short days; J. Liu and H. Kende, pers. commun). This is an important finding because the fact that CO is upregulated in both species in response to inductive photoperiods, even though the plants are of opposite flowering habits, suggests that the molecular mechanisms that are distinct between plants of varying photoperiodic responses lie genetically upstream of CO. In maize, ancestrally a short-day plant, the INDETERMINATE1 (ID1) gene promotes flowering in response to inductive photoperiods (Singleton 1946; Galinat and Naylor 1951). This is the only gene cloned to date that has unequivocally been demonstrated to be involved in flowering time in species other than Arabidopsis. ID1 encodes a protein containing zinc-finger motifs, suggesting that it binds DNA. It is expressed predominately in the leaf and influences flowering in a noncell-autonomous manner, and thus possibly regulates the production of a transmissible signal (Colasanti et al. 1998). There is no strong structural homology between ID1 and any of the Arabidopsis flowering-time genes that have been cloned to date, and the family of ID1-like genes that

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do exist in Arabidopsis (Colasanti et al. 1998) have not yet been reported to be involved in flowering. This could indicate a significant divergence in flowering mechanisms between Arabidopsis and maize. B. Non-photoperiodic Induction: The Autonomous Pathway As mentioned previously, loss of function of photoperiodic pathway genes does not prevent flowering but merely delays it, suggesting that at least one redundant pathway exists. Numerous flowering-time genes have been identified that are presumed to work outside of the photoperiodic control of flowering in the so-called autonomous pathway. LUMINIDEPENDENS (LD) was one of the first flowering-time genes identified (Redei 1962), and one of the first plant genes cloned through T-DNA mutagenesis (Lee et al. 1994a). In this technique, segments of DNA of known sequence are transferred into a plant by Agrobacterium, where they integrate into the genome at random locations. Interruption of a gene by the T-DNA often results in loss of gene function, and the corresponding gene sequence can be easily cloned by simple molecular techniques (Azpiroz-Leehan and Feldman 1997). The LD gene encodes a large protein containing two interesting structural features. First, a homeodomain—a nucleic acid-binding motif found in developmentally important proteins from yeast, plants, and animals—is found near the amino terminus. The homeodomain in LD is highly homologous to that found within the Drosophila Distal-less protein, which functions as a developmental switch to initiate limb formation (Cohen 1990). It also closely resembles the homeodomain found in Mata1, a yeast protein that acts as one component of a heterodimeric factor that represses expression of haploid-specific genes (Johnson and Herskowitz 1985). The other interesting structural feature is an acidic carboxyl-terminal region enriched in glutamine residues and containing short, homopolymeric glutamine stretches. These structural features are common to the activation domains of known transcriptional activators such as Drosophila Antennapedia and herpes virus VP16 (Gerber et al. 1994; Triezenberg 1995). Thus, it is possible that LD acts as a transcriptional regulator. Consistent with this proposed role, the LD protein contains nuclear localization signals and is localized to the nucleus. LD is expressed ubiquitously throughout the plant, with a concentration of mRNA expression in proliferating tissues, including the shoot, root, and floral apices (Aukerman et al. 1999). The function of LD may have diverged through evolution. An orthologous gene has been characterized from maize (van Nocker et al. 2000). The maize LD gene is highly homologous to its Arabidopsis counterpart,

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containing both the homeodomain and the potential transcriptional activator region, and exhibits an analogous mRNA expression pattern in the maize plant. However, when expressed in transgenic Arabidopsis containing an ld mutation, it does not complement the flowering-time defect, but instead causes developmental abnormalities associated with the shoot and floral meristems (van Nocker et al. 2000). What function this gene has been recruited for in maize is not presently known, but should be revealed by analyses of transposon-tagged lines. Although the activity of most plant genes studied to date seems to be controlled predominately at the transcriptional level, recent evidence suggests that posttranscriptional control may be an important factor in the regulation of flowering. The FCA gene was cloned and found to encode a large protein containing RRM motifs thought to mediate binding to RNA (Macknight et al. 1997). In support of the structural suggestion of function, the FCA protein binds to RNA in vitro, with a preference for G- and U-rich sequences (Macknight et al. 1997). The FCA gene produces multiple transcripts as a result of alternative splicing and transcriptional termination. Only one of these, which is a minority of FCA transcripts, would encode the presumed, full-length protein, and splicing to produce the full-length “active” FCA mRNA is likely to be regulated, as high-level expression of the genomic FCA sequence in transgenic plants resulted in only a minor increase in the amount of active mRNA (Macknight et al. 1997). Interestingly, it appears that FCA is able to promote flowering in a cell non-autonomous manner, because flowering is not delayed in periclinal chimaeras that express FCA only in the epidermal cell layers (Furner et al. 1996). In addition to the RNA-binding motifs, the presumed active form of the FCA protein contains a region designated the WW motif that contains two closely spaced tryptophan residues (Bork and Sudol 1994). This is potentially an essential component of the FCA protein, as it is excluded from the protein encoded by the strong fca-1 allele (Macknight et al. 1997). In other systems, WW motifs mediate interactions with protein partners containing proline-rich regions (Kay et al. 2000). Proteins or RNAs that interact with FCA have not been identified. One possibility is the protein encoded by FY. Mutations in FY do not further enhance the late flowering conferred by loss of FCA function (Koornneef et al. 1998a), suggesting that the two gene products operate in close proximity. In contrast, mutations in two other autonomous pathway genes, FPA and FVE, greatly enhance the lateness of fca (and fy) mutants. This suggests some redundancy in the mechanism of the autonomous pathway. However, as previously cautioned, this type of genetic analysis is contingent on mutations creating a complete loss of function, and even

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in cases where the gene has been cloned, this is difficult to demonstrate. FPA was recently cloned and, like FCA, encodes a protein containing RRM-type RNA-binding motifs (R. Amasino, pers. commun.). The cloning of FY, FVE, and another autonomous pathway gene, FLD, have not yet been reported. Semidominant mutations in the SHORT VEGETATIVE PHASE (SVP) gene confer photoperiod-sensitive early flowering (Hartmann et al. 2000). SVP encodes a MADS-box transcription factor that is expressed in the apical meristem during the vegetative phase, but apparently not in the inflorescence apical meristem. This expression pattern is consistent with its role as a repressor of flowering. SVP mRNA is also expressed in the early floral meristem, suggesting a role in flower development. However, loss of SVP function confers no gross floral defects, indicating that a function at this stage is redundant or minor (Hartmann et al. 2000). The genetic relationships between SVP and other autonomous pathway genes have not been characterized. C. Vernalization In many plants, flowering can be accelerated or induced by exposure to a long period of near-freezing temperatures. This is a commonly employed reproductive strategy that allows for flowering and seed production in the environmentally favorably period following natural winter. This phenomenon, termed vernalization, has been studied for decades at the physiological level but only recently at the molecular level. The lack of molecular work addressing vernalization is partly due to the fact that in Arabidopsis thaliana, the commonly utilized laboratory strains flower soon after germination, and extended cold treatments do little to further abbreviate the vegetative phase (Koornneef et al. 1998b). However, most natural ecotypes of Arabidopsis behave as winter annuals, flowering extremely late in the absence of cold, but very early when exposed to cold for extended periods. The flowering habit among natural ecotypes is largely determined by allelic variation at two loci, designated FRIGIDA (FRI) and FLOWERING LOCUS C (FLC); (Lee et al. 1993; Koornneef et al. 1998b). “Early” alleles at either loci behave similarly to presumed null alleles created by induced mutation, suggesting that natural early alleles have lost function (Michaels and Amasino 1999). FLC is expressed predominately in the vegetative apex and roots, but is absent from the inflorescence apex. Expression of FLC mRNA is apparently not significantly decreased as the plant proceeds through the vegetative phase, suggesting that repression of flowering by FLC can be overcome by developmental progression (Sheldon et al.

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1999). FLC encodes a MADS-box-containing protein (Michaels and Amasino 1999; Sheldon et al. 1999). Because other MADS-box proteins are known to work as heterodimers (Trobner et al. 1992), it is possible that FLC has a DNA-binding partner. One possibility is SVP, as both genes are expressed in the shoot apical meristem specifically during the vegetative phase. However, unlike those of FLC, mRNA levels of SVP are not diminished after extended cold treatments (Hartmann et al. 2000). The activity of FLC is semidominant, and transgenic plants containing extra copies of the FLC genomic sequence never flower without cold, acting in essence as biennials (Michaels and Amasino 1999; Sheldon et al. 1999). Importantly, these findings suggest that the difference in flowering habit between winter-annual plants and biennial plants could be quantitative rather than qualitative. The cloning of FRI has recently been reported; this gene encodes a protein that does not exhibit significant sequence identity to any other protein of known function (Johanson et al. 2000). In Arabidopsis, a genotype conferring the winter-annual habit can also be synthesized by impairing the function of the promotive autonomous pathway genes (Koornneef et al. 1991), and, like repression of flowering imposed by FRI, the block to flowering resulting from the loss of autonomous-pathway gene function is also dependent on FLC activity (Lee et al. 1994b; Koornneef et al. 1994; Sanda and Amasino 1996a,b). These data suggest that the flowering-repressive activity of FLC is both positively regulated by FRI and negatively regulated by autonomous pathway genes. Consistent with this idea, FLC mRNA expression is increased both in genotypes containing late FRI alleles, and in autonomous-pathway gene mutants (Michaels and Amasino 1999; Sheldon et al. 1999). FLC mRNA expression is decreased after extended cold exposures (Michaels and Amasino 1999), suggesting that vernalization involves molecular events “upstream” from FLC. The winterannual habit conferred by loss of autonomous-pathway gene function is not dependent on FRI, indicating that neither the activity of FRI nor the autonomous pathway genes is necessary for the vernalization response. Thus, although these genes set up a requirement for cold for flowering, they are unlikely to be directly involved in the associated cold signal transduction. Specific components involved in transmitting the signal from the cold stimulus to FLC expression have not yet been identified. Using a genetic approach, Chandler et al. (1996) identified at least two loci, designated VRN1 and VRN2, that could play such a role. These mutants were isolated in an fca mutant background based on a lack of vernalization response. FLC mRNA levels are only partially decreased after

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cold treatment in these mutants, consistent with the idea that VRN1 and VRN2 function upstream to regulate FLC expression (Sheldon et al. 2000). Ishitani et al. (1998) identified a recessive mutation, hos1-1, that constitutively activated gene expression from a cold-responsive promoter. hos1-1 plants exhibited accelerated flowering, suggesting that HOS1 might normally act as a negative regulator of vernalization. However, these results were difficult to interpret because hos1-1 conferred pleiotropic effects on growth, and the specific genetic background utilized (C24) is normally early flowering due to an “early” FLC allele (Sanda and Amasino 1996a). The well characterized cold-regulated (COR) genes involved in the process of acclimation probably have little or no role in vernalization, as freezing tolerance is not affected in the vrn mutants (Chandler et al. 1996), and constitutive expression of members of the CBF family of transcriptional activators upregulates COR gene expression in a winter annual line in the absence of cold, but has no effect on flowering time (J. Liu and S. van Nocker, manuscript submitted). Some characteristics of vernalization, including the requirement for cell division for the vernalized state to be attained and the stability of the vernalized state through mitosis, suggests an epigenetic mechanism (Wellensiek 1964). One possibility is the covalent modification of DNA through cytosine methylation. Evidence for the involvement of DNA methylation in the vernalization response has been presented by Burn et al. (1993) and Brock and Davidson (1994), who found that the promotion of flowering by extended cold in Arabidopsis and wheat, respectively, could be partially substituted for by exposure of plants to the ribonucleotide analog 5-azacytidine (5-azaC). Treatment with this compound results in demethylation of DNA. In the study by Burn et al. (1993), flowering was reportedly accelerated only in genotypes that are known to exhibit a strong vernalization response. Thus, the partial substitution for cold treatment conferred by 5-azaC apparently acted specifically upon the vernalization pathway. It was hypothesized that extended cold results in the selective demethylation and transcriptional activation of floral-promotive genes (Finnegan 1998). A further possible link between DNA methylation and vernalization was hypothesized by Finnegan et al. (1996), who reported that antisense expression of the METHYLTRANSFERASE1 (MET1) gene in transgenic Arabidopsis resulted in both decreased genomic DNA methylation levels and early flowering. This early flowering was apparently associated with decreased FLC mRNA abundance (Sheldon et al. 1999), again suggesting specificity for the vernalization pathway. In contrast to these results, Ronemus et al. (1996) found that MET1 antisense expression

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conferred highly pleiotropic effects, including slightly delayed flowering, in transgenic Arabidopsis. The apparent contradictions between these two reports could reflect differences in environmental conditions or genetic backgrounds used. However, neither group utilized a genetic background that exhibits a strong vernalization response. Other aspects of these results should also be interpreted with caution. Goto and Hamada (1988) and Chandler and Dean (1994) demonstrated that growth of plants on the nucleotide analog 5-bromodeoxyuridine, which does not result in reduced DNA methylation, could also accelerate flowering. D. Induction by Gibberellins The influence of GAs on flowering in many plants is well known (Lang 1965; Zeevaart 1983). In long-day rosette plants such as Arabidopsis, GAs generally have an inductive effect, and this is especially striking in Arabidopsis where flowering is delayed by growth in short days, or in winter-annual genotypes grown in the absence of cold. Consistent with this, flowering is delayed in the ga1 mutant that is defective in GA biosynthesis, and in the gai mutant, which is insensitive to GAs (Koornneef and van der Veen 1980; Koornneef et al. 1985). In addition, plants carrying mutations in the SPINDLY (SPY) gene, which exhibit a constitutive GA response, flower early (Jacobsen and Olszewki 1993). Exogenously applied GA is able to promote flowering in all late mutants studied, and mutations in GA biosynthesis or perception are interactive with all flowering-promotive genes studied, especially those grouped into the photoperiodic pathway (Putterill et al. 1995; Simpson et al. 1999; our independent observations). Consistent with the strongly interactive effect with photoperiod pathway genes, ga mutant plants are apparently unable to flower when grown in short days, and gai plants flower extremely late under such conditions (Wilson et al. 1992). Thus, it appears that the production of GAs represents an additional pathway to flowering that operates in parallel with the photoperiodic pathway, and, to some extent, the autonomous pathway as well. The role of GAs in vernalization is unclear. Although GAs are able to promote flowering in winter-annual genotypes (thereby bypassing the requirement for cold), winter-annual genotypes containing the ga or gai mutations still exhibit a normal vernalization response (R. Amasino, pers. commun.; Chandler et al. 2000). This would suggest that GAs are not necessary for vernalization. However, as GA production is not completely eliminated in the ga mutant (J.A.D. Zeevaart, pers. commun.), such results should be interpreted with caution. In addition, although GAI has an obvious role in GA signal transduction during vegetative

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growth, other yet unidentified GA-signaling components could be involved in the flowering response.

IV. ROLE OF CARBOHYDRATES Carbohydrates have long been known to play a key role in flowering (Bernier et al. 1993). The concentration of sucrose, the major translocated sugar in most plants, increases dramatically in phloem exudates upon photoinduction in both short-day and long-day plants, even when the photoinductive treatment does not result in a net increase in photosynthesis (Bodson and Outlaw 1985; Houssa et al. 1991; Corbesier et al. 1998). One of the earliest biochemically detectable changes in the shoot meristem upon photoinduction is the accumulation of sucrose (Bodson and Outlaw 1985) and labeling experiments suggest that this sucrose originates not from increased photosynthesis, but from mobilization of sugars from reserve carbohydrates such as starch in the leaves and stem (Bodson et al. 1977). Arabidopsis will flower in complete darkness if the aerial portion of the plant is supplied with sucrose or glucose (Redei et al. 1974; Goto 1982; Araki and Komeda 1993). Under such conditions, the lateflowering phenotype conferred by mutations in GI, CO, FCA, FPA, and FVE was complemented or nearly complemented. In contrast, flowering was not promoted by these conditions in plants carrying mutations in FWA or FT (Araki and Komeda 1993; Roldan et al. 1999). These surprising results suggest that the fundamental mechanism of both the photoperiodic and autonomous pathways could be the delivery of sugars to the shoot apex! Sucrose is synthesized in the cytosol from the products of photosynthesis or starch degradation, transported to and loaded into the phloem, translocated throughout the plant, unloaded from the phloem, and then transported from cell to cell. This complicated routing provides many opportunities for control of sugar transport, and thus it is likely that many genes are involved. Interestingly, mutations in the GI gene are pleiotropic in that mutants accumulate excess levels of starch in the leaves and stem (Eimert et al. 1995). At least one other Arabidopsis mutant that was originally identified as a starch accumulator, carbohydrate accumulation mutant1 (cam1), was found to flower late relative to wild-type plants, especially when grown under continuous light (Eimert et al. 1995). High starch content per se does not seem to be the direct cause of the flowering-time defect, because the flowering-time defect conferred by gi and cam1 was not rescued in genetic backgrounds where starch synthesis was dis-

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rupted (Eimert et al. 1995). Other mutants that lack starch, ADP-glucose pyrophosphorylase1 (adg1) and phosphoglucomutase1 (pgm1), and at least one other mutant that accumulates starch, starch-in-excess1 (sex1), also exhibit delayed flowering, but only under photoperiods of less than 16 h (Lin et al. 1988; Caspar et al. 1985; Caspar et al. 1991; Corbesier et al. 1998). The observation that both the overabundance of starch, and lack of starch, can affect flowering in a similar manner further suggests that flowering is not directly affected by starch content. In fact, the lack or excess of starch in the pgm1 and sex1 mutants, respectively, seems to disrupt carbohydrate metabolism in a similar manner, as in both mutants soluble sugars (including sucrose) accumulate to abnormally high levels (Caspar et al. 1985; Caspar et al. 1991). Thus, it seems probable that the predominant effector of flowering in these mutants is the levels of sugars. Arabidopsis plants grown at low temperature also accumulate soluble sugars, and this may be related to the delayed flowering seen under these conditions.

V. CONTROL OF MERISTEM IDENTITY The shoot and flower are, in spite of their radical difference in morphology, essentially analogous structures produced by the meristem. The fate of meristems—to generate flowers rather than shoots—is governed by a group of meristem identity genes, which are activated during the transition to flowering. This group of genes in turn controls expression both of the floral organ identity genes, which control the development of the floral organs, and cadastral genes, which regulate the boundaries of expression of the organ identity genes. The molecular biology of flower development is beyond the scope of this review, and the reader is referred to recent excellent discussions on this topic (Bowman 1997; Sessions et al. 1998). A. Meristem Identity Genes As with genes influencing the timing of flowering, genes involved in influencing meristem identity have been identified by screening for mutants in which meristem identity is altered. Such screens have identified genes that both positively and negatively regulate the shoot-toflower transition. In Arabidopsis plants homozygous for the recessive terminal flower 1 (tfl1) mutation, the normally indeterminate inflorescence terminates in a single flower, and lateral shoots develop as solitary flowers (Shannon and Meeks-Wagner 1991; Alvarez et al. 1992).

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Thus, a presumed function of TFL1 is to keep the inflorescence meristem in an indeterminate state. Plants lacking TFL1 activity also flower slightly early, suggesting that TFL1 functions during the vegetative phase as a repressor of the shoot-to-inflorescence transition (Shannon and Meeks-Wagner 1991; Schultz and Haughn 1993). TFL encodes a member of a small protein family exhibiting limited homology to mammalian Raf kinase inhibitor protein (RKIP). RKIP is a membrane-associated protein that regulates Raf-1 kinase, which is intimately involved in signal transduction cascades controlling cell proliferation and differentiation in mammals (Ferrell 1996). The amino-terminus of RKIP is cleaved off to form a small peptide hormone, leading to the speculation that TFL may in a similar manner be the progenitor of a small signaling peptide involved in flowering (Bradley et al. 1997). That intercellular signaling should be involved in flowering is expected, as the meristem must function as a unit to organize flower primordia even though it is composed of clonally unrelated cells (see below). Constitutive expression of the TFL1 gene in transgenic Arabidopsis confers a phenotype that is essentially opposite to that seen in tfl1 mutants—such plants exhibit delayed flowering, and produce secondary inflorescences that are not subtended by cauline leaves (Ratcliffe et al. 1998). Because Arabidopsis flowers are not normally found in association with leaves (bracts), such structures can be interpreted as a conversion of flowers to inflorescence shoots. Conversion of flowers to shoots is also seen in plants carrying loss-offunction mutations in a group of genes best typified by LEAFY (LFY). In plants carrying strong lfy alleles, early-arising (basal) flowers are completely transformed into shoots, whereas those that develop in more apical positions exhibit partial floral character. In plants carrying very weak lfy alleles, secondary shoots subtended by cauline leaves develop at the first few positions normally occupied by flowers (Schultz and Haughn 1991). That flowers eventually do develop even in the absence of LFY activity indicates that other genes function in a partially redundant manner to promote the inflorescence-to-floral switch. One of these is APETALA1 (AP1). Loss of AP1 function phenocopies very weak lfy alleles with respect to inflorescence structure, but dramatically enhances the phenotype of lfy plants, such that in lfy/ap1 double mutants, even the most apical nodes produce structures with strong shoot characteristics (Bowman et al. 1993; Huala and Sussex 1992; Weigel et al. 1992). Strong ap1 alleles also confer a striking floral phenotype—sepals found in the outer whorl of the flower exhibit leaf-like characteristics, and often subtend secondary flowers (Irish and Sussex 1990). This phenotype can be interpreted as a partial reversion of the flower into a shoot, further implicat-

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ing AP1 in meristem identity (Mandel et al. 1992). Strong constitutive expression of both LFY and AP1 in transgenic plants results in premature transformation of the shoot into a flower, mimicking loss of TFL function (Weigel and Nilsson 1995; Mandel and Yanofsky 1995). Incredibly, Arabidopsis LFY is able to accomplish this even in a divergent tree species, aspen, suggesting a high degree of conservation of meristem identity function during evolution (Weigel and Nilsson 1995). That loss-of-function mutations in genes such as LFY and AP1 exhibit additive phenotypic effects when combined is evidence that at least two pathways are normally involved in establishing the floral meristem (Shannon and Meeks-Wagner 1993). The genetic evidence indicating that these pathways are partially redundant is reinforced by experiments showing that the shoot-to-flower conversion conferred by 35S:AP1 is not dependent on LFY, and that mutations in AP1 cannot fully suppress this effect in 35S:LFY plants (Mandel and Yanofsky 1995; Weigel and Nilsson 1995). However, these two pathways are strongly interactive. In primordia destined to become flowers, LFY mRNA expression precedes that of AP1, and AP1 upregulation is delayed in lfy plants (Simon et al. 1996; Hempel et al. 1997; Liljegren et al. 1999). In addition, ectopic activation of LFY activity results in premature AP1 expression (Parcy et al. 1998). These findings suggest that LFY acts as a positive regulator of AP1. Conversely, LFY is expressed prematurely in primordia of 35S:AP1 plants, suggesting a reciprocal positive regulation between the two genes (Liljegren et al. 1999). The protein encoded by LFY does not resemble any other known protein (Weigel et al. 1992), but numerous lines of evidence indicate that it acts as a transcription factor. LFY protein is localized to the nucleus and is able to mediate transcriptional activation in yeast when fused to a suitable activation domain (Parcy et al. 1998). Consistent with the genetic evidence that LFY positively regulates AP1 expression, the LFY protein binds to a potential AP1 promoter element in vitro (Parcy et al. 1998). Moreover, Wagner et al. (1999) demonstrated through the use of an inducible LFY-GR fusion protein that LFY is able to rapidly activate AP1 expression even in the presence of cycloheximide, suggesting a direct interaction. Another gene with a promotive role in floral identity is CAULIFLOWER (CAL). The function of this gene is apparently completely redundant with that of AP1, such that in an otherwise wild-type genetic background, plants lacking CAL activity appear normal. However, in plants lacking both CAL and AP1 activity, meristems never develop determinate floral character and continue to proliferate, and inflorescences develop into structures that resemble tiny versions of the garden

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vegetable for which the gene is named (Bowman et al. 1993). The phenotypic similarity between Arabidopsis cal/ap1 double mutants and cauliflower led Kempin et al. (1995) to investigate functional conservation of CAL genes in the two species. In both Brassica oleracea, and its cauliflower derivative (Brassica oleracea, var. botrytis) CAL is expressed in a spatial and temporal pattern similar to that seen with CAL in Arabidopsis. However, the gene from var. botrytis encodes a protein that is significantly truncated and is probably nonfunctional (Kempin et al. 1995), suggesting a molecular explanation for what is probably the most popularly recognized natural inflorescence variation. Both AP1 and CAL encode proteins containing a MADS-box domain, consistent with roles as transcription factors (Mandel et al. 1992; Kempin et al. 1995). The essentially opposite phenotypes conferred by loss of TFL and LFY/AP1/CAL function indicates that these genes operate antagonistically, and molecular studies support this conclusion. Expression of both TFL and LFY mRNAs is maintained at a low level in the shoot apex during the vegetative phase, and is upregulated upon the transition to flowering. However, their expression is spatially separated, with TFL mRNA present in the center of the meristem, and LFY mRNA present only in emerging primordia (Bradley et al. 1997). In plants lacking LFY, AP1, or CAL activity, TFL expression extends into the lateral primordia (Ratcliffe et al. 1999), whereas in plants constitutively expressing LFY, TFL expression is greatly decreased (Ratcliffe et al. 1999; Liljegren et al. 1999). Like AP1 and CAL1, at least two other genes are known to have redundant roles in promoting floral identity. Mutations in APETALA2 (AP2), for example, do not confer strong flower-to-shoot conversion, but instead enhance both lfy and ap1 phenotypes (Shannon and Meeks-Wagner 1993). Plants carrying mutations in the UNIDENTIFIED FLORAL ORGANS (UFO) gene resemble weak lfy mutants in that basal inflorescence nodes exhibit some shoot identity (Levin and Meyerowitz 1995; Wilkinson and Haughn 1995). In addition, in short-day-grown ufo plants, the transition to an inflorescence meristem is incomplete, and reversion to a vegetative meristem can occur (Wilkinson and Haughn 1995). Like the aforementioned FKF1 and ZTL, UFO encodes an F-boxcontaining protein, implicating this factor in the selective elimination of other, possibly regulatory proteins (Ingram et al. 1995). The AGAMOUS gene, better known for its role in governing floral organ identity in the inner whorls, also functions to promote floral meristem identity by regulating meristem determinacy. The normally determinate floral meristem becomes indeterminate in ag mutant plants, and under short-day conditions can even completely revert to an inflorescence meristem (Yanofsky et al. 1990; Mizukami and Ma 1995, 1997).

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B. Integration of Flowering Pathways and Activation of Meristem Identity Genes Genes affecting the timing of flowering are obvious candidates for direct regulators of meristem identity genes, and recent genetic and molecular studies have demonstrated numerous interactions between the two classes of genes. Simon et al. (1996) utilized the inducible CO-GR expression system described above to examine the response of the LFY, AP1, and TFL1 genes to increased CO activity. Within 24 h of CO:GR activation in plants grown in inductive photoperiods, LFY and TFL1 mRNAs accumulated to detectable levels, and detectable AP1 mRNA upregulation occurred after ~72 h. The delayed expression of AP1 relative to that of LFY is in accordance with genetic evidence that LFY acts upstream of AP1 (above). These kinetics were similar to those seen upon the transfer of short-day-grown plants to inductive photoperiods (Simon et al. 1996). In contrast, when CO-GR was activated in short-day-grown plants, LFY and TFL1 were again activated within 24 h, but the delay in the upregulation of expression of AP1 was extended to ~120 h. The authors concluded that since inductive photoperiods were more effective than CO to activate AP1, an additional, unknown mechanism operating in inductive photoperiods is involved in the upregulation of AP1 (Simon et al. 1996). In fact, the flowering time genes FT and FWA (see below) may play a role that is redundant with that of LFY in activating AP1. Evidence for this was presented by Ruiz-Garcia et al. (1997), who showed that the phenotype conferred by a strong allele of lfy is greatly enhanced in an ft or fwa mutant background. It is known that GAs promote flowering at least in part through upregulation of LFY, because in the ga1 mutant, LFY promoter activity is reduced, and its upregulation in response to inductive photoperiods is delayed relative to wild-type plants. In addition, 35S:LFY expression can partially complement the flowering defect conferred by ga1 in short day conditions (Blazquez et al. 1998). Recently, Blazquez and Weigel (2000) demonstrated that distinct cis elements in the LFY promoter mediate the induction of LFY in response to GAs or inductive photoperiods. Thus, it appears that LFY represents an integration point of at least the photoperiod pathway and the GA pathway.

VI. COMPETENCY A multitude of physiological studies has indicated that flowering is dependent not only on the ability of the leaf to produce the floral stimulus, but also on the ability of the shoot apical meristem to respond to

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it (Lang 1965). The shoot apical meristem is a group of specialized cells found within the apex at the growing tip of shoots. In Arabidopsis, as in other plants, the meristem displays typical tunica-corpus organization, recognizable as early as the torpedo stage of embryogenesis (Long et al. 1996). The tunica layers (L1 and L2) are propogated through anticlinal cell divisions, and thus the L1, L2, and L3 tend to be clonally unrelated. The meristem can display an additional level of organization that is superimposed on the tunica-corpus structure. This consists of radially symmetric “zones” that are often distinguished by mitotic activity and cell size and density (Vaughan 1952; Steeves and Sussex 1989). The central initiation zone, at the summit of the meristem, is characterized by a group of large cells with prominent vacuoles that apparently divide very slowly. Flanking the central initiation zone is a ring of smaller, more densely cytoplasmic, proliferative cells termed the peripheral zone. In the peripheral zone, groups of cells are recruited into leaf or flower primordia where they may soon assume specialized roles. Immediately subtending the central initiation zone is a group of proliferative cells referred to as the rib, or file, meristem that produces the internal tissues of the plant stem. When given inductive photoperiods, the commonly studied annual genotypes of Arabidopsis flower soon after germination. Before the transition to flowering in such young plants, the apical meristem is small and without easily recognized cytological zonation. However, when the vegetative phase is extended (i.e., through growth in short-day photoperiods, or in winter-annual genotypes lacking cold treatment) the zonal pattern described above becomes more apparent (Vaughan 1952; Besnard-Wibaut 1981). The appearance of well-defined zonal organization in Arabidopsis has been correlated with the ability of the plant to exhibit a significant flowering response to applied GAs or long days (Besnard-Wibaut 1981), suggesting that appropriate meristem structure could constitute the morphological basis of competency. The molecular determinants of competency remain unknown. It is important to note that although transgenic Arabidopsis expressing LFY in a constitutive manner flower very early, they still progress through a short vegetative phase (Weigel and Nilsson 1995). That LFY is insufficient to force early-arising primordia into a floral fate suggests that genes controlling meristem competence act genetically downstream, or in a separate pathway, from LFY. Two candidates are FWA and FT. Mutations in these genes cause delayed flowering, and are epistatic to a 35S:LFY transgene [i.e., constitutive expression of LFY is unable to rescue that late-flowering phenotype conferred by fwa and ft mutations (Nilsson et al. 1998)].

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All known mutations in FWA are dominant. Dominance of mutations often indicates that the mutant gene product has gained activity. This could happen if the gene product were increased in abundance, or if it usually existed in the “off” position and were turned “on” by the mutation in a manner similar to the action of an upstream signaling molecule. Indeed, FWA mRNA levels were found to be increased in fwa mutants relative to wild-type plants, suggesting that the dominance of the mutation indeed results from increased FWA activity. Interestingly, it appears that the known mutant alleles of fwa result from epimutation, a class of mutation that does not disrupt the DNA sequence. In the case of fwa mutations, constitutive expression of the gene is associated with a reduction in methylation of DNA residues found in the promoter region of the gene (W. Soppe, pers. commun.). The FWA gene was recently cloned and found to encode a transcription-factor-like protein (M. Koornneef and W. Soppe, pers. commun,). Because a gain in function of FWA results in later flowering, the function of the wild-type FWA gene is likely to repress flowering. Alleles of FT conferring late flowering are recessive and likely result from decreased function, suggesting that FWA acts in a manner opposite that of FT. The Arabidopsis FT gene was recently cloned by activation tagging (Kobayashi et al. 1999; Kardailsky et al. 1999). In this approach, random plant genes are transcriptionally activated by the nearby insertion of T-DNAs containing strong enhancer sequences, and function of the activated gene is surmised based on the resulting phenotype. Activation tagging has become a powerful technique for the identification of genes whose products are normally limiting in a pathway affecting a given phenotype (Lindsey et al. 1998). The product of the FT gene is structurally related to that of TFL, suggesting that like TFL, the FT protein may function in cell-to-cell signaling (Kardailsky et al. 1999). FT mRNA is expressed throughout the aerial tissues of the plant, and is not localized to any specific domain within the shoot apex (Kobayashi et al. 1999; Kardailsky et al. 1999). This expression becomes evident only near the floral transition, and, consistent with being regulated by CO (see above), this upregulation of expression is delayed in the co mutant and in shortday conditions. However, upregulation of expression still occurs in plants lacking CO activity, indicating that FT is also regulated by another mechanism. As might be expected from a gene controlling meristem competence, constitutive expression of FT in transgenic plants results in a nearly complete elimination of the vegetative phase, as such plants form a flower after only ~2 leaves, which are embryonic in origin. Other genes that have been postulated to play a role in competence include EMBRYONIC FLOWER (EMF) 1 and 2. Plants carrying mutant

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alleles of these genes produce a modified flower upon germination (Sung et al. 1992; Bai and Sung 1995; Yang et al. 1995; Chen et al. 1997). Plants carrying strong emf1 alleles develop no leaves at all, indicating that the vegetative phase has been completely bypassed. Mutations in EMF2 confer a milder but similar phenotype, with a few small leaves produced on a modified inflorescence. The very early flowering associated with loss of EMF function suggests that the EMF genes normally act as strong repressors of reproductive development. It is possible that the two genes operate in distinct genetic pathways, as combining strong emf1 and emf2 alleles leads to severe developmental defects that could be considered an additive phenotype (Yang et al. 1995). Although cloning of the EMF genes has not been reported, some indication of their mode of action comes from examining interactions of emf mutations with other mutations affecting the timing of flowering. Mutations in GI or CO, for example, have no effect on emf1 or emf2 phenotype; that CO and GI are not required for the expression of the emf mutant phenotype suggests that CO and GI act as upstream regulators of the EMF genes. However, mutations in other flowering-time genes, including FCA, LD, FVE, FY, FHA, FPA, FWA, and FT resulted in a partial rescue of the early flowering emf phenotype, with mutations in FWA and FT having the greatest effect (Huang and Yang 1998; Page et al. 1999). These results suggest that the EMF genes, rather than having a general repressive effect on flowering, act specifically as downstream negative regulators of the photoperiodic pathway. Finally, it is interesting to note that constitutive expression of LFY was not sufficient to fully rescue the flowering defect conferred by the ga mutation in short days (Blazquez et al. 1998). This suggests that in addition to playing a role in LFY activation, gibberellins play a role in promoting competency. VII. CONCLUSION AND PERSPECTIVES Research into the molecular mechanisms of flowering is now entering its second decade. Many flowering genes have been identified in the model plant Arabidopsis thaliana, and most of these have now been cloned. In addition, the functions of most of the known flowering-time genes have been assigned within one of the multiple parallel pathways that promote flowering in this plant (Fig. 1.1). However, the research accomplished to date should be considered merely as a foundation for future work, as many aspects of flowering in this plant remain unexplored. For example, in many cases, the relationships and interactions among the genes in these pathways have been surmised based solely on

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florigen

COLD vernalization

molecular events at the shoot apex

sugars

VRN1 VRN2 LD, FCA, FPA, FVE, FLD

FT FWA FLC SOC1

FRI

AP1 LFY

GA molecular events at the leaf ATP5CS2

endogenous clock

LIGHT

TOC1, LHY, CCA1, ELF3, FKF1, ZTL, GI

phytochromes cryptochromes

starch

internode elongation

CO

sugars

ACS10

florigen ethylene production

Fig. 1.1. Schematic diagram of molecular pathways leading to flowering presumably operating in the leaf and in the shoot apex. Assignment of gene function to the leaf or apex is based predominantly on the reported mRNA expression pattern of the genes. Relationships among genes depicted here are based on genetic and/or molecular evidence. Arrows indicate a generally positive regulation, whereas lines with blocks indicate a repressive regulation. Not all of the genes referred to in the text are shown. The relationship between the CO gene, the hypothetical substance florigen, and genes acting downstream of florigen is especially speculative.

genetic evidence, and need to be confirmed with molecular and biochemical data. Some more general questions remain to be answered as well. For example, in spite of the wealth of physiological data suggesting that flowering is the result of a transmissible signaling substance (“florigen”), produced in the leaves and acting at the shoot apex, the molecular identity of this signal is still unknown. The possibility that flowering can be manipulated in species other than Arabidopsis through molecular methods is largely dependent on the degree to which flowering-time mechanisms have been conserved through evolution. In order to prove their utility in solving horticultural

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problems, the models proposed to describe floral initiation based on genetic and molecular studies in Arabidopsis will likely need to be evaluated in plants with dissimilar flowering habits. With very few exceptions (see above), this is an area of research that has remained largely unexplored. Probably the most attractive opportunity in this regard is maize, which has traditionally been the most popular monocot model for developmental studies, and has diverged significantly from Arabidopsis in terms of genetics, physiology, and anatomy. Current efforts underway to discover maize gene function by high-throughput, reversed-genetics approaches should greatly simplify this task (Martienssen 1998; Walbot, 2000). The apparent conservation of function of meristem identity genes among species offers some indication that the function of flowering-time genes may be similarly conserved. As the intriguingly complex pathways that constitute flowering become more completely characterized in Arabidopsis, this area holds much promise for future research.

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2 Floral Homeotic Gene Regulation Robert G. Franks and Zhongchi Liu* Dept. of Cell Biology and Molecular Genetics University of Maryland College Park, MD 20742

I. INTRODUCTION A. The ABC Model B. The MADS-domain Multi-gene Family C. The A Class Genes: AP2 and AP1 D. The B Class Genes: AP3 and PI E. The C Class Genes: AG, HUA1 and HUA2 F. Novel Class Genes: SEP1, SEP2, and SEP3 II. CONSERVATION OF THE ABC FUNCTIONS IN ANGIOSPERMS A. Dicotyledonous Species B. Monocotyledonous Species III. POSITIVE REGULATORS OF FLORAL ORGAN IDENTITY GENES A. Meristem Identity Genes: LFY and AP1 B. LFY, a Direct Activator of AP1 C. LFY, a Direct Activator of AG D. Two Phases of Regulation: Initiation and Maintenance of B Gene Expression E. UFO, a Coregulator of B Gene Expression IV. NEGATIVE REGULATORS OF FLORAL ORGAN IDENTITY GENES A. Temporal and Spatial Regulators of AG B. Repression of AP1 Expression in Floral Whorls 3–4 C. Restriction of B Gene Expression to Floral Whorls 2–3 V. SUMMARY LITERATURE CITED

*We thank Beth Krizek for photographs used for Figures 2.2E and 2.2F. Our research on Arabidopsis flower development is supported by the U.S. Department of Agriculture 9835304-6714 (Z.L.), U.S. Department of Energy 02-00ER20281 (Z.L.), and a NIH postdoctoral fellowship GM20426-01 (R.G.F.).

Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 41

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I. INTRODUCTION In the past decade, a major milestone in plant developmental biology has been the elucidation of the molecular genetic basis underlying floral organ specification. An elegant ABC model explains how three classes of genes (A, B, C classes) direct the development of four types of floral organs (Coen and Meyerowitz 1991; Weigel and Meyerowitz 1994). This model enables one to design and engineer the structure of flowers in a predictable manner by altering the expression of these ABC class floral homeotic genes (Jack et al. 1994; Krizek and Meyerowitz 1996a). Although the function of the ABC genes has been studied extensively, only more recently has the regulatory mechanism of their expression been elucidated. Both positive and negative regulators of ABC gene expression have been identified and, in some cases, significant progress has been made toward understanding the molecular mechanisms underlying the regulation of their expression. This review will focus on the regulation of ABC gene expression in Arabidopsis but will highlight similarities and differences among the ABC genes found in other plant species. For simplicity and uniformity, standard Arabidopsis nomenclature is used throughout this review even if a gene is from a different species. Specifically, uppercase letters identify wild-type genes or gene products and lowercase letters identify mutant genes or mutants. Additionally, the names of genes and mutants are shown in italic type, while the names of proteins are not italicized. For example, AGAMOUS (AG) refers to the wild type gene, agamous (ag) refers to the mutation or mutant, and AGAMOUS (AG) refers to the protein. A. The ABC Model The structure of the Arabidopsis flower is typical of many angiosperm flowers (Fig. 2.1A; Fig. 2.2A). It is made up of four types of floral organs arranged in four concentric circles or whorls. The sterile perianth organs, sepals and petals, comprise the outer two whorls while the reproductive organs, stamens and carpels, make up the inner two whorls. Despite dramatic variation in the number, color, and shape of floral organs in different species this arrangement of sepal, petal, stamen, and carpel, from the outermost whorl to the innermost whorl, is fixed in the majority of the angiosperm species. This cross-species similarity suggests that the molecular genetic systems responsible for patterning of floral organs are similar in the majority of flowering plants.

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A: A diagram of an Arabidopsis flower

B: Wild-type Whorls: 1

Whorls Sepal (Se)

1

Petal (Pe)

2

Se

2

3

4

Pe

Sta

Ca

A

Stamen (Sta) 3 Carpel (Ca)

B

4

C C: Class A mutants Whorls: 1

Ca

A B C

-

2 Sta

D: Class B mutants

3

4

1

2

Sta

Ca

Se

Se

A B C

3 Ca

E: Class C mutants

4 Ca

1

2

3

Se

Pe

Pe

4 Se

A

-

B C

-

Fig. 2.1. The ABC model. (A) A diagram of an Arabidopsis flower showing four sepals in whorl 1, four petals in whorl 2, six stamens in whorl 3, and two fused carpels in whorl 4. (B) In wild-type, the domains of the A, B, and C activities are indicated by the filled boxes. A class activity is only present in whorls 1–2; B class activity is in whorls 2–3; and C class activity is in whorls 3–4. (C) In A class mutants, such as ap2 or ap1, the A activity is absent. This results in the expansion of the C activity into all four whorls and the homeotic transformation of first whorl sepals to carpels and second whorl petals to stamens. (D) In B class mutants such as ap3 or pi, the B activity is absent. As a result, second whorl organs develop into sepals while third whorl organs develop into carpels. (E) In C class mutants, such as ag, the C activity is absent. This results in the expansion of the A activity into all four whorls and the homeotic transformation of third whorl organs into petals and forth whorl organs into sepals and another flower.

The analysis of mutations that alter the pattern of organ identity within the flower has generated a greater understanding of the molecular genetics of floral pattern specification. Floral homeotic mutations result in the substitution or replacement of one organ type by another organ type. (Fig. 2.2B–D; Bateson 1894; Acquaah et al. 1992). The isolation of floral homeotic mutations provided the initial clues to the genetic basis of floral patterning (Meyerowitz et al. 1989). It was the analyses of these floral homeotic mutants and the genetic interactions among them in Arabidopsis thaliana and Antirrhinum majus that led to

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Fig. 2.2. Microscopic photography of Arabidopsis flowers. (A) A wild-type flower with four sepals in whorl 1, four petals in whorl 2, six stamens in whorl 3, and two fused carpels in whorl 4. (B) A strong class A floral homeotic mutant, ap2-2. Two whorl 1 organs are carpel-like (arrow). The other two whorl 1 organs are still sepal-like. All whorl 2 organs are absent, and the whorl 3 consists of a single stamen. The whorl 4 carpels are similar to wild-type. (C) A class B floral homeotic mutant, pi-1. Organs in the outer two whorls are all sepals (arrowhead indicates a whorl 2 sepal). Organs in the inner two whorls are all carpels (arrow indicates a whorl 3 carpel). (D) A class C homeotic mutant, ag-1. Whorl 1 consists of four sepals; whorl 2 consists of four petals; whorl 3, however, is converted into six petals; and whorl 4 is a new flower with similar sepal, petal, petal arrangement (not shown in this picture). (E) A flower from a transgenic plant that ectopically expresses both B and C class genes: AP3, PI, and AG (Krizek and Meyerowitz 1996a). (F) A flower from a transgenic plant that ectopically expresses AP3 and PI and, at the same time, carries the ag-3 mutation (Krizek and Meyerowitz 1996a). Photos of (E) and (F) are gifts of Beth Krizek.

the establishment of the ABC model (Bowman et al. 1991b; Coen and Meyerowitz 1991; Weigel and Meyerowitz 1994). The ABC model places floral homeotic mutants into one of the three classes: A, B, or C and thus defines three classes of gene activities (Fig. 2.1B). In the outermost (first) whorl, the activity of the A class genes specifies the development of sepals. In whorl 2, where both A and B class genes are active, petal development is specified. In whorl 3, B and

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C activities together specify stamen identity, and in the innermost (fourth) whorl, C activity alone specifies carpel development. Thus, the A and B activities are predicted to overlap in whorl 2, while B and C activities overlap in whorl 3. In contrast, the A and C activities are predicted to be present in their respective domains without overlap. To account for this, the model predicts that the A and C activities are antagonistic to each other. The A activity in the first two whorls inhibits expression of C genes and vice versa. This tenant of the model is supported by the altered organ identity displayed in the A class homeotic mutants in which the pattern of organs in whorls one through four is carpel, stamen, stamen, carpel, respectively. This is consistent with the postulate that the C activity has spread to all four whorls (Fig. 2.1C). In C class mutants, the A activity is proposed to spread throughout all four whorls (Fig. 2.1E). Since the primary function of the ABC genes is to specify floral organ identity, the ABC class floral homeotic genes are also termed the “organ identity genes.” Molecular genetic analyses of homeotic mutations in the fruit fly Drosophila melanogaster indicated that homeotic genes encode master regulatory proteins. These transcriptional regulators control developmental programs that cooperate to generate a particular organ type (Gehring and Hiromi 1986). The molecular isolation of members of the ABC class floral homeotic genes indicates that these genes also function as master regulators of organ specific developmental programs (Weigel and Meyerowitz 1994). The ABC genes all encode DNA-binding transcription factors. Furthermore, in situ examination of their RNA expression patterns have largely supported the basic tenets of the ABC model. Their expression domains largely coincide with the domains of their activity as predicted by the ABC model. For example, the A class gene APETALA1 (AP1) is expressed in the first two whorls, while the C class gene AGAMOUS (AG) is expressed in the third and fourth whorls (Drews et al. 1991; Mandel et al. 1992b). Furthermore, the expression of the C class gene AG is expanded throughout all four whorls in the floral meristems of some A class mutants (Drews et al. 1991). Hence, the spatially restricted function of the ABC genes is largely regulated at the RNA level. There are, however, exceptions. For example, the A class gene APETALA2 (AP2) is expressed in all floral whorls although its activity is limited to whorls 1–2 (Jofuku et al. 1994). In this case, AP2 activity is likely regulated by post-transcriptional mechanisms. Clearly, one of the next major challenges in the field of flower pattern formation is to elucidate how the spatially and temporally restricted ABC activities are regulated. Currently, most investigations in this area of research are conducted in Arabidopsis, which will be the focus of this review.

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B. The MADS-domain Multi-gene Family With the exception of AP2, all other ABC genes encode a highly conserved 56 amino acid domain called the MADS-domain and thus are members of a multi-gene family (Riechmann and Meyerowitz 1997a). The name of the MADS-domain was derived from the four founding members: MCM1, yeast; AG, Arabidopsis; DEFICIENS (DEF), Antirrhinum; and SRF, human. The basic N-terminal half of the MADS domain is essential for DNA binding and the C-terminal half of the MADS domain is required for dimerization (Riechmann et al. 1996b). In the majority of plant MADS domain-containing proteins, a second conserved domain, the K box, was identified because of its similarity to the coiled-coil domain of keratin (Ma et al. 1991). The distinctive feature of the K box is the disposition of hydrophobic residues with a spacing that permits the formation of amphipathic α-helices (Ma et al. 1991; Pnueli et al. 1991). Between the MADS domain and the K box is a less strictly conserved linker (L) region. Amino acids in the L region and the K box have been shown to be important for the partner specificity in dimer formation (Riechmann et al. 1996b). MADS-domain proteins function as dimers and bind to a core consensus site CC(A/T)6GG, which is known as the CArG-box (SchwarzSommer et al. 1992; Wynne and Treisman 1992; Huang et al. 1993; Shiraishi et al. 1993). However, different MADS-domain family members can possess related but distinct DNA-binding specificity (Nurrish and Treisman 1995). Nevertheless, functional specificity (i.e. distinct organ identity activity) of the MADS-domain proteins is independent of their DNA-binding specificity. For example, hybrid genes were generated by swapping the amino terminal half of the MADS domain of the Arabidopsis proteins AP1, AP3, PI, and AG with the corresponding portion of human MEF2A or SRF proteins. Such hybrid proteins, having acquired the in vitro binding specificity of MEF2A or SRF, are able to perform the specific functions of the corresponding Arabidopsis genes in transgenic plants (Riechmann and Meyerowitz 1997b; Krizek and Meyerowitz 1996b). Thus, interactions between these MADS proteins with additional cofactors are probably crucial for the specific organ identity functions. Although AP3 and PI can each dimerize with AG or AP1 in vitro, these complexes do not bind to CArG boxes (Riechmann et al. 1996b). Only AP3/PI heterodimers can bind to the CArG boxes. This partner specificity of AP3 and PI suggests that the combinatorial mode of action between A and B genes in whorl 2 and between B and C in whorl 3 is not achieved through direct interactions between A and B proteins in

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whorl 2 or interaction between B and C proteins in whorl 3 (Riechmann et al. 1996a). For example, petal specific gene expression in whorl 2 does not appear to result from the activity of AP1/PI or AP1/AP3 heterodimers. Rather petal identity in whorl 2 is specified by the interaction of genes regulated by AP1 homodimers and genes regulated by AP3/PI heterodimers. C. The A Class Genes: AP2 and AP1 A class genes are defined as those required to specify sepal and petal identity. Interestingly, all A class genes appear to have other functions in addition to organ identity specification. AP2 has at least two functions: the specification of sepal and petal identity and the repression of AG RNA expression in whorls 1–2 (Bowman et al. 1991a; Drews et al. 1991). In strong loss-of-function ap2 mutants, when both functions of AP2 are defective, AG expression is extended to whorls 1–2, causing carpelloid structures in whorl 1 and staminoid petals or loss of petals in whorl 2 (Fig. 2.1C; Fig. 2.2B). Further, AG was found to be expressed precociously at earlier stages and at elevated levels, which may be responsible for the loss of floral organs in strong ap2 mutants (Fig. 2.2B). Unlike other ABC genes, AP2 is unique in that it encodes a novel, putative nuclear protein with two 68 amino acid repeat sequences, dubbed the AP2 domain. The AP2 domain has been predicted to perform functions of protein-protein dimerization and DNA-binding (Jofuku et al. 1994). AP2 is a member of a multi-gene family (Riechmann and Meyerowitz 1998). Studies with other family members such as the ethyleneresponsive element binding proteins (EREBPs) demonstrated that the AP2-domain recognizes and binds to DNA specifically in an 11-bp sequence (TAAGAGCCGCC), the GCC box (Ohme-Takagi and Shinshi 1995). Hence, AP2-domain containing proteins define a novel class of plant transcription factors. AP2 mRNA is detected in all floral whorls as well as in vegetative tissues, indicating that the spatially restricted activity of AP2 in whorls 1–2 must depend on post-transcriptional regulation. AP1 is another A function floral organ identity gene. Unlike AP2, AP1 does not negatively regulate AG expression in whorls 1–2 (GustafsonBrown et al. 1994). In ap1 mutants, whorl 1 organs are bract/leaf-like; whorl 2 organs are usually absent; whorls 3–4 are usually normal (Irish and Sussex 1990). Thus, AP1 activity is required for the development of sepals and petals. Unique to ap1 mutants is the formation of secondary flowers in the axils of first whorl bract/leaf-like organs, suggesting that ap1 mutant flowers partially adopt the fate of inflorescence shoots (Irish and Sussex 1990). This second defect of ap1 in flower/shoot

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transition will be discussed in a later section of this review. AP1 encodes a MADS box protein and AP1 mRNA is initially expressed throughout the floral meristem and later becomes restricted to whorls 1–2 (Mandel et al. 1992b). The spatially-restricted expression of AP1 at later stages is consistent with its role as an A class gene (Bowman et al. 1993; Mandel et al. 1992b; Gustafson-Brown et al. 1994). D. The B Class Genes: AP3 and PI APETALA3 (AP3) and PISTILLATA (PI) are the Arabidopsis B class genes. Mutations in either AP3 or PI cause similar homeotic transformations in whorls 2–3 such that second whorl organs develop as sepals and third whorl organs develop as carpels (Fig. 2.1D; Fig. 2.2C; Bowman et al. 1989; Hill and Lord 1989). AP3 and PI both encode MADS domain proteins that have been shown to bind DNA only as AP3/PI heterodimers (Jack et al. 1992; Goto and Meyerowitz 1994; Riechmann et al. 1996a; Riechmann et al. 1996b; Hill et al. 1998; Tilly et al. 1998). Obligatory heterodimer formation explains why both AP3 and PI are required to specify petal and stamen identity. Ectopic expression studies involve artificially expressing a gene in a new spatial or temporal domain, in which the gene is normally not expressed. The 35S promoter from the cauliflower mosaic virus (CaMV) is a constitutive plant promoter that is frequently used to drive the expression of genes in all tissues and at all developmental stages. If AP3 and PI together are sufficient to confer the B class activity, then ectopically expressing AP3 and PI in all four floral whorls would result in a flower of petals in the outer two whorls and stamens in the inner two whorls. Transgenic plants that constitutively and simultaneously express both AP3 and PI under the direction of the 35S promoter develop flowers that have petals in whorls 1–2 and stamens in whorls 3–4 (Jack et al. 1994; Krizek and Meyerowitz 1996a). Ectopic expression of both B class and C class genes led to the production of Arabidopsis flowers with stamens in all four whorls (Fig. 2.2E; Krizek and Meyerowitz 1996a). Thus, AP3 and PI together are both necessary and sufficient for the B activity within the context of a flower. AP3 and PI initially are not expressed in identical domains. AP3 mRNA is detected in whorls 2–3 plus in a small number of cells at the base of the first whorl (Weigel and Meyerowitz 1993; Tilly et al. 1998), while PI RNA is detected in whorls 2–4 (Goto and Meyerowitz 1994). At later stages of flower development, the expression of both genes is restricted to petals and stamens. Maintenance of this later expression in petals and stamens requires the functional activity of both AP3 and PI,

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suggesting a system of autoregulation (Jack et al. 1992; Goto and Meyerowitz 1994). Presumably, auto- and cross-regulation of AP3 and PI are responsible for the similar expression domains of AP3 and PI in later stages of flower development. E. The C Class Genes: AG, HUA1, and HUA2 AG was the first C class gene identified and isolated (Bowman et al. 1989; Bowman et al. 1991b; Yanofsky et al. 1990). AG plays a key role in specifying stamen and carpel identity. In ag loss-of-function mutants (Fig. 2.1E; Fig. 2.2D), the A activity is expanded into whorls 3–4, where stamens are replaced by petals, and carpels are replaced by a new flower. This results in a floral pattern of sepal, petal, petal, (sepal, petal, petal)n. The generation of flowers within a flower reveals a second role of AG: to maintain the determinacy of the floral meristem. AG encodes a MADSdomain containing protein (Yanofsky et al. 1990). As predicted by the ABC model, AG mRNA is detected in the inner two whorls during early floral stages. AG mRNA continues to be expressed in stamens and carpels during later stages and eventually becomes restricted to specific cell types within the stamens and carpels (Bowman et al. 1991a; Drews et al. 1991). Ectopic expression of AG under the 35S promoter in transgenic Arabidopsis or tobacco plants causes homeotic conversion from sepals into carpels and from petals into stamens (Mizukami and Ma 1992; Mandel et al. 1992a). Thus, AG appears not only necessary but also sufficient to specify stamen and carpel identity within the context of a flower. In summary, AG has at least three functions: repressing A class gene activity in whorls 3–4, specifying stamen and carpel organ identity, and maintaining the determinacy of floral meristems. For many years, it was thought that AG was the only C class gene because AG alone appeared sufficient to specify C activity, and several mutageneses only yielded additional ag alleles without identifying mutations in other genes that confer similar phenotypes. However, genetic redundancy and/or embryonic or seedling lethality may have prevented the isolation of mutations in additional C class genes. Chen and Meyerowitz (1999) searched for new C class genes by looking for mutations that enhanced the phenotype of the weak ag-4 allele. Flowers of ag-4 plants can still make stamens in the third whorl because of residual activity of the mutant ag gene product (Sieburth et al. 1995; Chen and Meyerowitz 1999). Mutations that enhance the ag-4 phenotype are expected to convert the third whorl stamens into petals, as is seen in the stronger ag alleles. Chen and Meyerowitz (1999) successfully

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isolated two new C class genes, HUA1 and HUA2 (Hua means “flower” in Chinese). Either one of the hua1 or hua2 mutations weakly enhances ag-4, while hua1 hua2 double mutants strongly enhance the ag-4 phenotype. Both hua1 and hua2 single mutant flowers are phenotypically normal. Flowers of the hua1 hua2 double mutants show a weak stamen and carpel phenotype. In the first few flowers, lateral stamens are petaloid, and the gynoecia are enlarged toward their tips and constricted along their sides. The lack of phenotype in hua1 and hua2 single mutants explains why they were only recovered in genetic screens when AG activity is compromised. Genetic analyses indicated that HUA1 and HUA2 share a redundant role with AG in all aspects of AG function: repression of A class gene expression, stamen and carpel identity specification, and regulation of floral determinacy. HUA1 and HUA2 act in parallel or together with AG in the specification of C activity because AG mRNA expression is not affected in the hua1 or hua2 mutants. Similarly, HUA2 expression is not altered in ag mutants. Since HUA2 encodes a novel protein that contains multiple nuclear localization signals and additional motifs, it is likely that HUA2 is a transcription factor and acts as a cofactor of the AG gene. F. Novel Class Genes: SEP1, SEP2, and SEP3 Recently, another class of floral homeotic mutants has been described (Pelaz et al. 2000). This new class is encoded by three genes: SEPALLATA1 (SEP1), SEP2, and SEP3. All three genes are MADS-box containing genes and were isolated based on their sequence similarity to AG. In fact, SEP1, SEP2, and SEP3 were previously named AGAMOUS-LIKE2 (AGL2), AGL4, and AGL9, respectively (Ma et al. 1991; Mandel and Yanofsky 1998). A reverse genetic approach was used to identify mutations in SEP1, SEP2, and SEP3. Reverse genetics refers to a variety of techniques that can be used to generate mutations in a particular gene whose sequence is known. Since we have now entered an era of genomics, the large number of DNA sequences available in Arabidopsis, rice, tomato, and a few other plant species allow the discovery of many genes and gene families. However, in a majority of the cases, the function of these genes remains unknown. Thus, reverse genetic approaches are now more frequently employed and are crucial to illuminate gene function. These approaches are labeled “reverse” because they lead from gene sequence to mutation in the opposite direction of the typical “forward” genetic approach that leads from mutation to gene sequence.

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In the reverse genetic approach employed by Pelaz et al. (2000), polymerase chain reaction (PCR) amplification was used to identify transposable element insertions within the SEP gene of interest. After generating individual mutations in each of the SEP genes, phenotypic analyses indicated that mutations in any one of the SEP genes alone cause only subtle phenotypes. However, sep1 sep2 sep3 triple mutants displayed a striking phenotype in which all floral organs in the first three whorls were sepals or sepal-like organs. The fourth whorl was converted into a new flower that repeats this same floral pattern (Pelaz et al. 2000). The phenotype displayed by the sep1 sep2 sep3 triple mutant is very similar to “bc” double mutants (such as pi ag or ap3 ag), suggesting that SEP1, SEP2, and SEP3 are required for B and C class gene expression or for their activity. Because of redundancy among SEP1, SEP2, and SEP3, removing one of these SEP genes by mutations normally would not reveal such a requirement. SEP1, SEP2, and SEP3 are all expressed just prior to the expression of the B and C class genes and are expressed throughout whorls 2–4 (SEP1 and SEP2 are also expressed in whorl 1 in young flowers) (Pelaz et al. 2000). The initial patterns of B and C class gene expression are not altered in the sep1 sep2 sep3 triple mutants, suggesting that SEP1, SEP2, and SEP3 are not required for the initiation of B or C class gene expression. Thus, it has been suggested that SEP1, SEP2, and SEP3 regulate B and C class genes post-transcriptionally. One possible mechanism for this post-transcriptional regulation is by a direct interaction between the SEP1, SEP2, and SEP3 gene products and the B and C class gene products. Yeast two-hybrid assays have revealed a number of interactions between SEP proteins and B and C gene products (Fan et al. 1997).

II. CONSERVATION OF THE ABC FUNCTIONS IN ANGIOSPERMS The ABC genes provide an excellent opportunity to understand the evolutionary conservation and divergence of floral development in angiosperms. Studies carried out in both dicot and monocot plants suggest that the basic genetic mechanisms that determine floral organ identity are conserved across angiosperms. For most of the ABC genes, cognate homologs (orthologs) can be identified in diverse angiosperm species. For some of these homologs, functional analogy to corresponding Arabidopsis genes has been confirmed (see Table 2.1) by studying loss-of-function mutants, transgenic plants ectopically expressing these

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Table 2.1. ABC genes in different plants. Only those genes are included that exhibit functional homology with corresponding Arabidopsis genes except ZAP1 and ZMM2 in Maize and BLIND in Petunia. Class

Arabidopsis

Antirrhinum

Petunia

Maize

Rice

A

APETALA1 APETALA2 APETALA3 PISTILLATA AGAMOUS

SQUAMOSA — DEFICIENS GLOBOSA PLENA

— BLINDy GREEN PETAL FBP1, PMADS2 pMADS3, FBP6

ZAPz — SILKY1 — ZAG1, ZMM2z

— — OsMADS16 OsMADS4 OsMADS3

SEP1, 2, 3

—

FBP2

—

LHS1

B C Novel (BC) z

Only sequence and expression data are available (Mena et al., 1995). Only genetic data are available (de Vlaming et al., 1984; Tsuchimoto et al., 1993).

y

homologs, transgenic plants expressing antisense genes to corresponding homologs, or functional complementation of Arabidopsis mutants with homologs isolated from different species. One major theme from these studies (mostly on the B and C class genes) is that organ identity genes in different species differ both in the number of genes involved (due to gene duplication) and in the distribution of functional roles among these duplicated genes. Because dicots and monocots are on separate branches of the angiosperm phylogenetic tree, the conservation of the ABC model in dicot and monocot species suggests that the ABC model represents an ancient regulatory network that in all likelihood is generally applicable to most angiosperms. In this review, we will focus on studies where functional data on ABC gene homologs are available. For a more extensive review of angiosperm flower development, see Irish and Kramer (1998). For reviews on grass species, see Schmidt and Ambrose (1998) and Ma and dePamphilis (2000). A. Dicotyledonous Species Like Arabidopsis, Antirrhinum majus is a dicot plant that has been extensively studied and has contributed greatly to the establishment of the ABC model. Although Arabidopsis (Brassicaceae) and Antirrhinum (Scrophulariaceae) are widely divergent dicot species and belong to different subclasses, the phenotype of the corresponding Antirrhinum mutants (Carpenter and Coen 1990; Schwarz-Sommer et al. 1990) are quite similar to Arabidopsis mutants (Coen and Meyerowitz 1991). DEF and GLO, orthologs of AP3 and PI, respectively, are both required for B function in Antirrhinum (Sommer et al. 1990; Trobner et al. 1992). How-

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ever, the expression domains of these orthologs are switched. In Arabidopsis, PI is expressed early in whorls 2–4, while AP3 is expressed in whorls 1–3. In Antirrhinum, DEF is expressed early in whorls 2–4, while GLO is expressed largely in whorls 2–3 (Schwarz-Sommer et al. 1992; Trobner et al. 1992). Despite the difference in expression, AP3 and DEF are functionally conserved as demonstrated by cross species complementation (Irish and Yamamoto 1995; Samach et al. 1997). Similar expression studies in several other species showed that the DEF expression pattern represents an ancestral condition, while the AP3 expression pattern represents an exception rather than the rule (Irish and Kramer 1998). Based on sequence similarity, there are two petunia PI-like genes, FBP1 and pMADS2 (Angenent et al. 1992, 1994; Kush et al. 1993). Consistent with it being a B function gene, FBP1 was shown to be required for petal and stamen development in co-suppression experiments (Angenent et al. 1992, 1994). However, pMADS2 appears to encode a redundant function, since loss of pMADS2 function has no phenotypic effect (van der Krol et al. 1993). The petunia AP3 homolog, pMADS1 is also called GREEN PETAL (GP), because mutations in this gene cause a homeotic conversion from petals to green sepals but do not affect stamen development (van der Krol et al. 1993). Furthermore, ectopic expression of pMADS1/GP under the control of the 35S promoter in wild-type plants resulted in partial conversion of sepals into petaloid organs, but had no effect on stamen identity (Halfter et al. 1994). The fact that pMADS1/GP is not required for stamen development suggests that petunia may have another, as yet unidentified, AP3 homolog specifying the B function in whorl 3. Thus, petunia differs from Arabidopsis and Antirrhinum both in an increased number of B class genes and in the limitation of B class gene activity to only one whorl. AG orthologs from various species are more conserved than are the B class genes, both with respect to DNA sequence and gene function. In Antirrhinum, PLENA (PLE) is considered the functional ortholog of AG (Table 2.1), as both the expression pattern and the function of PLE are similar to AG in Arabidopsis (Bradley et al. 1993). Petunia has two AGlike genes, pMADS3 and FBP6, both of which appear to have expression patterns consistent with a reproductive function (Tsuchimoto et al. 1993; Angenent et al. 1995; Kater et al. 1998). Ectopic expression of pMADS3 either via 35S promoter in transgenic plants or via gain-offunction alleles led to the homeotic conversion of whorl 1 and whorl 2 generating organs with carpelloid and staminoid features, respectively. However, ectopic expression of FBP6 did not lead to homeotic transformations (Kater et al. 1998).

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The Antirrhinum ortholog of the A class gene AP1 is SQUAMOSA (SQUA). SQUA, like AP1, plays a role both in meristem identity and organ identity; however, its role in organ identity determination is rather limited, as flowers of squa mutants are often nearly wild type (Huijser et al. 1992). The Antirrhinum mutant ovulata exhibits a phenotype similar to that of ap2. However, ovulata is a gain-of-function allele of the C class gene PLE that is caused by a transposon insertion into the second intron of PLE and may have disrupted binding sites for negative regulators (Bradley et al. 1993). Two genes STYLOSA (STY) and FISTULATA (FIS) together control the restriction of the C gene PLE to the inner whorls of the flower. Genetic and expression studies, however, indicated that the effect of STY and FIS on PLE expression is indirect and that STY and FIS are more general regulators of flower development (Motte et al. 1998). In petunia, the blind (bl) mutants exhibit a phenotype similar to A class mutants, which includes homeotic conversion from sepals to carpelloid sepals and petals into antheroid structures (de Vlaming et al. 1984; Angenent et al. 1992; Tsuchimoto et al. 1993). In addition, the petunia C class gene pMADS3 was ectopically expressed in bl mutants (Tsuchimoto et al. 1993), indicating that BL, like AP2, is a negative regulator of C class genes. However, the molecular nature of BL is unknown. Orthologs of Arabidopsis AP2 have not been identified in Antirrhinum, petunia, or other species (Table 2.1). The tomato TM5 gene and its petunia homolog FBP2 are expressed in the inner three whorls, all of which are defective when TM5 or FBP2 are inactivated by antisense constructs or co-suppression (Angenent et al. 1994; Pneuli et al. 1994). Although the phenotypes in tomato and petunia are slightly different, in both cases, petals are transformed into sepals or leaf-like organs, and additional whorls of organs or new flowers can develop in the center of the flower. This phenotype resembles the Arabidopsis sep1 sep2 sep3 triple mutants (Pelaz et al. 2000). Sequence analyses indicate that indeed SEP1, SEP2, and SEP3 are most closely related to TM5 and FBP2 (Purugganan et al. 1995). The presence of three redundant SEP genes in Arabidopsis suggests that tomato and petunia may also possess multiple genes for the same function. The antisense and co-suppression approaches used to knock out TM5 and FBP2 might have simultaneously abolished the activity of other redundant genes of TM5 or FBP2 due to their high levels of sequence similarity. B. Monocotyledonous Species Maize (Zea mays) is a monocot grass species that has been extensively characterized resulting in the development of a host of useful molecular genetic tools. In particular, transposon tagging and other reverse

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genetic approaches are available. A number of maize MADS box genes have been isolated and their functions have been assayed by reverse genetic, as well as forward genetic, approaches. Grass flowers have stamens and carpels, but lack obvious sepals and petals. Instead, grass-specific organs known as glumes, lemma, palea, and lodicules surround the stamens and carpels. Recent studies of a maize mutant silky1 provides compelling developmental evidence for recognizing lodicules as modified petals, and possibly, palea and lemma as modified sepals (Ambrose et al. 2000). SILKY1 encodes an AP3-like gene in maize. Homeotic transformations of stamens to pistils and lodicules to organs resembling lemma/palea are exhibited in silky1 mutants. In situ hybridization indicates SILKY1 is localized to lodicule and stamen primordia. Thus genetic, morphological, sequence, and expression data all support that SILKY1 is a B function gene (Ambrose et al. 2000; Ma and dePamphilis 2000). The maize C class gene ZAG1 was identified first by sequence homology to AG (Schmidt et al. 1993). Subsequently, reverse genetic approaches created a putative null allele of zag1 (Mena et al. 1995). While the ABC model would predict a loss of both reproductive organ development and floral meristem determinacy, only the later phenotype was evident, with supernumerary carpels being reiterated within the zag1 florets. Although ZAG1 is expressed in both stamens and carpels, the zag1 mutation does not affect stamen development. One possible explanation is the existence of partially redundant C class genes in maize. Indeed, ZMM2, a gene closely related to ZAG1, has been isolated (Mena et al. 1995; Schmidt and Ambrose 1998). The expression pattern, sequence, and map position of ZMM2 all suggest that it is a duplicate gene with activities that are non-identical, but partially overlapping with those of ZAG1. Consistent with the genetic analyses, ZAG1 is more highly expressed in carpels and ZMM2 more highly expressed in stamens. Thus, it is highly likely that in maize the AG-like activity is shared by two genes. Several MADS box genes that play important roles in controlling flower development in rice have also been studied. Using antisense experiments, Kang et al. (1998) demonstrated that the rice MADS box genes OsMADS3 (Oryza sativa MADS box gene 3) and OsMADS4 are putative orthologs of AG and PI respectively. OsMADS16 gene has been proposed as a homolog of AP3 based on its amino acid sequence similarity to AP3, its expression pattern, and its interaction with OsMADS4 in yeast (Moon et al. 1999). The leafy hull sterile 1 (lhs1) mutations display mutant phenotypes similar to transgenic plants expressing a dominant-negative mutant form of OsMADS1, suggesting that the lhs1 mutation may be defective in the OsMADS1 gene (Kinoshita et al. 1976; Jeon et al. 2000). The fact that

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wild-type OsMADS1 can rescue lhs1 mutants further indicates that OsMADS1 and LHS1 encode the same gene (Jeon et al. 2000). Strong lhs mutants exhibit leafy palea and lemma, and partial transformation from lodicules to leafy palea and lemma. Additional carpels and a new flower may be generated. This mutant phenotype and the sequence similarity between LHS1 (OsMADS1) and TM5 and FBP2 suggests that LHS1 may encode a functional homolog of TM5, FBP2, and the Arabidopsis SEP genes. However, the phenotype of lhs also suggests that LHS1 has an additional role during the development of palea and lemma during late stages of flower development. In this respect LHS is similar to AP1.

III. POSITIVE REGULATORS OF FLORAL ORGAN IDENTITY GENES A. Meristem Identity Genes: LFY and AP1 At the beginning stages of Arabidopsis development, the primary shoot produces leaves with axillary second order shoots. Later, at the transition to reproductive phase, the primary shoot switches to producing flowers. Two genes LEAFY (LFY) and AP1 are necessary and sufficient for this developmental switch (Irish and Sussex 1990; Huala and Sussex 1992; Weigel et al. 1992; Bowman et al. 1993; Mandel and Yanofsky 1995; Weigel and Nilsson 1995). Loss-of-function mutations in these two genes cause the conversion (to varying degrees) from flowers to second order shoots. Conversely, constitutive expression of either LFY or AP1 cause the conversion from shoots to flowers. Thus LFY and AP1 are referred to as “meristem identity genes.” Specifically, in lfy mutants, flowers are replaced by leaves and second order shoots. In ap1 mutants, (leaf-like) bracts develop in the first whorl and secondary flowers develop in the axils of first whorl floral organs. Most strikingly, ap1 enhances the defects of lfy. In lfy ap1 double mutants, leaf-like organs arise in a spiral fashion (a feature of shoot) rather than whorled fashion (a feature of flowers), and all flowers are replaced by shoot-like structures. Thus, LFY and AP1 have partially redundant roles in floral meristem identity specification, with LFY playing a more prominent role than AP1. For more specific reviews regarding floral inductive pathways leading to the activation of LFY and AP1, see Yanofsky (1995), Koornneef (1997), Weigel (1997 and 1998), Ma (1998), and van Nocker (2001). Although the LFY protein does not share sequence homology with other known families of DNA-binding proteins, LFY can bind DNA in a sequence specific manner and stimulate transcription in yeast cells

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when fused to a heterologous activation domain (Weigel et al. 1992; Parcy et al. 1998). Thus, LFY may activate the floral program by transcriptional activation of target genes involved in flower development. This hypothesis is consistent with LFY RNA and protein expression that precedes the transcriptional activation of ABC genes. Specifically, LFY mRNA is expressed in early floral meristem, and is transiently expressed in sepal, petal, stamen, and carpel primordia (Weigel et al. 1992). The LFY protein is expressed in a similar pattern and localizes to the nucleus, consistent with a role in transcriptional activation (Parcy et al. 1998). To further understand the regulatory relationship between meristem identity genes and ABC genes, Weigel and Meyerowitz (1993) examined ABC gene expression in lfy and ap1 single and double mutants. They found that LFY and AP1 are positive regulators of ABC gene transcription. In lfy mutants, early AG expression is delayed, and the initial expression domain is smaller than in wild-type flowers. The expression of AG mRNA in ap1 mutants is relatively normal, however, AG RNA entirely fails to accumulate in the center of ap1 lfy double mutant flowers. Therefore, AG expression is more strongly affected in the ap1 lfy double mutants than in either single mutant. Similarly, both the amount and the domain of expression of AP3 and PI are severely reduced in strong lfy mutants. The function of AP1 in activating AP3 and PI only becomes obvious when LFY activity is reduced or eliminated as shown in lfy ap1 double mutants. Like B and C class genes, the expression of AP1 is delayed and reduced in lfy mutants (Ruiz-Garcia et al. 1997; Liljegren et al. 1999). Since AP2 RNA is detected in a variety of non-floral tissues, including leaves and stems, AP2 transcription is likely regulated independently of the meristem identity genes (Jofuku et al. 1994). B. LFY, a Direct Activator of AP1 The dual roles of AP1 as a meristem identity gene and an A class organ identity gene correlate well with its two phases of expression. AP1 is initially expressed in the entire floral meristem and later becomes restricted to the first two whorls (Bowman et al. 1993; Mandel et al. 1992b; Gustafson-Brown et al. 1994). This expression pattern correlates well with an early function of AP1 in meristem identity specification and a later function of AP1 in sepal and petal identity specification. LFY is an obvious candidate activator of AP1 expression as AP1 RNA expression in the floral meristem is initiated soon after LFY is first detected and AP1 RNA expression is delayed and reduced in lfy mutants (Ruiz-Garcia et al. 1997; Liljegren et al. 1999).

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Evidence that LFY can directly activate AP1 comes from experiments in which constitutive and ectopic expression of LFY under the 35S promoter led to precocious expression of AP1 in young seedlings (Parcy et al. 1998). Thus the role of LFY in activating AP1 can be separated from its role in floral meristem specification. Consistent with the idea that AP1 expression is directly regulated by LFY, in vitro DNA-binding assays showed that a high affinity binding site for LFY is present in a minimal AP1 promoter. Additionally, LFY, upon fusion to a strong transcriptional activation domain, can activate the expression of a reporter gene in yeast, which is under the control of an AP1 promoter (Parcy et al. 1998). Using a post-translational inducible system, Wagner et al. (1999) demonstrated that AP1 is an immediate target of LFY. Specifically, a steroid hormone-inducible LFY switch was constructed. This construct (35S::LFY-GR) uses the CaMV-35S promoter to express the LFY coding sequence that has been fused to a glucocorticoid receptor (GR) hormone binding domain. In the absence of the steroid hormone dexamethasone, the LFY-GR fusion protein is held in the cytoplasm and is non-functional. In the presence of dexamethasone, the LFY-GR fusion protein moves to the nucleus and is able to perform its function as a transcriptional activator. As the translocation of the LFY-GR protein into the nucleus does not depend on protein synthesis, a direct effect of LFY on its target gene transcription can be evaluated in the presence of cyclohexamide (a protein synthesis inhibitor). The LFY-GR protein was able to rescue defects of AP1 expression at early stages even in the presence of cyclohexamide, indicating that LFY directly activates AP1 at early stages. However, the ability of LFY-GR to rescue defects of AP1 expression during later stages is dependent on protein synthesis. Thus, the two phases of AP1 expression appear to be controlled by different regulatory mechanisms. While LFY directly activates AP1 expression during early stages of flower development, the effect of LFY on AP1 expression is indirect at later stages. C. LFY, a Direct Activator of AG Despite the knowledge that LFY and AP1 are required for AG expression, it has been difficult to determine if the effect of LFY and AP1 on AG expression is direct or indirect. For example, a failure to activate AG might simply result from the fact that the shoot structures formed in lfy or lfy ap1 mutants never acquire any floral identity. Fortunately, a gainof-function LFY protein LFY:VP16 is able to separate the role of LFY in meristem identity specification from the role of LFY in regulating AG

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(Parcy et al. 1998). Specifically, LFY:VP16 is a fusion protein between LFY and a strong transcriptional activation domain from the Herpes Simplex Virus (HSV) VP16 protein, generating a form of LFY that is constitutively active with respect to transcriptional activation. The LFY:VP16 fusion protein was expressed in developing plants from the endogenous LFY promoter, and thus the expression pattern of LFY:VP16 mimics the expression of the wild-type LFY protein. Transgenic plants harboring the LFY:VP16 construct initiate floral meristem formation normally; however, the floral organs display homeotic transformations, where sepals are transformed into carpels in whorl 1 and petals are transformed into stamens in whorl 2. In situ hybridization showed that these LFY:VP16 plants exhibit both ectopic and precocious AG RNA expression. When the LFY:VP16 plants were crossed to the strong loss-of-function ag-1 mutant, the floral organs in whorls 1–2 were largely restored to normal sepals and petals. Hence, ectopic and precocious AG expression is chiefly responsible for the abnormal floral organs formed in these LFY:VP16 plants. When the LFY: VP16 was ectopically expressed in developing seedlings under the control of the 35S promoter, seedlings were growth-arrested and AG was ectopically expressed (Parcy et al. 1998). The induction of ectopic AG expression in pre-flowering seedlings by LFY:VP16 further indicates that LFY:VP16 is able to activate AG in non-floral tissues. Thus, the effect of LFY:VP16 on AG is rather direct and does not require proper floral meristem formation. However, a 35S::LFY construct that drives the ectopic expression of the wild-type LFY protein is not sufficient to generate pre-flowering seedling expression of AG; rather the presence of the strong VP16 activation domain is required (Parcy et al. 1998). Two models were proposed to explain this observation. The first model postulates that there is a repressor that is present in whorls 1–2 of the floral meristem and in the vegetative tissues that normally prevents LFY from activating AG expression. In this model the enhanced transcriptional activity of the LFY:VP16 protein overcomes this repressor activity. Alternatively, in wild-type plants, the LFY protein is assisted by the action of a co-activator only expressed in the center of the floral meristem. Hence, AG is only activated by LFY in the center of a wild-type flower. According to this model, the strong transcriptional activity of LFY:VP16 can activate AG transcription independently of any co-activators. Analysis of the AG cis-regulatory sequences revealed that sequences within the second AG intron are necessary and sufficient for the wildtype AG expression pattern (Sieburth and Meyerowitz 1997; Busch et al. 1999). At least two redundant enhancers within the intronic sequences

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mediate the expression of AG (Busch et al. 1999). These enhancers are LFY responsive, as they respond appropriately to expression of the LFY:VP16 construct or to the loss of LFY activity in the lfy mutant. Using deletion analysis, one functional enhancer was found within a 230 base pair (bp) fragment of the intron. Within this fragment, two LFY binding sequences, that are 31 bp apart, were detected by gel shift analysis. These sequences are similar to each other and to previously defined LFY binding sites in the AP1 promoter (Parcy et al. 1998). Alterations to the binding site sequence that prevent the binding of LFY in vitro also cause the enhancer fragments to lose their responsiveness to both wild type LFY as well as LFY:VP16 proteins in vivo (Busch et al. 1999). These results suggest that LFY-dependent stimulation of AG expression requires the direct binding of LFY to the enhancer sequences of AG. In addition, two CArG boxes have been identified within the 3′ activation cis-element where a LFY binding site is also located (Deyholos and Sieburth 2000). These CArG boxes could serve as binding sites for MADS-domain proteins such as AP1. D. Two Phases of Regulation: Initiation and Maintenance of B Gene Expression The regulation of AP3 and PI can be divided into two phases: the early initiation phase and the later maintenance phase. These two different phases are regulated by distinct sets of genes. The early phase of AP3 and PI expression are positively regulated by meristem identity genes LFY and AP1 (Weigel and Meyerowitz 1993; Goto and Meyerowitz 1994). AP3 and PI expression is significantly reduced in strong lfy mutants, whose flowers lack petals and stamens. Hence, LFY is required to initiate AP3 and PI expression. Although, ap1 mutations alone have little effect on B class gene expression, lfy ap1 double mutants display no detectable AP3 and PI expression (Weigel and Meyerowitz 1993), suggesting that AP1 does play a role in B class gene activation. Activation of AP3 by LFY apparently relies on a mechanism different from those for the activation of AG and AP1. AP1 and AG can be activated in ectopic tissues in response to the ectopic expression of wild type LFY (in the case of AP1) or LFY:VP16 (in the case of AG). However, ectopic and constitutive expression of LFY by 35S::LFY or LFY::VP16 failed to activate AP3 ectopically. Apparently, B class genes require additional factors for their activation. Results from several experiments (see below) indicate that the activation of the B class genes requires the UNUSUAL FLORAL ORGANS (UFO) gene. When LFY and UFO are both constitutively expressed in seedlings (35S::LFY and 35S::UFO), they

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can induce AP3 expression in seedlings (Parcy et al. 1998). Thus, activation of AP3, like that of AP1 and AG, is not dependent on the formation of floral meristems. Similarly, flower-independent activation of PI is observed in seedlings where both LFY and UFO are expressed constitutively (Honma and Goto 2000). LFY-GR experiments that directly test the effect of LFY and UFO on AP3 or PI expression in vivo in the presence of protein synthesis inhibitors will be required to determine if LFY and UFO are immediate upstream activators of AP3 and PI. The maintenance (late) phase of AP3 and PI expression is dependent on the activity of AP3 and PI. The autoregulatory role of AP3 and PI is observed in ap3 and pi mutants where both AP3 and PI late phase expression is reduced while the early phase expression is not affected (Jack et al. 1994; Goto and Meyerowitz 1994). Three CArG boxes were identified between –90 to –180 of the AP3 promoter (Hill et al. 1998; Tilly et al. 1998). These same elements are necessary for maintaining AP3 expression in petals and stamens (Hill et al. 1998). AP3/PI heterodimers can bind to CArG box 1 and 3 in vitro in a sequence specific manner (Hill et al. 1998; Tilly et al. 1998). In addition, AP3-GR, which is activated by steroid hormone, can induce AP3 expression in the absence of de novo protein synthesis (Honma and Goto 2000). Thus, direct interaction between AP3/PI and the CArG boxes in AP3 promoter is responsible for maintaining late phase AP3 transcription. Interestingly, the promoter or intron sequences of PI do not contain any CArG box. This raised the possibility that AP3/PI heterodimers may not directly bind to PI cis-elements. Indeed, electrophoretic mobility shift assays (EMSA) failed to detect binding of AP3/PI to the proximal promoter element of PI. Using a similar AP3-GR system, it was found that the ability of AP3/PI heterodimer to activate PI transcription requires de novo protein synthesis (Honma and Goto 2000). Thus, in contrast to the direct autoregulation of AP3 by AP3/PI, autoregulation of PI transcription by AP3/PI is indirect and requires de novo synthesis of additional factor(s). Using a promoter fusion to the uidA reporter gene encoding bglucuronidase (GUS), the cis-regulatory region of AP3 and PI promoters have been dissected. Minimal promoters of about 727 bp for AP3 (Hill et al. 1998) and 498 bp for PI (Honma and Goto 2000) were identified that can direct the wild-type pattern of respective gene expression. Hence, these fragments contain all necessary cis-regulatory elements. Within 727 bp of the AP3 minimal promoter, multiple cis-acting elements were identified that control temporal and spatial subsets of the AP3 expression. Two elements for initiating early stage AP3 expression were identified; one is located proximally and the other is located

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distally within the promoter of AP3. The three CArG boxes described earlier exist within the proximal early element (Hill et al. 1998). In addition, a petal-specific element and two stamen-elements were identified that direct AP3 expression during maintenance phase (Hill et al. 1998; Tilly et al. 1998). Interestingly, genomic sequences of the AP3 and PI promoters do not show any sequence similarity (Honma and Goto 2000). Within the 498 bp PI promoter, multiple, discrete cis-regulatory elements were identified. The elements located in the distal region of the PI promoter are generally involved in the early initiation phase of PI expression while the element more proximal to the PI promoter is involved in the maintenance phase regulation. Thus, while cis-elements required for initiation and maintenance are located in distinct, separable regions of the PI promoter, the cis-elements required for initiation and maintenance of AP3 expression overlap. E. UFO, a Coregulator of B Gene Expression As described earlier, UFO activity is essential for B gene expression and ufo mutants exhibit floral organ identity defects that are similar to partial loss-of-function lfy and to B class mutants (Wilkinson and Haughn 1995; Levin and Meyerowitz 1995). In ufo mutants, AP3 and PI RNA level is reduced (Wilkinson and Haughn 1995; Levin and Meyerowitz 1995; Hill et al. 1998). Constitutive expression of UFO under the control of 35S promoter results in precocious and ectopic activation of AP3 and PI in flowers, confirming a positive regulatory role of UFO for B gene expression (Lee et al. 1997; Honma and Goto 2000). Consistent with its role as an upstream activator, UFO RNA accumulates in the floral meristem before the onset of AP3 expression. UFO is expressed initially throughout the entire floral meristem, but later is restricted to whorls 2–3 (Ingram et al. 1995; Lee et al. 1997). Thus, the presence of both LFY and UFO in whorls 2–3 might be necessary to initiate B gene expression. What is the relationship between LFY and UFO in activating AP3? Clearly, the simple hierarchical models of UFO acting downstream of LFY or LFY acting downstream of UFO are inconsistent with the following observations. First, LFY and UFO expression is activated independently (Levin and Meyerowitz 1995; Lee et al. 1997). Second, constitutive expression of LFY fails to rescue ufo mutants and, conversely, constitutive expression of UFO fails to rescue lfy mutants (Weigel and Nilsson 1995; Lee et al. 1997). In other words, the ability of the 35S::UFO construct to activate B class gene expression is dependent on wild-type LFY activity (Lee et al. 1997) and vice versa. Based on these

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data, a model has been proposed in which UFO and LFY are coregulators of B genes. LFY is responsible for general floral-specific activation of B gene transcription and UFO confers regional specificity to whorls 2–3 (Lee at al. 1997). The UFO gene encodes a protein with a F-box motif (Ingram et al. 1995). F-box proteins are part of the heteromeric ubiquitin ligase complex known as SCF (Skp1, Cdc53/cullin, F-box), which plays a key role in the degradation of a variety of regulatory proteins (Patton et al. 1998). The F-box protein in each SCF complex probably acts as a receptor to recruit specific protein targets for degradation. UFO has been shown to interact in vitro with Arabidopsis Skp1-like proteins ASK1 and ASK2 (Samach et al. 1999), supporting the idea that UFO may be a component of the SCF complex. In addition, ask1 mutants exhibit mosaic organs in their flowers similar to those seen in ufo mutants (Zhao et al. 1999), suggesting a role of Skp1-like proteins in B class gene regulation. Thus, LFY and UFO might act by distinct mechanisms to coordinately regulate B class gene expression. For example, LFY binds to AP3 promoter and activates AP3 transcription, whereas UFO might act as a member of the heteromeric ubiquitin ligase complex that specifically removes repressors of AP3 transcription via ubiquitin-mediated protein degradation.

IV. NEGATIVE REGULATORS OF FLORAL ORGAN IDENTITY GENES It has become evident that combined activities of both positive regulators, such as LFY, and negative regulators are necessary to specify the proper temporal and spatial domains of ABC gene expression. A number of negative regulators of the ABC genes in Arabidopsis have been identified that play crucial roles in delimiting ABC gene expression to specific domains. Many of these negative regulators have been identified through genetic screens for floral mutants that exhibit partial or complete homeotic transformation from one floral organ type to another. Based on the ABC model, one can interpret the mutant phenotype by predicting an ectopic expression of a particular class or classes of the ABC genes. The predictions can be easily tested with in situ hybridization experiments to examine the specific A, B, or C gene expression in the newly isolated mutant background. Once confirmed, these newly isolated mutations define negative regulators of the ABC genes. Despite the identification and molecular cloning of a large number of the negative regulators of the C class gene AG, the mechanism of negative regulation is still not well understood.

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Work to date suggests that a number of negative regulators work in a coordinated fashion to initiate and maintain AG repression. A. Temporal and Spatial Regulators of AG 1. APETALA2 (AP2). AP2 is both an A class organ identity gene and a repressor of AG, specifically in whorls 1–2. The DNA binding motifs found in AP2 suggests that AP2 could directly interact with DNA and AP2-domain containing proteins have been shown to bind to DNA elements termed GCC boxes (Ohme-Takagi and Shinshi 1995). However, the absence of any GCC box in the cis-regulatory elements of AG (Deyholos and Sieburth 2000) indicates that AP2 may mediate negative regulation indirectly, via other intermediate steps. Thus far, no direct DNA binding of AP2 to the AG cis-elements has been reported. Bomblies et al. (1999) examined the cis-regulatory sequences of AG that mediate the repressive action of AP2. They also tested if the repressive effect of AP2 on AG depends on the activity of LFY. Sequences within the second intron of AG that have been shown to be required for activation of AG expression by LFY (Busch et al. 1999) are also required for the repression of AG expression by AP2 (Sieburth and Meyerowitz 1997; Bomblies et al. 1999). The two independent LFY responsive enhancers identified within the large intron (Busch et al. 1999) are also involved in mediating the repressive effect of AP2 (Bomblies et al. 1999). However, additional experiments suggest that AP2 may regulate AG expression through both LFY-dependent and LFY-independent mechanisms. It is possible that the LFY-independent mechanism might be mediated by AP1 (Deyholos and Sieburth 2000). 2. LEUNIG (LUG). Since AP2 is expressed throughout the developing floral meristem, the spatially restricted activity of AP2 in repressing AG in the first two whorls must depend on additional levels of regulation (Jofuku et al. 1994). This spatially restricted activity of AP2 was initially thought to be conferred by the presence of other co-regulators that are only present in the first two whorls. One candidate is LUG, which was identified in a screen for enhancers of a weak ap2 allele (Liu and Meyerowitz 1995). Plants with mutations in the LUG gene exhibit homeotic transformations similar to, but less severe than, ap2 mutants (Komaki et al. 1988; Liu and Meyerowitz 1995). These lug mutants also display ectopic and precocious expression of AG RNA, suggesting that LUG is required for proper repression of AG. Furthermore, lug ap2 double mutants exhibit more severe homeotic transformations than either single mutants. In situ hybridization experiments indicated that the

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mechanism of this enhancement is through the increased ectopic expression of AG. The enhancement of defects seen in the lug ap2 double mutants suggests that LUG and AP2 share partially redundant functions. In addition, dominant interactions were observed between strong ap2 alleles and lug mutations, suggesting either that these two gene products might interact directly or that an activity composed of both LUG and AP2 is required above a threshold level for proper AG repression. LUG was recently cloned (Conner and Liu 2000) and found to encode a nuclear protein that has an overall domain structure similar to a class of functionally related transcriptional co-repressors including Tup1p of yeast and Groucho of Drosophila (Hartley et al. 1988; Williams and Trumbly 1990). A common structure shared by all members of this class includes Q-rich regions near the N-terminus of the protein and 6–7 WD repeats at the C-terminus. The WD repeats [named so because the repeat often ends with the amino acids tryptophan (W) and aspartic acid (D)] have been shown to mediate protein-protein interactions and are found in proteins with a wide variety of biochemical functions (Neer et al. 1994; Smith et al. 1999). The mechanism of this class of transcriptional co-repressors has been extensively studied in yeast and Drosophila. Several mechanisms were implicated such as interfering with the interaction between activators and the general transcriptional machinery (quenching); interacting with the general transcriptional machinery (direct repression) or by affecting chromatin organization. The Tup1p protein, although it cannot bind to DNA on its own, can interact with a variety of DNA binding transcription factors and mediate transcriptional repression through any of the above mechanisms. In particular, it was shown that Tup1p can organize repressive chromatin structure through direct interaction with the N-terminal region of histones H3 and H4 (Edmondson et al. 1996). LUG may function similarly by interacting with AP2 or other unidentified DNA-binding transcription factors to bring about transcriptional repression of AG. In situ hybridization showed that LUG mRNA is ubiquitously expressed in all floral whorls (Conner and Liu 2000). Hence, like AP2, additional factors or post-transcriptional modifications are needed to limit the activity of LUG to whorls 1–2. Recently, two additional genetic enhancers of lug were identified, SEUSS (SEU) and LARSON (LSN) (R. Franks, X. Bao and Z. Liu, unpublished data). The seu mutants display a phenotype that is very similar to, albeit weaker than, lug mutants. Furthermore, seu lug double mutants display an enhanced phenotype that is characterized by strong precocious and ectopic expression of AG and enhanced homeotic transformations of floral organs, particularly in the first two whorls. lsn mutant

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does not have an obvious phenotype on its own but shows strong enhancement of lug in floral organ identity specification. Both SEU and LSN, together with LUG and AP2, are candidates for members of a transcriptional repression system that regulates AG expression. 3. AINTEGUMENTA (ANT). Mutations in ANT cause narrower floral organ shape and a decrease in floral organ number (Elliott et al. 1996; Klucher et al. 1996). Furthermore, ant mutants are female sterile due to failure of integument initiation and megasporogenesis. Although ant single mutants rarely show homeotic transformation of organ identity, ant dramatically enhances the organ identity defects of ap2 and lug (Elliott et al. 1996; Krizek et al. 2000; Liu et al. 2000). This enhancement correlates with an increased ectopic AG expression in ant ap2 and ant lug double mutants. Hence, ANT is likely another redundant repressor of AG. ANT encodes a member of the AP2 family of DNA-binding transcriptional regulators (Elliott et al. 1996; Klucher et al. 1996); the sequence similarity between ANT and AP2 may underlie their functional redundancy in AG repression. 4. CURLY LEAF (CLF). The clf mutants are characterized by narrow and curled rosette and cauline leaves as well as short stem internodes (Goodrich et al. 1997). The clf flowers display narrow petals, and partial homeotic transformations in whorls 1–2. These phenotypes resemble those reported for plants in which the AG gene was ectopically expressed (Mizukami and Ma 1992). RNA gel blot and in situ analysis indicated that AG was ectopically expressed in leaves and in developing petals of clf mutants at later stages of flower development. Double mutant analyses with ag-3 indicate that the ag mutation is epistatic to clf and thus the clf phenotype in leaves and flowers was dependent upon AG activity. Hence, CLF is another repressor of AG expression with primary roles in leaves, stems and, to a lesser extent, flowers. CLF encodes a protein with extensive sequence similarity to the product of a Drosophila polycomb group gene, ENHANCER OF ZESTE (Goodrich et al. 1997). Drosophila polycomb group genes appear to form multimeric complexes that interact with DNA to bring about heritable maintenance of transcriptional states and there is indirect evidence that they do so by modifying chromatin structure (Carrington and Jones 1996). Like the polycomb group genes, CLF appears to be required for the maintenance of repression and not for the initiation of repression of AG. This interpretation is consistent with the observation that ectopic AG expression was only detected at later stages of flower development. CLF is expressed in 8-day-old seedlings throughout the apical meristem,

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leaf primordia, and leaves (Goodrich et al. 1997). It is also expressed in the infloresence meristem and in all four whorls of the flower. The fact that AG and CLF RNA are co-expressed suggests that CLF RNA expression is not sufficient to repress AG expression. 5. STERILE APETALA (SAP). Flowers of sap mutants resemble those of ap2 or lug mutants with carpelloid sepals and loss of petals (Byzova et al. 1999). In sap mutants, AG RNA is not only detected ectopically in floral whorls 1 and 2, but also in inflorescence meristems. SAP is thus another negative regulator of AG expression. Consistent with a role in AG repression, mutations in SAP enhance the organ identity defects of ap2. To test the regulatory relationship between SAP and AP2, AP2 RNA expression was examined in sap mutants and vice versa. AP2 RNA expression was unaltered in sap mutants and SAP RNA expression was not altered in ap2 mutants. Thus, SAP and AP2 do not appear to regulate each other at the transcriptional level. Like other negative regulators of AG, SAP appears to possess additional functions as revealed by defects of sap mutants in female gametophyte development and by defects of sap ag double mutants in meristem identity determination. SAP encodes a protein with serine-rich and glycine-rich domains that are often found in eukaryotic transcriptional regulators. 6. FILAMENTOUS FLOWER (FIL). The effect of the fil mutation is complex; genetic analyses of fil mutants indicated that FIL is required for the maintenance and growth of inflorescence meristems, floral meristems, and floral organs (Sawa et al. 1999a; Chen et al. 1999). More relevant to this review are the findings that AG is ectopically expressed in fil mutants in floral whorls 1 and 2 and that homeotic transformations in whorls 1–2 are enhanced in ap2 fil and lug fil double mutants (Chen et al. 1999). These data suggest that FIL is yet another member of the AG negative regulators. FIL encodes a nuclear protein that contains a zinc finger and an HMG box-like domain, suggesting a role in transcriptional regulation (Sawa et al. 1999b). Unexpectedly, FIL RNA expression is restricted to the abaxial side of the developing leaves and floral organs, and 35S::FIL plants display an abaxialization of leaves (Sawa et al. 1999b). Thus, FIL controls the identity of the abaxial side of lateral organs. B. Repression of AP1 Expression in Floral Whorls 3–4 In wild type, AP1 is initially expressed in the entire floral primordia, but at later stages AP1 RNA is restricted to whorls 1–2 (Mandel et al. 1992b; Gustafson-Brown et al. 1994). The inhibition of AP1 expression in

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whorls 3–4 results from negative regulation by AG that becomes expressed in whorls 3 and 4 at this stage. In ag loss-of-function mutants, AP1 RNA is expanded to all four whorls (Gustafson-Brown et al. 1994). In the ap2 or lug single mutants, when AG expression is expanded to all four whorls, AP1 RNA is absent from all four whorls (Gustafson-Brown et al. 1994; Liu and Meyerowitz 1995), suggesting that the ectopic AG in whorls 1–2 represses AP1 transcription. However, the mechanism by which AG brings about repression of AP1 transcription is presently unclear. The C class genes HUA1 and HUA2 also participate in the negative regulation of AP1 (Chen and Meyerowitz 1999). In hua1 hua2 double mutants, ectopic AP1 expression was seen in late stages in carpel walls and occasionally in stamens. HUA1 and HUA2 may act in parallel with AG to repress the expression of AP1 in whorls 3–4, possibly as transcriptional co-regulators. C. Restriction of B Gene Expression to Floral Whorls 2–3 1. SUPERMAN (SUP). The sup mutants exhibit supernumerary stamens interior to the third whorl stamens at the expense of carpels (Schultz et al. 1991; Bowman et al. 1992). In situ hybridization experiments indicate that the B class genes AP3 and PI are ectopically expressed in whorl 4 in sup mutants. Additionally, ap3 sup and pi sup double mutants exhibit phenotypes similar to the ap3 or pi single mutants. From these molecular and genetic experiments, SUP was originally thought to function as a negative regulator of B class genes in whorl 4. SUP encodes a nuclear protein with a single zinc finger and a putative basic leucine zipper motif, suggesting a role in transcriptional regulation (Sakai et al. 1995). However, in situ hybridization revealed that SUP is expressed in whorl 3, not in whorl 4. Further, SUP RNA expression is, in fact, dependent on AP3 (Sakai et al. 1995). Initial AP3 expression precedes SUP RNA expression, and ectopic AP3 expression under 35S promoter causes ectopic SUP expression. In addition, SUP RNA is much reduced or absent in ap3 mutant flowers, while the onset of AP3 expression in the sup mutant is normal. These new findings do not support the earlier hypothesis that SUP represses B gene expression in whorl 4. Two alternative models were proposed (Sakai et al. 1995); in one SUP functions to prevent the spread of AP3 activity from whorl 3 to whorl 4, while in the second model SUP functions to limit the extent of cell proliferation in whorl 3. In either model, SUP acts to maintain a boundary between whorls 3 and 4.

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2. LEUNIG (LUG) and CURLY LEAF (CLF). In addition to their roles as negative regulators of AG, LUG and CLF also negatively regulate B class gene expression. Patches of ectopic AP3 and PI expression detected in whorl 1 organs of lug mutant flowers suggest that LUG represses B class gene expression in whorl 1 (Liu and Meyerowitz 1995). Similarly, ectopic AP3 expression was detected in leaves of clf mutants, suggesting a role of CLF in repressing AP3 expression in leaves (Goodrich et al. 1997). Whether these effects reflect a direct action of LUG or CLF on B class genes is unknown. These studies, however, suggest that the regulation of B genes employs at least two mechanisms: a region-specific coactivator such as UFO and negative regulators such as LUG.

V. SUMMARY The initial activation of the ABC genes in a flower specific fashion is dependent upon meristem identity genes such as LFY and AP1. Later, ABC gene expression is spatially refined by a combination of other positive regulators, such as UFO, and negative regulators, such as AP2, LUG, ANT, CLF, and SAP. The combined activity of both positive and negative regulators insures proper spatial and temporal expression of the ABC genes and thus the stereotypical structure of a given flower. Now that many of the key regulatory molecules have been identified and isolated, the challenge for the future is to further clarify the molecular mechanisms underlying ABC gene regulation. Clearly a variety of mechanisms are employed in this process. Evidence to date suggests that both transcriptional and post-transcriptional mechanisms are employed and that generating the proper ABC expression domains likely requires targeted degradation of specific repressors, autoregulatory enforcement mechanisms, and recruitment of transcriptional co-repressors. The identification of an increasingly large number of genes involved in ABC gene regulation suggests that the mechanism of ABC gene regulation is rather complex and many questions remained unanswered. For example, what is the molecular or biochemical basis underlying the genetic enhancement or dominant interaction among these mutants? Do these ABC regulators physically interact directly? Do they regulate each other’s expression? Future experiments involving immunoprecipitation assays and/or yeast two-hybrid assays will allow us to test physical interactions among these ABC regulators. Examination of the expression of these genes by RNA in situ hybridization and immunolocalization in different mutant backgrounds will illuminate the regulatory

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relationship among these genes. For genes that encode DNA-binding domains, gel-shift assays may help to identify their target ABC genes. Further deletion analyses of the promoters of the target ABC genes and testing these deletion constructs using gel-shift and transgenic reporter assays will help to locate sequence motifs that are crucial for ABC gene expression. Similarly, site-directed mutagenesis and domain-swamping —for example, between AP2 and ANT—may help to assess their DNAbinding and transcriptional activation potential and target-site specificity. Thus far, many of the expression studies have only looked at RNA expression. It is now necessary to examine the protein expression of these genes to determine if translation of the RNA molecules is spatially or temporally regulated. Alternatively, regulation of the subcellular localization of regulatory proteins may explain the whorl-specific gene activity. These studies will lead to a more complete picture of the molecular hierarchy responsible for ABC gene regulation. This review has focused on the genetic basis of ABC gene regulation and has treated floral organ identity specification as independent from environmental events. As we further clarify the molecular mechanisms of floral organ identity, we may be able to better predict the effects of environmental influences, such as the effect of photoperiod, hormone, and temperature on floral development. Understanding the interaction between the genetic programs and environmental factors will be critical for controlling traits of agricultural varieties in the field. As our understanding of the genetic mechanisms of organ identity specification grows, so does our ability to engineer new floral variants. One can envision a multitude of applications in the areas of horticulture and agriculture. For example, to engineer environmentally friendly ornamental types (such as pollen-free or fruit-free cultivars), or to facilitate outcrossing and simplify breeding programs, novel methods of generating male-sterile plants will be highly desirable. By specifically repressing the B or C class genes in whorl 3 through transgenic techniques, one may create flowers whose stamens are converted into carpels or petals. Alternatively, our knowledge of organ identity genes could be used to increase the visual diversity of floral variants. Replacing reproductive structures with petals leads to showier flowers, as is seen in a number of presently existing “double flowers” (Acquaah et al. 1992) and in Arabidopsis transgenic plants (Fig. 2.2F). New chimeric organs that result from a partial homeotic transformation may have useful or visually interesting properties: stamenoid petals that are more tubular or sepalloid petals that are more resistant to wilting and thus increase the life of the cut flower. Increasing the proportion of a given organ within the flower may enhance the yield of certain floral-derived products. Yields

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of saffron, derived from stigmatic tissue of the crocus flower, might be improved if ectopic expression of C class genes is employed to generate additional carpels. As we continue to illustrate the “blueprints” of floral development, we hope to enable “agricultural architects” of the future to rationally design floral types to better meet societal needs. In addition to the technical hurdles that lie ahead, we also face the multi-disciplinary challenge of managing these new variants such that environmental and socioeconomic effects are carefully considered.

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3 Lingonberry: Botany and Horticulture Inger Hjalmarsson The Nordic Gene Bank, Smedjevägen 3, P.O. Box 41, S-230 53 Alnarp, Sweden Rodomiro Ortiz The Royal Veterinary and Agricultural University, Department of Agricultural Sciences, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark

I. INTRODUCTION II. HISTORY A. Early B. 18th and 19th Centuries C. 20th Century D. Horticultural Research in the Northern Hemisphere 1. Nordic Region 2. Other Sites in the Northern Hemisphere III. BOTANY A. Taxonomy and Geographic Distribution B. Morphology 1. Vegetative 2. Reproductive C. Ecology D. Other Vaccinium Species in Scandinavia 1. Bilberry 2. Bog Bilberry 3. Small-fruited European Cranberry 4. Dwarf Cranberry IV. MANAGEMENT OF NATURAL STANDS A. Photosynthesis B. Biomass Production C. Seed Ecology and Regeneration D. Berry Production E. Effects of Forestry Management F. Experiments in Natural Habitats Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 79

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V. HORTICULTURE A. Propagation 1. Plants Collected Directly from the Wild 2. Shoot Cuttings and Rhizomes 3. Seedling Plants B. Frost Protection C. Culture 1. Soil 2. Nutrients 3. Mycorrhiza 4. Mulching 5. Irrigation D. Plant Pathology 1. Weed Control 2. Diseases E. Crop Improvement 1. Early Studies of Ecotypes 2. Breeding at Balsgård 3. Description of Swedish Cultivars 4. Description of North American Cultivars VI. SUMMARY AND FUTURE PROSPECTS LITERATURE CITED

I. INTRODUCTION Lingonberry (Vaccinium vitis-idaea L., Ericaceae) is a perennial, evergreen dwarf shrub that is indigenous to Scandinavia, where the peasized, bright-red fruit is picked from wild stands. Lingonberry is known as puolukka in Finland, as tyttebær in Norway and Denmark, and as lingon in Sweden. Lingonberry jam, with or without sugar, may be eaten with porridge, potatoes, bread, pancakes, cow and reindeer milk, herring, black blood pudding, meatballs, and steak among other foods. The berries have also been used for soups and beverage. Retzius (1806) recommended lingonberry drinks for fever patients. Furthermore, lingonberry has been used as an anti-scorbutic (Nyman 1868), and because of its richness in glycosides (Bandzatiene 1999), as a diarrhea medication (Stodola and Volak 1986). Folk medicine recommends that lingonberry tea, derived from leaves, be used against rheumatism (Henriksson 1923b) and as a remedy for urinary tract infections (Nielsen 1978). Recent reports suggest that lingonberry may have anticancer attributes due to high anthocyanin content (Bomser et al. 1996). Lingonberry jam is a traditional delicacy and, although considered a luxury today, it was once one of the few staples available to poor people. While no longer necessary for survival, berry picking has become a recreational activity for many and jam making has moved from homes

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to processing plants. Industry requires a continuous supply of the berries and shortages of labor have led to the start of domestication of the crop. Lingonberry has also been grown as an ornamental plant or shrub in Scandinavian gardens and landscapes since the 17th century (Adelswärd 1994; Balwoll and Weisaeth 1994; Lundqvist 1995) and is an important component of Christmas decorations. Since the 1960s, cultural studies have been carried out in Sweden, Finland, and Norway but published information is scattered. This review article focuses on Nordic lingonberry and includes botany, management of natural stands, cultural practices, and breeding. Information about lingonberry research in Germany, the former Soviet Union, Poland, and North America has also been included.

II. HISTORY A. Early There is little documented information about berry utilization in Scandinavia before Linnaeus. Remnants of lingonberry wine in Danish graves from the Bronze Age are the first proof of its use in the home (Brøndegaard 1987). The Icelandic law books (Grágrás) of the 13th century stipulated that berry-picking on other people’s land must be limited to what can be eaten on the spot, thereby indicating the importance of lingonberry as human food (Armfelt Hansell 1969). There are a few other published reports of wild lingonberry in the Middle Ages (Eriksson et al. 1979). The Italian diplomat Magalotti (1674) wrote the first thorough description of wild lingonberry in Sweden after his journey through the country in the 17th century. By that time lingonberry was also mentioned in Nordic gardening books. In 1651, André Mollet, the French gardener working for Queen Christina, published Le Jardin de Plaisir and suggested the use of lingonberry for parterres de broderie (hedge gardens) instead of the less adapted box model (Adelswärd 1994; Lundqvist 1995). The first Norwegian gardening book, written in 1694 by Christian Gartner, recommended planting lingonberry for culinary and medical purposes (Balwoll and Weisaeth 1994). B. 18th and 19th Centuries Botanists such as Linnaeus (1748) and Retzius (1806) described lingonberry during the 18th and 19th centuries. The economist Fischerström (1779) also discussed lingonberry in his dictionary about Swedish

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households and natural science. Furthermore, lingonberry recipes were published in the cookbooks of the time. In addition, lingonberry was included in crofters’ (tenants’) contracts and mentioned in local horticultural records. For example, Waern (1834) reported on the cultivation of white lingonberry, Vaccinium Vitis idaea fructu albo, in the garden of Baldersnäs (Sweden). Lingonberry was also considered in ethnology studies by Hyltén-Cavallius (1868) and in diet investigations by Keyland (1919) and Grøn (1942). Although lingonberry has been considered among the most important of the fruit jellies (Retzius 1806), no evidence exists about its importance in the diet of earlier times. Eriksson et al. (1979) indicated that berry harvest was most valuable to poor people, especially in years of bad crops when lingonberry could mean survival. Crofters’ contracts in the 19th century often stated that the crofter’s family should pick a certain quantity of berries for further delivery to the estate (Armfelt Hansell 1969). Women and children did most of this work and they also picked the family supply. The berries were originally picked by hand, but Fischerström (1779) described the earliest picking tool. The use of these tools became more widespread and, at the beginning of the 20th century, a debate started about whether this practice was harmful. However, experiments at the Royal Swedish Academy of Agriculture concluded that the tools were harmless to the plants (Sylvén 1918). Johansson (1983), von Zabeltizt (1989), and Dale et al. (1994) have described the development of other lingonberry harvesting aids. Throughout the history of this region, lingonberry has been important as a supplier of energy and vitamins. Lingonberry fruit differs from most of the other wild berry species owing to its long-term storage potential. Consequently, berries are kept from one year to the next without sugar, a product that was rare in most Scandinavian homes until the 19th century (Kuuse 1982). Traditionally, berries are placed in jars and preserved by pouring clear water over them to produce a dish known as “water-lingon” in Swedish cuisine (Retzius 1806). The storage ability of the berries depends primarily on their benzoic acid content, with up to 65 mg benzoic acid per 100 g of berries (Karlsson and Malmberg 1974). Lingonberry fruit also contains large amounts of aroma compounds and anthocyanins (Anjou and von Sydow 1967; Andersen 1985), negligible amounts of proteins, and only small amounts of minerals, although lingonberry fruit provides 4 mg vitamin C, 0.02 mg carotene, and 67 kcal of energy per 100 g (Statens Livsmedelsverk 1978). According to Fuchs and Wretling (1991), lingonberry fruit has 7 g sugar/100 g and 24 g titrable acids/L fruit juice.

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Lingonberry has a long history of commerce in the Nordic countries. Linnaeus (1732) described how farmers in northern Sweden sent their berries to the markets in Stockholm. The moor farmers of Jylland (Denmark) traded lingonberry with Copenhagen in the 1800s (Brøndegaard 1987). The first reference to export of lingonberry from Norway was dated 1835 (Valset 1976; Graff 1991). However, the “days of glory” for lingonberry began at the turn of the century, in conjunction with the development of the railway. In 1902, a record quantity of 20 million kg of lingonberries was exported from Sweden (Wikmark 1907). The Swedish berries were mainly sold to Germany, where they competed with berries from Norway and Finland. C. 20th Century Taking care of wild lingonberries captured people’s interest in Finland, Norway, and Sweden at the beginning of the 20th century. For example, Norwegian committees were established to make berry picking more efficient (Valset 1976; Graff 1991), whereas Swedes started an industry with dried lingonberry and blueberry (Lind 1916). According to official statistics, the exports remained high throughout the 1930s (Eriksson et al. 1979; Graff 1991). Finnish exports during this decade varied between 2.4 and 6.7 million kg yearly (Anon 1983). The demand for lingonberry continued to be high during World War II, when people were encouraged by their governments to create contingency stocks in case there were shortages of other fruits. Lingonberry exports never surpassed earlier levels after World War II (Eriksson et al. 1979). But commercial lingonberry harvest has been reported from Alaska, Nova Scotia, and Newfoundland in North America (Holloway 1984). The commercial lingonberry harvest in Finland from 1977 to 1985 was estimated to vary between 1.7 and 10.2 million kg annually, of which 35 to 80% was exported (Hiirsalmi and Lehmushovi 1993). Swedish exports during the same period fluctuated between 1.5 and 4.2 million kg (Holmberg 1987). In 1985 the highest economic return (85 million Swedish Krone) was realized. Finland and Sweden import some wild berries but remain net exporters of lingonberries. In Norway, however, more fruit are imported than exported. The Norwegian food industry uses about 1 million kg of lingonberry yearly and an additional 0.2 to 0.4 million kg are sold on the fresh market. The majority of these berries are imported (Nes 1994). Foreign trade in wild lingonberry remains important in Scandinavia (Statistics Sweden 1994). Per capita consumption of wild small fruits (mainly lingonberry and blueberry) in Sweden was estimated to be 0.6 kg in 1990 (Statens Jordbruksverk 1994).

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D. Horticultural Research in the Northern Hemisphere 1. Nordic Region. Lingonberry is a healthy component of the Scandinavian diet and is considered an exotic fruit by many people outside the region, which suggests that this crop will always have a market. However, the question persists whether the crop should be picked solely from native stands or through commercial production, or both. Interest in commercial harvest from wild stands has decreased but the industry demands a reliable annual supply. Sweden. AB Bjäre Industrier, the Swedish producer of lingonberry jam, initiated the first lingonberry plantings at Vången (Skepparslöv) in 1962 and another 5 ha were planted at the Ottarp farm (Ryssby) from 1966 to 1968 (Teär 1972). Additional small plantations were started later in both middle and northern Sweden, and supplementary experiments were carried out in the Department of Pomology at the former Agricultural College of Sweden in Alnarp (Fernqvist 1977). A Nordic symposium about domestication of wild berries was held in 1974 at Karlstad (Fernqvist 1974). Most of this early research was published in Swedish, but English abstracts were printed for the ISHS International Symposium on Vaccinium Culture (Fernqvist 1977; Hjalmarsson 1993). Since the late 1970s, most lingonberry research has been carried out at the Department of Horticultural Breeding in the Swedish University of Agricultural Sciences (SLU) in Balsgård. Pilot plantations are run parallel to the plant breeding experiments and provide continuous experience on practical cultivation. The first plantations were planted with micropropagated plants of ‘Sussi’ and ‘Sanna’. Plant spacing was 40 cm in a zig-zag pattern that aimed to create a dense carpet of lingonberry vegetation (Eckerbom 1990). Currently a row system with plant spacing of 25–30 cm × 80 × 100 cm is recommended (Nilsson and Rumpunen 1997). Finland. The first cultivation experiments began in 1968 at the Institute of Horticulture in Piikkiö (southwest Finland). The broad research program involved experiments in natural habitats, field studies on plant material, soil, liming, fertilization, and shading among others. Research results were reported in Finnish journals, and review articles were also written (Lehmushovi 1977b; Hiirslami 1989). Current research is carried out by the Department of Plant Production of the University of Helsinki and focuses on weed control (Saario 1998). Norway. The first commercial lingonberry field in Norway was planted in the mid-1960s. In 1974 public research began at Kise Research Station, where the experiments focused on plant material, plant establish-

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ment, water requirement, and weed control (Nes 1994). In 1983, a group of farmers in southeastern Norway started a lingonberry project; however, it did not lead to any extensive production (Vestrheim et al. 1994). Seven years later, a private farmer imported Dutch and German cultivars for field planting. At the same time, the Agricultural University of Norway (NLH) started cultivar trials, which are still going on (Vestrheim et al. 1994). 2. Other Sites in the Northern Hemisphere Eastern Europe. Former Soviet researchers started their research on lingonberry domestication in the mid 1960s (Paal 1992) owing to a rising demand and low productivity of wild lingonberry. Propagation methods, seed germination, factors influencing root development in cuttings, mulching, phenology, somatic embryogenesis, and micropropagation were included in their research agenda (Butkus et al. 1989; Labokas and Budriuniene 1989; Audrina 1996; Banner 1996, 1998; Bandzaitiene 1998; Kutas 1998; Kutas and Sidorovitch 1998). In Poland, mulching was also investigated (Pliszka and Scibisz 1985). Plant growth was enhanced in mulched plots but it did not improve fruit yield. Testing foreign lingonberry germplasm to identify new high-yielding cultivars has also been an important activity in Eastern Europe. A high second harvest was reported for Dutch and German cultivars in Byelorussia (Pavlovsky and Ruban 1998). However, this second harvest of foreign cultivars was low in Latvia (Audrina 1996) and Russia (Tiak and Cherkasov 1998). Four promising selections were made from the local material in Latvia (Audrina 1996) and two Russian cultivars (‘Kostromskaya Rozovaya’ and ‘Kostromichka’) were released in the mid1990s (Tiak and Cherkasov 1998). It seems that seedlings from northern regions have earlier growth and faster development than those from southern sites (Reier and Paal 1998). Plant breeding through chromosome doubling (CD) with colchicine was unsuccessful in Byelorussia. The CD-derived plants did not improve fruit yield because of few flowers and pollen sterility (Morozov 1998). Wild tetraploid lingonberry accessions collected in Magadan (Russia) were crossed with other Vaccinium species, but the hybrid seeds did not always germinate. Germany. Lingonberry research was started in 1973 by Prof. G. Liebster at the Institute of Fruit in Weinhenstephan (Liebster 1977, 1984; Müller 1982). Morphology and physiology research led to an efficient method for vegetative propagation of the Dutch cultivar ‘Koralle’ and the German

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cultivars (‘Erntedank,’ ‘Erntekrone,’ and ‘Erntesegen’). The German cultivars are selections from wild lingonberry stands (Zillmer 1985). Further investigations at Wilhelm Dierking Beerenobst included soils for lingonberry cultivation, mulching, and fertilizers (Dierking and Krüger 1984; Dierking 1985). The University of Hannover started research on lingonberry nutrition in the 1980s to determine the correlation between yield and growth with nutrient levels using the leaf analysis (Krüger and Naumann 1984a,b,c; Krüger 1985). There were some attempts by German scientists to obtain hybrids between lingonberry and cranberry (Vaccinium macrocarpon Ait.) (Christ 1977), as well as in vitro propagation of lingonberry clones (Gebhardt and Friedrich 1986). Tissue culture has been confirmed as a means for rapid mass propagation of lingonberry (Riechers and Bünemann 1989) and to facilitate fruit picking, a harvest machine was developed by von Zabelitz (1989). Nowadays, about 35 ha are grown commercially in Germany (Dierking and Dierking 1993). North America. Wild lingonberry fruit are collected commercially in Alaska, Nova Scotia, and Newfoundland (Holloway 1984; Hendrickson 1997). Since 1965 researchers at Fairbanks (Alaska) have been working in lingonberry improvement. Early investigations included assessment of substrates for lingonberry cultivation, influence of light intensity on growth, gibberellic acid effect on fruit set, chilling temperature requirements, factors affecting rooting of stem cuttings, and seed propagation (Hall and Bell 1970; Holloway et al. 1982a,b; Holloway et al. 1982; Holloway et al. 1983; Holloway 1985). In Newfoundland, lingonberry researchers are studying crop establishment, maturity dates, pests affecting the crop, and the best cultural practices to enhance earliness and fruit yield (Penney et al. 1997). Researchers working in the Department of Horticulture at the University of Wisconsin–Madison have investigated physiology (photoperiod response) and cultural practices (weed control, humus application) needed to introduce lingonberry as a new fruit crop in the northern United States (Stang et al. 1993a,b, 1994; Stang 1994). They have established small-scale demonstration plots and determined fruit processing requirements. Cuddy (1998) has reviewed advances in lingonberry cultivation at Wisconsin. North American scientists have tested cultivars and selections from the wild (Penney et al. 1977; Estabrooks 1997). In the mid-1990s, the University of Wisconsin–Madison released two lingonberry cultivars for commercial production (Stang et al. 1994), ‘Splendor’ and ‘Regal’, both

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selections from Finnish exotic wild germplasm. Vaccinium reticulatum has been crossed with lingonberry because this diploid evergreen wild species, native to Hawaii, has a large fruit size among other interesting characteristics (Zeldin and McCown 1997). Some North American researchers (Hosier et al. 1985; Serres et al. 1994) have investigated micropropagation methods including basal media, plant growth regulators, and culture conditions. III. BOTANY A. Taxonomy and Geographic Distribution The genus Vaccinium includes approximately 400 species (Galletta and Ballington 1996) dispersed from the arctic to subtropical regions and mountainous tropics (Hutchinson 1969). Five Vaccinium species occur in Scandinavia: V. vitis idaea (2n = 24), V. myrtillus L. (2n = 24), V. uliginosum L. (2n = 24, 48, 72), V. oxycoccus L. (2n = 24, 48, 72), and V. microcarpum (Turcz.) Hook. (2n = 24) (Hylander 1955) (Plate 3.1A). The chromosome numbers in brackets are those reported by Luby et al. (1990). The rare hybrid Vaccinium × intermedia Ruthe was described by Ritchii (1955a) as an intermediate between V. vitis-idaea L. and V. myrtillus L. Although this hybrid has been seldom reported in the Nordic Region, it has been seen sometimes in Sweden (Scania and Stockholm) and in two locations at Jylland, Denmark (Lagerberg 1948). Triploid forms of Vaccinium vitis-idaea have been reported in Sweden (Ising 1950) and in Finland (Ahokas 1971). Hultén and Fries (1986) have mapped the distribution of the circumpolar lingonberry (Fig. 3.1), and Hultén (1971) provided details about the abundance of lingonberry in the forests of Finland, Norway, and Sweden and its sparse occurrence on the calcareous soils of southern Sweden and the Danish islands. Lingonberry is also common on the moors of Jylland (Brøndegaard 1987; Hultén 1971) and grows at 1800 m above sea level in Jotunheimen, Norway (Hultén 1958; Lagerberg 1948). Lingonberry seems to be a European crop (Plate 3.1B), although the Pacific Northwest in the United States has observed a significant planting of lingonberry in recent years (Finn 1999). Vaccinium vitis-idaea is the only species in the section Vitis-idaea (Moench) Koch (Galetta and Ballington 1996). Hultén (1949) divided lingonberry into two subspecies: subsp. vitis-idaea L. and subsp. minus (G. Lodd.) Hultén. Both subspecies are found in the arctic mountains of Norway. Subspecies vitis-idaea predominates in Eurasia, while subsp.

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Fig. 3.1. Lingonberry in the Northern Hemisphere (From: Hultén and Fries 1986, with kind authorization from Koeltz Scientific Books).

minus prevails in the mountains of North America. Apparently, the two subspecies hybridize in Scandinavia (Hultén and Fries 1986). B. Morphology 1. Vegetative. Lingonberry is as an evergreen, small shrub with subterranean rhizomes, and aerial shoots varying from 5 to 30 cm tall (Hultén 1958) (Fig. 3.2). The subsp. minus is shorter and has smaller leaves and berries than subsp. vitis-idaea. The leaves of subsp. vitis-idaea possess conspicuous venation, while the venation is inconspicuous in subsp. minus (Hultén 1949). The leathery leaves of lingonberry are ovate with a thick, glossy upper surface and a pale, glandular lower surface. These characteristics allow the plant to survive the cold and windy winters of the North without desiccation. The lingonberry plants typically have leaves ranging in length from 4 to 29 mm and width from 2 to 16 mm (Teär 1972).

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Lingonberry plants collected from the Nordic forest.

2. Reproductive. Floral initiation in Vaccinium species starts in late spring or early summer (Bell and Burchill 1955; Eck 1966b). Flowering occurs for one month between May and June in the southern part of Scandinavia, and four weeks later in Lapland (Hultén 1971) (Fig 3.3). In these northern latitudes, secondary flowering on current year shoots is rare, as noted in middle Europe by Hegi (1927) and Ritchii (1955b). At Wisconsin, flower initiation occurs at 8 to 12 h day length and a minimum of 8 weeks seems to be needed for maximum flower induction (Stang et al. 1993a). It is possible to distinguish between floral and vegetative buds of lingonberry in August at northern latitudes. The vegetative buds are larger (2–3 mm) and wider (1 mm) and they have a tendency to bend downward. The inflorescence is a slightly pendulous raceme of 4 to 6 flowers (Teär 1972). The lingonberry flowers (Plate 3.1C) are white to pinkishred, 4 to 6 mm in length, urceolate, and possess 4 to 5 petals (Knuth 1899). These flowers are hermaphroditic and epigynous with 4 to 5 locules per ovary and 15 to 20 ovules per carpel. The flower has 8 stamens that open by pores (also known as porandrous). The form of their sepals, hairiness of their filaments, and length of their style (Hultén

Fig. 3.3.

Lingonberry plants in blossom.

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1949) differentiate the flowers of the two subspecies. The dark red, globular berry of the subsp. vitis-idaea ripens in September and has, on an average, a diameter of 6 to 8 mm (Teär 1972). The style is closely surrounded by anthers; the stigma and anthers mature nearly simultaneously but the anthers may mature later. The stigma rises outside the corolla, which suggests that wind might be important for pollination (Hagerup 1954). However, most authors (Haslerud 1974; Lehmushovi 1977a) consider entomogamy essential for fruit set. Bumblebees (Bombus terrestis L. and B. pratorum L.) were the principal pollinating species at Ottarp (Eriksson 1975). However, some years the bumblebee workers were not ready until the last half of the flowering period (Ängeby 1978). Nectaries, hidden beneath the stamens, produce a large amount of nectar that attracts insects. Cross-pollination is effected as the insect passes the stigma, depositing pollen, and in the process of getting to the nectaries, pollen from the anthers is transferred to the insect. The anthers (Plate 3.1D) are touched afterwards, thus releasing pollen grains to the surrounding environment. Pollen grains of lingonberry are arranged in tetrads, and the rate of germination exceeds 80% (Eriksson 1975; Lehmushovi 1977a). Pollen tubes required five days to grow through the style. Different botanical varieties of lingonberry are mentioned in the literature. Among them, variety ovata J. Henriksson, exhibiting oblong-ovate berries, occurs in the county of Dalsland (Sweden) and in northern Norway (Henriksson 1923a; Jørstad 1960). Swedish botanists have also noted lingonberry stands with white berries (Nyman 1868; Lagerberg 1948). Temperatures between 15 and 20°C enhance pollination, while temperatures above 25°C lower fruit set (Eriksson 1975; Hjalmarsson 1997). Fruit set was greatest following artificial cross-pollination (64%) and open-pollination (58%), whereas fruit set after self-pollination and in isolated flowers were only 28% and 2%, respectively, in wild stands. Seed set ranged from 10.9 per berry in open-pollinated plants to 4.1 in isolated plants. There were 8.6 seeds per berry after cross-pollination, while 3.8 seeds were obtained after self-pollination. Similar results were obtained with the cultivars ‘Sanna’ and ‘Sussi’ (Hjalmarsson 1997). C. Ecology Teär (1972) was the first Nordic scientist to thoroughly investigate vegetative and reproductive growth of wild lingonberry. His research was aimed at obtaining information about plant ecology to facilitate a predictable process of domestication. Since then botanists and foresters

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have further investigated native lingonberry populations together with other common understory species in the forests of northern Scandinavia. The effect of light on the annual growth differed according to species (Kellomäki 1977). In lingonberry the relationship between the amount of photosynthesis and productivity was nonlinear. Maximum growth was reached at low photosynthetic light flux. Bilberry and lingonberry were abundant after the closure of the canopy at the expense of more light-dependent species. Kellomäki argued that lingonberry possesses phenotypic and reproductive plasticity, which is common among vascular plants in ground cover communities. Branching occurred on one-year-old shoots in young ramets as well as from buds on older shoots after rejuvenation in deciduous bilberry and evergreen lingonberry in habitats of northern Finland (Tolvanen 1995). Lingonberry has a predominantly monopodial growth habit, but shoot growth stopped in terminal inflorescences after a few growing seasons. This indicates a sympodial branching system, i.e., older ramets grew more horizontally than younger ramets. Terminal buds mainly developed into vegetative shoots in the northern Finnish forest understory, whereas a great number of lateral buds were activated in open habitats. This, however, did not lead to any change in the total number of new shoots. Instead, flower production was greatest in open habitats. Differences in growth habits between the two sites indicated high morphological plasticity, allowing the species to respond rapidly to changing environments. D. Other Vaccinium Species in Scandinavia Berry crops have always been important components of human diets, although some of the berry species have remained economically important only locally. Lingonberry has been considered among the major new berry crops (Finn 1999), but other Vaccinium species indicated below have been locally harvested and may become economically important new crops in the Nordic region. Most of their fruit continues to be harvested from wild stands, although owing to their attributes some of these species are attracting the attention of the industry either for processing (e.g., for juices or jams) or for pharmaceutical purposes. As expected in this process, lingonberry and other Vaccinium species are being domesticated to shift the harvest from wild or native stands to their commercial cultivation in farmers’ fields, which will ensure a stable fruit supply for the industry (Finn 1999). Hence, potential markets for these new crops are playing an important role in the process of domestication of lingonberry and other Nordic Vaccinium species.

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1. Bilberry (V. myrtillus). This species is a perennial low bush whose leaves fall off in autumn (Mossberg et al. 1992). The angular twigs remain green during the winter. The leaves are not leathery as with lingonberry, but are thin and pointed with denting edges. Bilberry flowers between May and June, and the flowers grow one by one. The flower is almost round and pale green with a reddish tone. Berries ripen in July to August and most commonly they are blue and covered with dew. However, they may also be black and shiny and are called “shoemaker’s berry.” Bilberry is common in most of Scandinavia, is harvested from the wild and eaten fresh, as jam, cream, or soup. Bilberries, which have a long history in European folk medicine (Morazzoni and Bombardelli 1996), are still used as a medical treatment against diarrhea. 2. Bog Bilberry (V. uliginosum). This small bush (10–75 cm tall), which flowers between May and June in Scandinavia, stretches across circumboreal Northern Hemisphere regions (Finn 1999). It may be seen as a common under shrub, especially where heath-lands are turning to swamp and along lakeside forest. The berries are blue, oval, covered with dew and harvested from the wild plants (Mossberg et al. 1992). Some accessions have been crossed with V. corymbosum to improve winter hardiness and obtain early harvest in highbush blueberry. The cultivar ‘Aron’ was developed following this breeding approach (Hiirsalmi 1989; Hiirsalmi and Lehmushovi 1993). The fruit juice has no color (opposite to bilberry), and the taste is often described as flat and stale. 3. Small-fruited European Cranberry (V. oxycoccus). This species is similar to the American cranberry, but is much smaller (4–8 cm tall). The leaves (6-8 mm long) are evergreen (Mossberg et al. 1992). The plant flowers between June and July, and each flower cluster has 2 to 4 flowers. The round-shaped berry diameters are 8 to 10 mm, and the berry ripens late. Hence, berry picking is preferred after the first winter frost. This small-fruited European cranberry is common in Scandinavia, although not in the very northern mountain area. The berries are used for jam or alcoholic beverage. 4. Dwarf Cranberry (V. microcarpum). This Vaccinium species is even smaller than the small-fruited European cranberry and extends to the very north of Scandinavia (Mossberg et al. 1992). The leaves are 3 to 8 mm long, and the plant flowers between June and July. The flowers grow one by one or two together, and the berries (5–6 mm) are more oval than those of the small-fruited European cranberry.

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IV. MANAGEMENT OF NATURAL STANDS A. Photosynthesis Seasonal carbon dioxide assimilation per unit leaf mass was three times higher in the deciduous bog bilberry (V. uliginosum) than in evergreen lingonberry in sub-arctic environments (Karlsson 1982). However, lingonberry was light saturated for a longer period, i.e., 80 to 86% of the time in July and 60 to 70% in early August. Lingonberry leaves are largest on the mid-position of each yearly shoot segment, and have axes with uniform and horizontal angles. Old lingonberry leaves are important for a rapid CO2 assimilation in the spring (Karlsson 1982). Old leaves need two weeks to build up to full photosynthetic capacity. Similarly, lingonberry plants are able to extend their growing season late in autumn. Increased leaf age, however, affects maximal photosynthetic capacity. Leaves will only retain 2/3 of their original capacity during the second growing season, and in the following years an additional 10% will be lost annually (Karlsson 1982). Hence, lingonberry leaves need four growing seasons to assimilate the same amount of carbon dioxide as bog bilberry leaves assimilate during one season. Current year shoot growth in lingonberry was mainly supported during early summer by photosynthetic products of older leaves (Karlsson 1982). Photosynthesis in bog bilberry and lingonberry was similar. In response to drought, lingonberry has a great ability to survive in dry environments. The two species respond differently to light utilization and water economy, and occupy distinct niches in natural subarctic environments. B. Biomass Production There were, on average, 101 to 231 m rhizomes per m2 in a large number of selected lingonberry plots in natural habitats in Sweden (Teär 1972). Annual production was calculated at 13 to 55 new rhizomes per m2. New rhizomes typically grew 15 to 20 cm, and never had a growth rate exceeding 40 cm. Rhizomes had buds, which may develop new shoots under or above ground. A total of 150 dormant buds were found per m of rhizome. New plants were usually developed in groups near where the rhizomes originated, and only about 15% of these originated from buds at the terminal ends. Every year 10 to 25% of the old plants were replaced by new plants through rejuvenation. The average plant age was 4 years, but plants as old as 11 years were found. The total amount of dry biomass varied between 153 and 579 g per m2 (Teär 1972). About

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half the biomass was underground and rhizomes accounted for 10 to 20%. The above ground biomass was equally divided between shoots and leaves, with the latter constituting 78% of the annual production of biomass. A lingonberry plant had on average 3.3 shoots, and on average produced 1.7 new shoots per plant every year, but more than half the plants had only one new shoot. Most of the shoots were produced from vegetative one-year-old shoots. Large plants produced more shoots than small plants, though the ratio (new shoots/total number of shoots) was lower. Furthermore, flowering shoots were shorter than vegetative shoots, and produced new shoots from stems that flowered late in the growing season. On average there were 8 to 9 leaves per shoot. Flowering shoots had more leaves than those that were strictly vegetative. At the beginning of the summer, current-year leaves accounted for 70% of the total leaf number (Teär 1972). Leaves growing on shoot tips were more pointed than others growing below the shoot tips. The number of plants with flower buds increased with the age of the plants. Plants with 5 to 6 older shoots had mostly new flowering shoots (2.8–3.1). However, the share of new fertile shoots within a plant decreased as it grew in size. Most fertile shoots were in clear felled areas, while the opposite occurred in forests with Norway spruce. On average, there was one flower bud per flowering shoot. The number of flowers per cluster varied from 4.1 to 5.9, while there were, on average, 3 to 4 berries and a maximum of 14 berries per cluster. Increased shoot growth and higher levels of nitrogen, phosphorous, and potassium were noted after irrigation and fertilization, while the photosynthetic rate was the same in controls and treatments (Karlsson 1985). These changes were more conspicuous in lingonberry than in bog bilberry. Lingonberry had fewer old leaves per shoot after the treatment. These results suggested that lingonberry, as previously reported in other evergreen species, has decreased leaf longevity when nutrient levels are increased in the substrate. C. Seed Ecology and Regeneration Fruit set was lower after self-pollination than after open-pollination (Fröborg 1996). A few lingonberry seeds were recorded in the seed bank of a sub-arctic pine-birch forest in Lapland, Finland (Vieno et al. 1993). Vaccinium species lack developed seed banks even though they are known to have high seed production (Eriksson and Fröborg 1996). The work of Eriksson and Fröborg focused on “windows of opportunity,” i.e., spatially and temporally unpredictable conditions in which seedling

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recruitment was possible. Lingonberry seedlings favor moist soil with high organic content. Likewise, sudden optimal conditions at a specific micro-site may lead to establishment of new seedlings within stands of adults. Extensive production of well dispersed seeds but no extended dormancy will take advantage of such favorable situations. Furthermore, recruitment through “windows of opportunity” could explain unexpected high genetic variation within populations of persistent clonal plants. Regeneration of understory species after fire was investigated in Northern Sweden (Schimmel 1989, Schimmel and Granström 1996). To survive or escape a forest fire, plant material must either be able to withstand high temperatures or be deeply buried. Rhizomes of lingonberry were killed after 10 minutes at 55° to 59°C. The main part of the subterranean runners grew in the middle or a little below the middle of the humus layer known as mor, which occurs by decomposition in the superficial soil layers instead of its surface. A small proportion of rhizomes in the mineral soil occurred in thick humus layer. The total bud bank ranged from 470 to 950 shoots per m2. Slightly burned plots produced more sprouts than clipped plots that weren’t burned. Sprouting from rhizomes in the upper horizon increased after burning. Improved nutrient status in the soil could explain this phenomenon. If fires were limited to the moss layer, pre-fire coverages were reached within 2 to 4 years. The most severe fires, however, eliminated lingonberry as well as other Vaccinium species. Tolvanen et al. (1995) mapped the recovery of lingonberry after removal of annual branches or ramets in a boreal forest. Recovery proceeded unexpectedly high even after the most severe treatment (100% removal of ramets). When whole ramets were removed the percentage of new growth emerging from basal buds increased. About 42 to 112% of the above ground biomass and 60 to 70% of the coverage were regained after three growing seasons. D. Berry Production The National Forestry Survey did the first research project regarding wild berry production in Swedish forests (Eriksson et al. 1979; Kardell 1980). Production of bilberry, raspberry, and lingonberry were analyzed on 44,000 sites during three years (1974 to 1977), and berries were counted and weighed. Lingonberry occurred on 1.2 million ha out of 23.5 million ha of productive Swedish forests. The occurrence of lingonberry varied little with the density of the forest. Coverage was about 5% on clear-cut areas and in young forests, while about 7% were

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recorded for old established forests. In northern Sweden, lingonberry was more common than bilberry and raspberry. In this region, coverage was highest (8–9%) in stands of Scots pine, while it was 4% for Norwegian spruce. Productivity was relatively high on clear-cut areas, in young and very old forests, whereas productivity was low on 80 to 90% of the forests. Most of the berries (70–80%) were produced in the forests of northern and central Sweden. Between 70 and 80% of the total production was considered available for harvest. The National Forest Survey for 1978 through 1980 investigated the occurrence of cloudberry, small-fruited cranberry, and lingonberry (Kardell and Carlsson 1982; Kardell 1986). The inventories involved both forests and bogs. The latter covered 5.1 million ha with average lingonberry coverage of 1.1%. The results indicated that lingonberry preferred mineral soil, where the coverage increased from south to north, while the opposite occurred in bogs. A negative relationship was reported between lingonberry coverage and site elevation. The total mean berry production during this investigation was 209 million kg yearly. Only 4% of this yield was from bogs. The yield as a whole was widely distributed over the country. The highest yields (13.8 and 17.3 kg /ha) were noted from forests and bogs in central Sweden. Graff (1991) calculated the total Norwegian lingonberry production, which varied between 44 and 115 million kg per year. Raatikainen (1988) and Raatikainen et al. (1984) noted that variation in berry production depended on different factors, such as tree-canopy density and lingonberry coverage in Finland. The total annual production was estimated at 180 to 200 million kg, with an average yield of 8 kg/ha in the forests. About 80% of these berries were considered harvestable. However, lingonberry yield varies from one year to another due to night frost in June (Kardell and Carlsson 1982). About 50% of the flower buds, flowers, and green fruits are killed at temperatures below –1.5°, –3.1°, or –3.5°C, respectively (Teär 1972). There was also an association between snow cover and yield. Lingonberry buds were unable to withstand minimum temperatures, which varied between –25° and –32°C in January (Raatikainen and Vänninen 1988). After the exceptionally cold winter of 1985, highest yields were harvested from plants that had overwintered below a thick snow cover. The annual production of lingonberry in the Nordic countries can be estimated at 500 million kg, of which 80% is considered available for harvest. However, only 2 to 11% of the total production is collected. Through a mail survey, Hultman (1983) found that 38% of the Swedish population picked 15 L of lingonberry per person annually. People in northern Sweden gathered more berries than those in the south. The per-

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centage of berries picked was 28% in the south and 7% in the north, i.e., 11% of the production was harvested in Sweden. Only 3 to 5% of the total lingonberry production was picked in Norway (Graff 1991), and 2 to 7% of lingonberry production was harvested in Finland (Saastamoinen 1981). During the 1970s the average picker gathered 14 L per year (Hultman 1983). Saastamoinen and Lohiniva (1989) studied lingonberry picking in the Rovaniemi region of Lapland. About 70% of the households participated in the harvest. The total quantity of wild berries collected was 2 to 4 times larger than the national average. This is considered typical for rural communities in northern, eastern, and central Finland. Approximately 86% of the families in five communities in central Finland picked lingonberry and 9 to 44% of the total production in this area was gathered (Rossi et al. 1984). E. Effects of Forestry Management As lingonberry picking is a popular family pastime, there is an apprehension that modern forestry could threaten berry production. Consequently, the section of Environmental Forestry of the Swedish University of Agricultural Sciences (SLU) started a series of experiments at 27 testing sites across Sweden in 1976 with the aim of studying the relationship between silviculture and the development of grand cover vegetation. The original status of the sites was mapped during the first year. Different treatments that included thinning, clear cutting, fertilization, and soil disruption were carried out. Results from these experiments were reported after 5, 10, and 15 years (Kardell and Eriksson 1983, 1990, 1995). Lingonberry biomass was reduced by 15 to 20% directly after thinning of the overstory trees. Recovery was slow over time but thinning was positive for vegetative lingonberry development and, after 15 years, the coverage was slightly higher than in the control plots. Additionally, thinning resulted in a 3- to 4-fold yield increase. Clear cutting also led to decreased lingonberry biomass in the beginning, but after 15 years the plants had completely regained their positions. Lingonberry thus has good competing capacity on clear cuts and takes advantage of extra light. Clear cuts may be exposed to spring frosts, but in general these are good areas for berry production. On average a 4-fold yield gain was recorded. Development after fertilization with 150 kg N/ha was negative, as lingonberry coverage and yield decreased by 12 and 45%, respectively, following fertilization. Soil disruption caused a 20% biomass reduction after clear cutting and reduced berry production over 14 years by 39%.

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During a six-year period, removal of branches in clear-cut areas doubled yield, while stump removal had the opposite effect (Kardell and Wärne 1981; Kardell 1992). Pinus contorta forests allow less light penetration and lingonberry yields were 46% lower in those forests than those growing with P. silvestris (Kardell and Eriksson 1989, 1990). Kardell and Eriksson (1990) predicted that lingonberry production in Sweden could decrease by 10%, owing to soil fertility, mechanical soil disruption, and the widespread introduction of Pinus contorta. In contrast, minimal use of fertilizers and machinery, thinning, and clear cutting followed by dead branch removal appears to benefit lingonberry growth and production. Flavor of berries harvested from plants grown in fertilized plots was judged by a test panel to be slightly inferior to those plants grown in unfertilized plots (Kardell et al. 1981; Åkerstrand et al. 1988). The berries from fertilized plots had higher N levels during the year of treatment. Furthermore, molds and yeast were common in the samples from fertilized plots, resulting in reduced storage life. However, the effects of fertilization were considered small in comparison with those associated with environment and post-harvest handling. F. Experiments in Natural Habitats A prerequisite for successful field planting is the knowledge of the conditions prevailing in natural habitats (Teär 1972; Lehmushovi 1977a). This point was considered for ecological research by Teär as well as by Finnish researchers, who focused their investigations on wild stands from 1968 to 1976. Soil characteristics, temperature, light, and moisture were investigated to elucidate the factors promoting vegetative and reproductive development. The effects of fertilizer applications were also studied. One of the most important factors influencing yield is the weather during flowering. Frost, severe drought, or abundant rain may lead to 60 to 100% loss of buds, flowers, and unripe berries (Lehmushovi 1977a). In the years with favorable weather, this loss ranged from 30 to 60%. Crosspollination by insects and good light conditions are also essential for fruit set. The results of fertilizer trials in natural populations in forests are contradictory. A 2- to 3-fold gain in fruit yield after fertilization has been reported (Lehmushovi and Hiirslami 1972; Lehmushovi 1977a), but if the natural habitat included competing grasses and broad-leaved herbs, there was no benefit. Teär (1972) found a lower percentage of flowering shoots in fertilized plots than in unfertilized plots. The fertilizers did not

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affect the number of flower buds per shoot; however, their use resulted in slight increase in the production of rhizomes, new shoots, and leaves.

V. HORTICULTURE A. Propagation 1. Plants Collected Directly from the Wild. The cheapest way to propagate lingonberry is by digging up plants from natural habitats. Plants may be dug early in the spring, before growth starts, or late in the autumn when it has ceased. If homogeneous clumps of lingonberry were chosen and subdivided before planting, 73% survived when there were four plants per division, while only 18% survived when there were 15 plants per division (Teär 1972). Material collected from native stands should be planted with only the shoots above ground, because the lingonberry plants develop roots in the uppermost soil layer. These plants were susceptible to drought and frost heaving due to their fine, fibrous, and shallow root system. Plant survival ranged from 30 to 90% (Öster 1974). Plants could be established more reliably if the plants were first grown in humid peat nursery beds. After three growing seasons, plants that were first grown in nursery beds had better coverage (80%) than those directly planted from the forest (50%), though these coverages were 80% and 90%, respectively, the following year (I. Hjalmarsson, unpublished). After 3 to 5 years with an initial density of 80 to 100 plants per m2, an even cover was achieved with 400 to 600 plants per m2 (Teär 1972). Plant materials from sunny locations were more difficult to establish than those from shaded locations (Öster 1974). Selected plants had stronger sympodial growth, shorter shoots, greater number of leaves, twice as many flower buds per flowering shoot, and 3 to 4 times more shoots per plant than those from native stands (Teär 1972). In a 5- to 6-year-old lingonberry field, more than 1000 flower buds per m2 were observed, while there were only 300 to 500 flower buds per m2 in the forest (Lehmushovi 1975). The major drawbacks with wild plantlets are that they have a poor rate of establishment, are heterogeneous, and are difficult to handle. 2. Shoot Cuttings and Rhizomes. Propagation by shoot cuttings has been successful (Öster 1974; Lehmushovi 1975). The best results were obtained with mature spring and autumn shoots, while soft wood shoots taken during the summer were the most difficult to root (I. Hjalmarsson, unpublished). The shoots required about eight weeks for rooting and the

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most favorable substrate was peat. An average rooting percentage of 85% was achieved when mist and peat beds were used (Lehmushovi 1975). Pre-treatments with auxins also promoted rooting and almost 100% rooting was obtained if cuttings were over-wintered in non-heated greenhouses. The shoots were taken from September to October, placed in moistened peat, and wrapped with transparent plastic. By March or April the plants were ready to be transplanted in the field. The best shoot cuttings of ‘Sanna’ were collected in spring and late summer (Gustavsson 1998). However, cuttings from the current year growth were more uniform than mature shoots. Thus, softwood cuttings taken in late summer were recommended. Rooting was more successful in outdoor plastic tunnels than in the greenhouse for ‘Sanna’ (Gustavsson 1998). Plants from cuttings generally grow well in the field and tend to crop early. However, their rhizomes develop slowly. Plants raised from micro propagation produce rhizomes more easily. Rhizomes can propagate lingonberry, therefore shallowly planting 5 to 10 cm rhizome pieces in boxes with moist peat has been recommended (Öster 1974). One or two shoots normally develop at the terminal end, while roots are produced basically. Best results were achieved during spring and late summer, when 60 to 80% of the material was rooted. Similar results were reported by Lehmushovi (1975), who, nevertheless, concluded that rhizomes were difficult to procure and use for propagation. Lack of strong rhizomes with well-developed buds may result in weak plants. Also, rhizomes are sensitive to drought and therefore impossible to plant directly in the field without first growing in a nursery. 3. Seedling Plants. Lingonberry is easily propagated by seeds. The berry contains many seeds that can be cleaned by pulverizing the berry with water in a blender. Well-developed seeds will sink to the bottom and the rest of the mix is then decanted. Seeds germinate well directly after harvest but can also be cold-stored. About 72% seed germination (in dry seeds that were kept in a refrigerator for six months) has been observed after two weeks in damp sand (Lehmushovi 1975). Results from experiments with whole berries, pulverized berries, or direct seed sowing in the field at Ottarp were not encouraging. The acids in the fruit flesh inhibited seed germination (Karlsson and Malmberg 1974), and seedling plants were very susceptible to drought immediately after sprouting. The most preferred sowing appears to be in boxes with fertilizer-free peat and a top layer of sand during winter (Hjalmarsson 1993). The seeds are not covered because light is necessary

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for germination, which begins in about three weeks at 20° to 25°C. Seedlings are watered once a week with a complete nutrient solution, given additional light, and then transplanted as soon as they can be handled. After hardening-off at the beginning of July, they are transplanted to the fields. In greenhouse experiments, the number of leaves increased from 5 to 23 after three months, and shoot length from 1 to 7 cm (Hjalmarsson 1977). The development of leaves and shoots was simultaneous. However, the burst of leaves at the terminal end occurred in flushes and 3 to 4 leaves were born at a time, with 10 days between each flush. When the plants had reached 4 cm in height (11 leaves), the first lateral shoots became visible just above the cotyledons. Seedlings grown outdoors produced dense stands; after two years, for each m2 there were 2755 current year shoots, 330 flowering shoots, 430 flower clusters, and 28 m of rhizomes, which accounted for 9.4% of the total biomass dry weight (Hjalmarsson 1977). Lingonberry seedlings also develop rhizomes when they are very young. In addition, seedlings offer a quick way of producing a large quantity of plants, but fruit production could be delayed by 2 to 3 years due to juvenility and seedlings are genetically diverse. B. Frost Protection An experimental field at Ottarp was consistently exposed to spring frosts. Consequently a frost protection experiment was established in 1976 (in 4 blocks of 12 m2), which consisted of gravel mulch, covering with transparent plastic film, and spruce twigs along with a control. The transparent plastic was laid on wooden frames and, as with the spruce twigs, was removed during warm days. In the sandy soil at Ottarp the gravel mulch increased the temperature near the surface by 0.5°C, whereas the temperatures for plastic film and spruce twigs were 1.4° and 1.6°C greater than ambient temperatures. In 1978, a year with four frosts during spring, all plots were harvested and it was observed that all treatments increased yield. The best result was obtained with plastic film, where a 4-fold yield gain was obtained compared to the control. In mulched plots lower temperatures were observed than in control plots at Balsgård (Gustavsson 1993). This was especially noted in sawdust plots, where the difference was 1.8°C. Transparent plastic covers during the night raised the temperature in the control 1.4°C higher, as in the Ottarp experiment. The positive response to transparent plastic film covering was less pronounced with peat and sawdust.

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C. Culture 1. Soil. In nature, lingonberry grows most abundantly in conifer forests, where the environment is characterized by leached soils with low pH, low base saturation, and low calcium content. Nutrient turnover and nutrient availability is low. Rhizomes and roots grow shallowly, mainly in the humus horizon, which has a porous structure and is considered the principal source for nutrients (Teär 1972). The effects of different soil types were investigated in a replicated trial that included eight different types of soils varying in the amount of clay, humus, and loamy sand plus two organic soils with a high percentage of humus and pure peat at an outdoor frame-yard. The pH ranged from 5.8 in the silt loam to 4.2 in the peat substrate. Within each frame an area was photographed every autumn and spring to record plant development and rhizome spreading. With the aid of these photos the surface coverage of lingonberry shoots and leaves was assessed. The best and fastest plant development was recorded in the pure peat and loamy sands with moderate humus content. These results, supported by Finnish researchers, documented superior growth and coverage in peat (Lehmushovi and Hiirsalmi 1973; Lehmushovi and Säkö 1975). Plants grown in peat, in which coverage was 95%, had greater biomass, of which 24% was accounted for by rhizomes and roots. One quarter of the total shoots from plots in peat were fertile, whereas in the silty loam only 2% were fertile. The highest fruit yields were recorded in plots with the best vegetative development. However, length of the juvenile phase and the beginning of fertility varied among different seedlings. In another experiment the effects of soil types, nitrogen fertilizer rates, and peat mulch were investigated (Hjalmarsson 1980). Fertilizer was added at four nitrogen levels. The organic soil provided the best environment for vegetative growth and the silt loam the poorest. Plant analyses at the end of the experiment indicated that nutrient uptake was similar in the different soil types. However, the percentage of calcium was significantly higher in plants grown in the calcium-rich silt loam. The first-year fruit yield was only 10 g per plot, except for plots with sand, where the yield was 40 g. The following two years the sandy soils were superior for yield, whereas those plants grown in the silt loam had the lowest yield. It was speculated that vigorous plants grown in the organic soil showed delayed flower bud initiation but would have higher productivity at an older age.

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2. Nutrients. Nutrition research in lingonberry has concentrated on nitrogen fertilization; however, the effects of phosphorous, potassium, and lime have also been investigated. Nitrogen fertilization is beneficial to vegetative growth and up to 5 g N per m2 has enhanced growth without a decrease in berry quality. At rates greater than 5 g N per m2, berry production was negatively affected. In these studies nitrogen was applied in a solution that contained 100 g N (NH4–N/NO3–N 40/60), 13 g P, 65 g K, 9 g S plus trace elements, but no calcium. Leaf analysis showed that nitrogen and potassium concentration or content increased and calcium content decreased concomitantly to increased fertilizer rates. Studies of the effect of dolomitic lime (6 and 12 kg per m2) and a balanced fertilizer (NPK 11-11-22 at 5 or 10 g/m2) showed that liming slightly decreased spreading, shoot height, and significantly lowered berry production (Lehmushovi and Hiirsalmi 1973; Lehmushovi and Säkö 1975). In contrast, the greatest ground coverage, tallest shoots, and the highest yields were recorded in plots with fertilizer, although berry size was small in these plots. Joint application of lime and fertilizer negatively affected all characteristics. In another experiment, nitrogen, potassium, and phosphorus were applied singly or in mixtures with two or three elements, and with or without trace elements on a mineral soil (Lehmushovi 1977a). Yields were low because the plants did not grow well in the mineral soil with clay. Nevertheless, fertilizers in small amounts increased fruit yield, while larger application rates did not increase yields. In the investigations with a range of nitrogen fertilizer rates and saltpeter plus sulfate of potassium-magnesium, the best growth was in the control and at the lowest nitrogen application (Sakshaug 1974; Hjalmarsson 1980). Soil analyses in autumn showed that the higher the fertilizer rates, the lower the soil pH and the higher total salt concentration. Nitrate also increased, while ammonium, phosphorous, and potassium were more stable in the soil. Yield responses could not be evaluated due to annual frost, although berry size was consistent over years and across treatments. Slow release sources of nitrogen on sandy soils have not proven to increase growth or yield of cultivars when compared to standard fertilizers. In solution, ammonium uptake was faster than nitrate uptake and lethal levels were reached at 400 mg/L (Ingestad 1973, 1974). Based on growth studies, a 40 ammonium/60 nitrate ratio was recommended. The nutrient requirements to grow lingonberry seedlings were 100 N, 50 K, 13 P, 7 Ca, and 8.5 Mg mg/L (Ingestad 1973, 1974). With the above

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combination of nutrients, maximal growth with lingonberry seedlings was achieved at pH 4.5 to 5.0 and conductance 0.4 to 0.5 mS, which are typical for calcifuges. Potassium regulates water movement in plants and may be less critical in xeromorphic lingonberry. Lingonberry seedlings grown in nutrient solution have the same mechanism regulating potassium uptake as spring wheat, cucumber, birch, pine, and spruce (Jensén and Pettersson 1978). However, in contrast to these species, lingonberry did not appear to be able to accumulate potassium ions. Uptake was limited to what was needed for current growth. A remarkable characteristic of lingonberry was its rapid absorption of calcium (Ingestad 1973, 1974). Such a phenomenon is also typical of species that are adapted to acid soils, but still have a physiological calcium requirement like the other species. Calcium was taken up together with nitrate. Lower growth rates and yellowing leaves were noted in the treatments with high nitrate concentrations, indicating that inhibitory levels of calcium had been reached. The chlorosis was also ascribed to low mobility and activity of iron within the plant tissues due to internal increase of pH. Ingestad (1973) pointed out the practical consequences of his findings. First, the potassium/calcium ratio may have a bearing on water economy, especially if lingonberry is grown in soils with high calcium availability. Low potassium level may lead to poor water-holding capacity, and leaves with lime-chlorosis are more drought-sensitive than normal green leaves. Second, sensitivity to high total salt concentration indicates the importance of small but frequent fertilizer applications and the need to avoid drought. 3. Mycorrhiza. All the members of Ericales are characterized by ericoid mycorrhizae and lack of root hairs (Lihnell 1974; Önner 1977). The mycorrhizae are endotrophic and the fungal hyphae penetrate the epidermal cells of the roots, resulting in a root system in which a high proportion of the biomass is composed of fungal material. Young active roots tend to be infected by mycorrhizae, while they are not found in older roots that have lost their epidermis. Some American scientists such as Goulart et al. (1993) ascribed the ericoid mycorrhizae as most important in their role as nitrogen supplier, though they may also enhance phosphorous uptake. Interactions with heavy metals and soil-borne root diseases were also discussed by these researchers. The fungal species Hymenoscyphus ericae (Read) Korf and Kernan (formerly called Peziella ericae Read) was found on lingonberry (Önner 1977).

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Experimentally it has been shown that lingonberry seeds can germinate and develop normally under sterile conditions (Lihnell 1974), consequently the symbiosis is not obligatory. Mycorrhizae were observed in all living epidermal root cells from all samples collected in wild stands (Önner 1977). There were no differences in degree of infection between samples from wild stands or from those of experimental fields. Weak growing plants tended to have fewer epidermal cells and therefore less mycorrhizae; nevertheless, this did not explain the weak growth of lingonberry plants. Önner (1977) claimed that mycorrhizae are very important for lingonberry in wild stands characterized by low fertility, especially in dry environments. The symbiosis appears to be less important when water and nutrient levels are not the limiting factors. The fact that most of the hyphae are found inside the root could be ascribed to the low pH in the soil, which inhibits the growth of mycorrhizae. It has, therefore, been suggested (Goulart et al. 1993) that the mycorrhizae can assist the host to adapt to a slightly higher soil pH. 4. Mulching. Mulches have been established for frost protection and for other purposes. During the 1970s, the aim was to imitate the humus horizon in natural habitats to obtain a better soil substrate. Later experiments focused on weed control. The growth of wild plants dug from the forest was studied following addition of different amounts of peat mulch (Sakshaug 1974; Hjalmarsson 1980). In addition, different levels of nitrogen in combination with the peat mulch were tested. The nitrogen fertilizer (3 g N/m2) was split into six applications of 0.5 g N/m2 per year—the first three times as ammonium sulfate, thereafter as calcium ammonium nitrate. In addition sulfate of potash-magnesium was included for the first three times. The plots were mulched with wet peat at the end of May 1972. The peat was limed with 1 kg of dolomitic lime/m2 and the peat mulching was repeated in 1974. Weeding was performed by hand in the first year. The following years herbicides, which had been tested at Ottarp previously, were used. Furthermore, fungicides were applied against “leaf falling disease.” In the first year after application of peat, if the peat layer was 4 cm greater, shoot growth was damaged. However, upon application of the mulch two years later, there was no negative effect. Plants mulched with 4 cm of mulch produced twice as many new shoots as the unmulched control. When nitrogen was added in the peat, the fertilized plots with 2 cm peat had a similar positive effect as the treatment with 4 cm peat. Generally the treatments without peat mulch showed the

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weakest growth, and there was a trend that mulched plots with fertilizer had more shoots than their unfertilized counterparts. No effects of mulch or fertilizer on survival of frost at bloom were noted, but differences between individual years were observed and associated with frost damage. Peat mulch caused a decrease in salt concentration, nitrate, phosphorous, and potassium contents, which may explain the more vigorous growth in those plots. Other organic (peat, sawdust, pine needle) and non-organic mulches (gravel, plastic) were also tested (Gustavsson 1998, 1999). Vegetative growth and fruiting were consistently superior with peat mulch. Winter frost damage was highest on the organic mulch treatments, while the plots with plastic mulch and gravel were unaffected. Growth in sawdust mulch was poor and the experimental plots were scored for fungal disease. Fungal disease symptoms were most severe with gravel mulch and control, and least severe with pine needle mulch. In summary, genetic growth habit affects the outcome of soil surface treatments. Low-growing and rapidly spreading cultivars like ‘Sussi’ grow well without mulch, while bush-like cultivars like ‘Sanna’ tend to suffer from broken branches on open soil (Saario and Voipio 1997). Covering the soil with plastic film mulch is not recommended for rhizomeforming cultivars like ‘Sussi’. 5. Irrigation. Irrigation provides a means to achieve consistently high yields and high fruit quality in most small fruit crops. Lingonberry grows best with a combination of irrigation and peat mulch (Hjalmarsson 1980). More shoots are observed where extra water has been provided. Increasing irrigation from 40% of field capacity to 100% field capacity of loamy sand soil increased rhizome number by 37% and shoot number per plant by 39% (Stang et al. 1993a). Peat mulch favors shoots developing from rhizomes. D. Plant Pathology 1. Weed Control. Weeds were a major problem in the early attempts to grow lingonberry. Young plants are unable to compete against weeds and the rhizomes close to the surface are easily damaged by mechanical cultivation. Herbicides (Andersson 1974, 1976; Fernqvist 1977; Hjalmarsson 1980), mulching (Gustavsson 1993, 1996; Saario and Voipio 1997), and allelopathy (Saario 1998) have been studied as possible ways to suppress weeds. A number of herbicide trials were performed at Ottarp early in the 1970s (Andersson 1974, 1976; Fernqvist 1977; Hjalmarsson 1980).

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Promising results were obtained with lenacil and linuron for annual weeds and with dichlobenil for perennial weeds in the Nordic Region of Europe. However, berries from untreated plots had the best taste (Ingelög et al. 1977). None of the herbicides or dosages in various experiments were harmful to lingonberry unless summer applications were used; therefore, they should be avoided. In Wisconsin, terbacil, oryzalin, and simazine herbicides provided effective weed control at application rates recommended for other perennial crops (Stang et al. 1993a). At present there are no herbicides registered by official authorities for use in cultivation of lingonberry in Scandinavia or in the United States. In studies comparing mulching materials (peat, wood chips, and sawdust) and herbicide treatments (glyphosate and propyzamide), the latter eradicated perennial weeds during winter, but propyzamide was no longer inhibiting weed germination in spring and summer. By summer the applied herbicides were no longer effective and the plots had many weeds. Sawdust mulch has consistently and reliably given the best weed control. Mulching with peat and wood chips can also be effective. Herbicides can reduce berry numbers, even though the plants look healthy. The general recommendation for successful weed control, safe plant development, and clean berries involves a combination of herbicides and mulching (Gustavsson 1999). 2. Diseases. Disease attacks have not yet caused damage of economic importance in lingonberry plantings (Gustavsson 1997). Nilsson (1974) described the pests and types of damage found in domesticated lingonberry and divided them into four classes: deformed plants, leaf spots, berry symptoms, and dead shoots. In the first group is lingonberry tumor caused by the fungi Exobasidium vaccinii Fuckel (Woronin), which causes leaves to form thick reddish knobs. However, tumors are more common in the forest than in the field, probably due to humidity. A more serious pest is the little leaf disease, which was found in the field at Ottarp. This disease is spread by cicada and caused by a phytoplasma (Tomenius and Åhman 1983). The risk of infection, which may result in stunted plants and dwarf leaves, can be reduced by always using healthy plant material and eliminating suspected plants in the surroundings. There are two main foliar diseases; one of them, mostly seen in forests, is thought to be caused by the fungus Mycosphaerella stemmatea (Fr.: Fr.) Romell (Magnusson 1976). The infection is characterized by dark spots (4–5 mm in diameter) surrounded by red-brown edges. “Leaf falling disease” is another condition that causes spots that appear in the autumn. These spots have diffused edges and cover large areas, and the

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infected leaves fall off during autumn and spring. In addition, infected plants are thought to be less winter hardy. Magnusson (1976) tried unsuccessfully to isolate the causal fungus. Nilsson (1974) tested fungicides, commonly used in orchards, to control “leaf falling disease” and good results were obtained when fungicide applications started in June (suggesting that the fungi disperse spore early). Monilinia urnula (Weinm.) Whetzel, whose symptoms are grey and hard berries, may have a negative economic impact in the future (Magnusson 1976). The same is true of Godronia cassandrae Peck, which has been found in plantations of highbush blueberry (V. corymbosum) (Magnusson 1976). It has also been reported on wild lingonberry in Sweden, Finland (Eriksson 1970), and Norway (Gjaerum 1969). E. Crop Improvement 1. Early Studies of Ecotypes. During the first attempts to domesticate lingonberry, wild germplasm was collected for comparative studies. A collection of 500 accessions from all over Sweden were planted from 1962 to 1965 and studied by Teär (1972), who observed variation in fertility, number of clusters per shoot, and number of flowers per cluster. About 10 promising ecotypes were propagated for further studies and sent to Öjebyn Experimental Station in northern Sweden. The same year (1969) the station also received a large collection of unselected accessions from southern Sweden. Unfortunately, the ecotypes were collected as sods and it was therefore uncertain whether they consisted of one or more clones (Öster 1974). The material varied considerably in growth behavior and yield. The Finnish wild material has also been surveyed at Piikkiö. A field experiment was established with 88 clones from all over the country in 1969–1970 and evaluated in 1973–1974. The original clones were divided into ten geographic areas to facilitate statistical analysis (Lehmushovi 1986). Data on shoot characteristics, flowering, fruit set, yield, and berry weight were collected. The comparison indicated a trend of increasing shoot height from south to north. The flowering period was found to be rather long, on average, 30 and 33 days. Bloom was shortest (23–26 days) in the northern clones and longest (40–45 days) in the southern clones. Coastal clones were more fertile than the inland clones. The total number of flowers was highest in the southern material and lowest in the northern. Fruit set varied between 23 and 75%. The lowest percentage was noted on plants from Åland and the highest on plants from southeastern locations. In addition the accessions from Åland proved to be least productive, while plants from a southeast

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area yielded most. Berry size increased towards the north. Lehmushovi (1986) suggested that tall shoots, large berries, and late but short bloom are important characteristics for new commercial cultivars. 2. Breeding at Balsgård. The first two Nordic cultivars, ‘Sussi’ and ‘Sanna’, were introduced at Balsgård in 1987 and 1988, respectively (Trajkovski and Sjöstedt 1986; Eckerbom 1988). Both cultivars originated from open-pollinated seed samples collected in Småland (Sweden) forest from large stands of high-yielding genotypes by the late Professor Dr. Sven Dalbro of the Royal Veterinary and Agricultural University (KVL), Denmark. Seedlings were selected for vegetative growth, fruiting habit, and plant health in Balsgård. Four populations originating from the above-mentioned material were studied in a growth chamber at Alnarp (Hjalmarsson and Ortiz 1998). The results suggested that in wild lingonberry spreading ability (i.e., number of rhizomes), growth, plant height, and number of vegetative shoots and flowering shoots are genetically controlled. In Balsgård, lingonberry breeding was intensified in 1990 (Gustavsson 1992, 1993, 1996, 1997). Breeders at that time had 50 genotypes that had been selected for high yield, large berry size, concentrated maturity, and resistance against “little leaf disease” from seedling plants originating in Småland and northern Sweden. Outstanding clones were propagated for comparative studies together with other known cultivars as well as some Latvian and Lithuanian selections. To further broaden the genetic base for breeding, seedlings from Fennoscandia, the Baltic States, Russia, and Japan were raised in 1992 and 1993. Three years later there were 84 lingonberry accessions from different natural populations in Balsgård, from which second and third generation seedlings were planted out for assessment (Gustavsson 1997). Plant height, size of leaves and berries, precocity, plant vigor, and amount of rhizomatous growth, productivity, fruit ripening time, tendency towards off-season flowering, winter hardiness, and disease resistance were evaluated. Crosses began in 1993 to determine the best breeding method. A modification of the method described for blueberry (Galletta 1975) was identified as the best. In 1994 and 1995 several crosses were made using ‘Sussi’ and ‘Sanna’ as well as two American cultivars, ‘Splendor’ and ‘Regal’ that were derived from a Finnish seed lot and released by the University of Wisconsin at Madison (Stang et al. 1994). Gustavsson (1997) found large variation in fruit set and seeds per berry between crosses and years. In some crosses very few berries were obtained, and these berries sometimes contained only non-viable seeds.

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Gustavsson postulated that this phenomenon might be caused by sterility barriers existing between certain cultivars. The first lingonberry cultivars on the market were selections from wild stands in Germany and The Netherlands (Zillmer 1984). The German cultivars ‘Erntedank,’ ‘Erntekrone,’ and ‘Erntesegen’ were developed by A. Zillmer and released in 1975, 1978, and 1981, respectively (Zillmer 1984, 1985). These cultivars and the Dutch ‘Koralle’ were the first plants whose fruit were harvested commercially in Europe (Dierking and Dierking 1993). Today there are about a dozen lingonberry cultivars worldwide selected from wild material. The crosses undertaken at Balsgård appear to be the first in which several known cultivars were combined to obtain improved germplasm (Gustavsson 1997). 3. Description of Swedish Cultivars. The descriptions are based on information included in the catalogue of the Swedish Elitplantstation (Nilsson and Rumpunen 1997) as well as other publications (Gustavsson and Trajkovski 1999). Lingonberry growers in Germany are interested in early Scandinavian cultivars and their ability to shorten the harvest time (Dierking and Dierking 1993). ‘Ida’. Released in 1997. The plant is average in size (0.1–0.2 m) and rather dense. Production is about 140 g per plant (three-year-old stands). This cultivar may be harvested twice in southern Sweden (middle of August and October). The berries are large (0.6 g per berry) and it is popular as an ornamental plant because of its beautiful leaves and abundant and repeated flowering periods. ‘Linnea’. Released in 1997. The plant is upright (0.15–0.25 m) and rather dense with a few rhizomes. Production is about 150 g per plant (threeyear-old stands) and the very good quality, medium-sized berries (0.4 g) ripen in the late season. ‘Sanna’. Released in 1988. The plant is upright and its height ranges from 0.2 to 0.3 m. Production varies between 200 and 600 g per plant or 5 to 10 metric t ha–1 (four-year-old plants). Fruit ripen in midseason, i.e., from the middle of August to the beginning of September. The bright red berries are larger than the average size of wild accessions (see Plate 3.1B), and are excellent for processing, particularly jam making. ‘Sussi’. Released in 1986. The plant habit is low growing (0.15–0.25 m) and the rhizomes spread rapidly. Production ranges from 200 to 300 g

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berries per plant (four-year-old stands). Fruit mature in mid-season (around August 20), and are dark red berries that are larger than the average wild berries. ‘Sussi’ is excellent for processing, especially for jam production. 4. Description of North American Cultivars. The University of Wisconsin–Madison named and released two new cultivars (‘Splendor’ and ‘Regal’) derived from open-pollinated seed collected in southwest Finland in the mid-1990s (Stang et al. 1994). ‘Splendor’. Released in 1994. The plants are precocious and show moderate spreading and plant height (0.15–0.19 m) at maturity. Fruit yield is about 25 g per plant. The brilliant carmine red fruits are medium (0.41 g) and ripen in mid to late September. ‘Regal’. Released in 1994. The plants are precocious and show moderate spreading and plant height (0.18–0.22 m) at maturity. Fruit yield is about 29 g per plant and the fruit are small (0.33 g), bright red, and the best harvest time is in late September.

VI. SUMMARY AND FUTURE PROSPECTS The Vaccinium genus comprises many interesting berry species. Among them are the American blueberry and cranberry, which were successfully introduced as commercial crops during the last century. There is reason to believe that the European lingonberry has a similar potential. Today large quantities are harvested from the wild and there is an important worldwide trade. Other Vaccinium species might be considered for cultivation as well. Vaccinium plants in general have certain highly appreciated qualities. The plants are easy to grow and can be grown on marginal land with a minimum of fertilizers and pesticides. The berries, consumed either as fresh or processed, possess an attractive taste and healthful properties. In addition they are suitable for machine harvest and have good keeping qualities. Through breeding, it is also possible to combine unique and valuable properties from different species. For example, one challenge for breeders would be to combine the high levels of antioxidantia in the European bilberry with the high productivity of its North American counterparts. Most certainly continued research in Vaccinium will lead to an

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increased number of species and hybrids in cultivation, an expansion of the cultivated area both in acreage and geographic distribution, and new functional food products on the market. This review has emphasized Nordic research on lingonberry and has provided information on the design and outcome of Swedish attempts for domestication of this species. Additional research has been reported in Germany (Liebster 1984; Dierking and Krüger 1984) and in the former Soviet Union (Paal 1992). More recently researchers in Alaska (Holloway 1984), Wisconsin (Stang et al. 1990, 1993b, 1994), and Canada (Estabrooks 1997) have also focused on the domestication of lingonberry as a new crop. According to Galletta and Ballington (1996), lingonberry may extend Vaccinium culture further north (in North America and Europe) and south (in South America). However, weed control and the high cost of planting material remain the two major constraints for lingonberry cultivation (Zillmer 1998). As with the highbush blueberry in the United States (Eck 1966a), the first Nordic experiments with lingonberry were aimed at determining soil and environment requirements for its cultivation, followed by an emphasis on breeding. Both organic soils and sandy soils with moderate to high content of humus were preferred by lingonberry, while soils with high clay content inhibited plant development. A well-drained acid substrate is essential for the cultivation of this species. Application of peat mulch increases vegetative growth and berry yield. In addition field experiments indicated the importance of irrigation in dry periods. Furthermore, although contradictory results were sometimes obtained, the experiments suggested that application of nitrogen fertilizer at low levels (5–10 g/m2) positively affected the vegetative lingonberry growth. Experiments with hydroponics confirmed the sensitivity of this species to high salt concentrations. The plasticity of lingonberry was noted in a number of different investigations. There is improved adaptation to open and sunny fields through sympodial growth, and an increased number of flowers as compared to cultivation in shaded areas. Increased yields were also noted in open habitats unless the plants were affected by spring frost. The source of plant material affected performance and clonally propagated wild plants, dug from the forest, cropped early and had good rhizome development compared to seedling propagules. Seedlings were adequate producers of daughter plants, but were not as precocious. Currently propagation by cuttings is the method used for commercial production of lingonberry, whereas plants dug from native stands and derived from seedlings remain important for genetic enhancement.

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The ability to spread through rhizomes has been considered a problem rather than an asset in row cultivation. However, the early establishment of a dense carpet of vegetation offers some advantages. A mixture of genotypes in the field through the use of seedlings may insure cross-pollination, which will increase fruit set and yield. It is also likely that such a plantation system would be a long-term investment because rhizome systems of Vaccinium species have a long life, e.g., up to 100 years (Sjörs 1989). According to Galletta and Ballington (1996) there has been interest in establishing lowbush blueberry fields by using seedling progenies from elite clones. This idea may also be applied to lingonberry cultivation. Pioneering work was carried out by Lehmushovi (1986), who investigated wild plant material collected from across Finland in an experimental field in Piikkiö. Bloom length was one of the characteristics that changed according to origin and genetic background. However, all the Finnish clones exhibited one distinct flowering period, while lingonberry originating in central Europe tended to flower twice. Germplasm exchange between regions may therefore significantly affect plant biorhythms (Paal 1992). Collection, characterization, and evaluation of clones as well as the development of broad base germplasm are essential to achieve success in lingonberry breeding. Lingonberry, being a new crop, offers a unique challenge to breeders and genebank curators. Germplasm collections must be enriched to meet the need for the genetic enhancement of this species. Luby et al. (1990) recommended comprehensive seed collections of native forms as well as field genebanks comprised of elite wild clones and cultivars for the conservation of Vaccinium genetic resources. Based on the results reported in this review, we recommend that further research also focus on the uptake and utilization of nutrients by lingonberry. Xeromorphic leaves, symbiosis with mycorrhiza, and enhanced adaptability to survive in distinct environments create a complex system that is not fully understood. The results of such research may affect breeding strategy and crop husbandry of lingonberry. In this process, highest priority should be given to develop an ecologically friendly cultivation system for lingonberry. The development of enhanced methods for pollination, weeding, and rejuvenation of lingonberry should be emphasized. Pests are another area that needs further study. The fungus causing “leaf falling disease” has not yet been identified and its potential interaction with decreased leaf longevity at high nutrient levels has not been investigated.

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Despite 40 years of lingonberry research and production, the industry associated with this crop is still in its infancy. One factor that has hindered its expansion is the economic return, which tends to fluctuate according to the size of the wild lingonberry harvest rather than market demand. Thus aggressive marketing of domesticated lingonberry appears to be crucial. Demand for lingonberry products will not increase unless the unique taste and health attributes of this species are promoted. The industry should consider products other than the traditional jam. In Finland the aroma of lingonberry is already appreciated in baby food, yogurt, ice cream, and liquor (Hiirsalmi and Lehmushovi 1993). Cranberry juice has become a popular drink in the United States, owing (partially) to its ability to prevent infections in the urinary tract (Avorn et al. 1994). Folk medicine suggests that the lingonberry juice may have similar qualities. In addition, recent research indicates that several Vaccinium species, among them lingonberry, contain anti-cancer compounds (Bomser et al. 1996). Lingonberry may also have an expanded future as a new berry crop in home gardens and as an ornamental plant. LITERATURE CITED Adelswärd, G. 1994. Vad menade André Mollet? Ljung eller lingon? Lustgården 74:47–60. Ahokas, H. 1971. Notes on polyploidy and hybridity in Vaccinium species. Ann. Bot. Fennica 8:254–256. Andersen, M. Ø. 1985. Chromatographic separation of anthocyanins in cowberry (lingonberry) Vaccinium vitis-idaea. L. J. Food Sci. 50:1230–1232. Andersson, C.-R. 1974. Kemisk ogräsbekämpning i lingon odling. Swedish Univ. of Agr. Sci. Konsulentavdelningen. Trädgård 71:33–36. Andersson, C.-R. 1976. Herbicide trials in lingonberries (Vaccinium vitis-idaea) during 1971–75. Unpublished report. Agr. College of Sweden, Dept. Pomology, Alnarp. Anjou, K., and E. von Sydow. 1967. The aroma of cranberries. Acta Chem. Scand. 21:945–952. Anonymous. 1983. Finland, the encyclopedia. Holger Schildts förlag, Helsingfors. Armfelt Hansell, Ö. 1969. Bärboken. P.A. Nordstedt och Söners Förlag, Stockholm. Audrina, B. 1996. The first results of cowberry breeding in Latvia. p. 48–56. In: K. Buivids (ed.), The Baltic Botanical Gardens in 1994–1995. National Botanic Gardens of Latvia, Salaspils. Avorn, J. M., J. H. Gurwitz, R. J. Glynn, I. Choodnovskiy, and L. A. Lipsitz. 1994. Reduction of bacteria and pyuria after ingestion of cranberry juice. J. Am. Med. Assoc. 271:751–754. Balwoll, G., and G. Weisæth. 1994. Horticultura. Norsk hagebok frå 1694 av Christian Gartner. Landbruksforlaget, Otta, Norway.

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Raatikainen, M., E. Rossi, J. Huovinen, M.-L. Koskela, M. Niemela, and T. Raatikainen. 1984. The yields of the edible wild berries in Central Finland. Silva Fenn. 18:199–219. Raatikainen, M., and I. Vänninen. 1988. The effects of the 1984–85 cold winter on the bilberry and lingonberry yield in Finland. Acta Bot. Fennica 136:43–47. Reier, U., and T. Paal. 1998. Germination of Vaccinium vitis-idaea and Rubus chamaermorus seeds originating from different latitudes. In: T. Paal (ed.), Wild berry culture: an exchange of western and eastern experiences. Estonian Agr. Univ., Forest Research Inst., Tartu. Forest Studies 30:147–156. Retzius, A. J. 1806. Försök til en Flora Oeconomica Sveciae eller Swenska Wäxters Nytta och Skada i Hushållningen. Lund, Sweden. Riechers, Ü., and G. Bünemann. 1989. Micropropagation of lingonberry (Vaccinium vitisidaea). Erwerbsobstbau 31:129–132. Ritchii, J. C. 1955a. A natural hybrid in Vaccinium: I. The structure, performance and chorology of the cross Vaccinium intermedium Ruthe. New Phytol. 54:49–67. Ritchii, J. C. 1955b. Biological flora of the British Isles. Vaccinium vitis-idaea L. J. Ecol. 43:701–708. Rossi, E., M. Raatikainen, J. Huovinen, M.-L. Koskela, and M. Niemala. 1984. The picking and use of edible wild berries in Central Finland. Silva Fenn. 18:221–236. Saario, M. 1998. Allelopathy of lingonberry? In: T. Paal (ed.), Wild berry culture: an exchange of western and eastern experiences. Estonian Agr. Univ., Forest Res. Inst., Tartu. Forest Studies 30:157–161. Saario, M., and I. Voipio. 1997. Effects of mulching and herbicide on weediness and yield in cultivated lingonberry (Vaccinium vitis-idaea L.). Acta Agr. Scand., Sect. B., Soil and Plant Sci. 47:52–57. Saastamoinen, O. 1981. Bär ger arbete och inkomster. Finlands Natur 40:164–167. Saastamoinen, O., and S. Lohiniva. 1989. Picking of wild berries and edible mushrooms in the Rovaniemi region and Finnish Lapland. Silva Fenn. 23:253–258. Sakshaug, K. 1974. Gödslings- och torvtäckningsförsök med lingon i Ottarp. Agricultural College of Sweden. Konsulentavdelningen. Trädgård 71:29–32. Schimmel, J. 1989. Regeneration of some common understorey species in northern Sweden after fire of different severity. M.Sc. thesis. Swedish Univ. Agr. Sci. Dept. Forest Site Research, Umeå. Schimmel, J., and A. Granström. 1996. Fire severity and vegetation response in the boreal Swedish forest. For. Ecol. 77:1436–1450. Serres, R. A., S. Pan, B. H. McCown, and E. Stang. 1994. Micropropagation of several lingonberry cultivars. Fruit Var. J. 48:7–14. Sjörs, H. 1989. Blåbär, Vaccinium myrtillus—ett växtporträtt. Svensk Bot. Tidskr. 83:411–428. Stang, E. J. 1994. Lingonberry cultivars—building blocks for an industry. Fruit Var. J. 48:3–6. Stang, E. J., M. D. Anderson, S. Pan, and J. Klueh. 1993a. Lingonberry cultural management research in Wisconsin, USA. Acta Hort. 346:327–333. Stang, E. J., B. A. Birrenkot, and J. Klueh. 1993b. Response of ‘Erntedank’ and ‘Koralle’ lingonberry to preplant soil organic matter incorporation. J. Small Fruit Viticulture 2:3–10 Stang, E. J., J. Klueh, and G. Weis. 1994. ‘Splendor’ and ‘Regal’ lingonberry: New cultivars for a developing industry. Fruit Var. J. 48:182–184. Stang, E. J., G. G. Weis, and J. Klueh. 1990. Lingonberry: potential new fruit for the northern United States. p. 321–323. In: J. Janick and J. E. Simons (eds)., Advances in new crops. Timber Press, Portland, OR.

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Statens jordbruksverk. 1994. Livsmedelskonsumtionen 1990–1993. Jordbruksverkets rapport 6. Jönköping, Sweden. Statens livsmedelsverk. 1978. Livsmedelstabeller. Energi och vissa näringsämnen. Uppsala, Sweden. Statistics Sweden. 1994. Foreign trade 1993: Imports and exports. Commodities according to harmonized system. Official Statistics Sweden. SCB Tryck, Örebro. Stodola, J., and J. Volak. 1986. Tidens stora bok om Läkeväxter. (B. Wahlin, trans.) Tidens förlag, Stockholm. (Original work published 1984). Sylvén, N. 1918. Jämförande försök med maskin och handplockning av skogsbär. Kungl. Lantbruksakademiens handlingar och tidskrift 57:62–76. Teär, J. 1972. Vegetativ och fruktifikativ utveckling hos vildväxande och odlade lingon. Ph.D. diss. Alfa-Lavals Offsettryckeri, Tumba, Sweden. Tiak, G., and A. F. Cherkasov. 1998. Experiences in growing Vaccinium vitis-idaea L. in European Russian central parts. In: T. Paal (ed.), Wild berry culture: an exchange of western and eastern experiences. Estonian Agr. Univ., Forest Research Inst., Tartu. Forest Studies 30:198–200. Tomenius, K., and G. Åhman. 1983. Mycoplasma-like organisms in the phloem of little leaf diseased plants of Vaccinium vitis-idaea and V. myrtillus in Sweden. Swedish J. Agric. Res. 13:205–209. Tolvanen, A. 1995. Aboveground growth habits of two Vaccinium species in relation to habitat. Can. J. Bot. 73:465–473. Tolvanen, A., K. Laine, T. Pakonen, and P. Havas. 1995. Recovery of evergreen clonal dwarf shrub Vaccinium vitis-idea after simulated microtine herbivory in a boreal forest. Vegetatio 116:1–5. Trajkovski, V., and B. Sjöstedt. 1986. Breeding of lingonberries and lowbush blueberries. Swedish Univ. Agr. Sci. Div. Fruit Breed.—Balsgård. Rep. 1984–1985:49–51. Valset, K. 1976. Ville vekster—nyttige frukter. Det norske hageselskap og Grøndahl and Søns Forlag, Oslo. Vestrheim, S., K. Haffner, and L. Øyre. 1994. Dyrking av tyttebær. Norsk Landbruk 113:9. Vieno, M., M. Komulainen, and S. Neuvonen, S. 1993. Seed bank composition in a subarctic pine-birch forest in Finnish Lapland: Natural variation and the effect of simulated acid rain. Can. J. Bot. 71:379–384. von Zabeltitz, C. 1989. Entwicklung einer Erntemaschine fur Kulturpreiselbeeren. Erwerbsobstbau 31:165–168. Waern, C. F. 1834. Underrättelser för år 1833, om Baldersnäs Trädgård, i Norra Dalsland. Svenska Trädgårdsföreningens Årsskrift 1:66–74. Wikmark, E. 1907. Om en svensk framtid och om ett fosterländskt företag: Några ord. Göteborg, Sweden. Zeldin, E. L., and B. H. McCown. 1997. Intersectional hybrids of lingonberry (Vaccinium vitis-idaea, section vitis-idaea) and cranberry (V. macrocarpon, section oxycoccus) to Vaccinium reticulatum (section macropelma). Acta Hort. 446:235–238. Zillmer, A. 1984. Beschreibung von fünf Preiselbeersorten. Erwerbsobstbau 26:282–283. Zillmer, A. 1985. Account of my three types of Vaccinium vitis-idaea—‘Erntedank’, ‘Erntekrone’, ‘Erntesegen’. Acta Hort. 165:295–297. Zillmer, A. 1998. Some thoughts about the cultivation of lingonberries Vaccinium vitisidaea L. In: T. Paal (ed.), Wild berry culture: an exchange of western and eastern experiences. Estonian Agr. Univ., Forest Research Inst., Tartu. Forest Studies 30:198–200. Åkerstrand, K., L. Kardell, and T. Möller. 1988. Undersökning av blåbär och lingon från kvävegödslade provytor. Vår Föda 40:259–270.

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Ängeby, O. 1978. Apis mellifera as pollinator of Vaccinium myrtillus and Vaccinium vitis idaea. Proc. 4th Int. Symp. on Pollination. Maryland Agr. Expt. Sta. Spec. Misc. Publ. 1:165–170. Önner, B. 1977. Studier av mycorrhizaförekomsten hos vildväxande och odlade lingon. M.Sc. thesis. Swedish Univ. Agr. Sci. Dept. Pomology, Alnarp. Öster, H.-E. 1974. Försök med odling av lingon. Swedish Univ. Agric. Sci. Konsulentavdelningen. Trädgård 64.

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4 Caper Bush: Botany and Horticulture Gabriel O. Sozzi* Departamento de Biología Aplicada y Alimentos Facultad de Agronomía, Universidad de Buenos Aires Avda. San Martín 4453. C 1417 DSE—Buenos Aires, Argentina.

I. INTRODUCTION A. History B. World Production II. BOTANY A. Taxonomy and Distribution B. Morphology and Anatomy C. Floral Biology and Seed Dispersal III. ECOPHYSIOLOGY A. Environmental Requirements B. Growth and Flowering C. Adaptations to Water Stress and Poor Soils IV. HORTICULTURE A. Biotypes B. Propagation 1. Seed 2. Vegetative C. Cultural Practices 1. Plant Establishment 2. Intercropping 3. Pruning 4. Plant Nutrition 5. Irrigation 6. Weed Control 7. Pests and Diseases 8. Harvest and Yield

*I am grateful to Prof. John M. Labavitch for a critical review of the manuscript, to Prof. Jules Janick for his helpful comments, and to María M. Quiroga for her support during the preparation of this review. Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 125

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V. POSTHARVEST TECHNOLOGY A. Capers 1. Handling and Curing 2. Industrial Treatment and Packaging B. Caperberries VI. COMPOSITION AND UTILIZATION A. Composition B. Utilization 1. Food Use 2. Ornamental Use 3. Medicinal and Cosmetic Value VII. INTERNATIONAL TRADE VIII. CONCLUDING REMARKS LITERATURE CITED

I. INTRODUCTION The caper bush (Capparis spinosa L., Capparidaceae) has been introduced as a specialized culture in some European countries during the last three decades. The economic importance of caper plant (young flower buds, known as capers, are greatly favored for seasoning and different parts of the plant are used in the manufacture of medicines and cosmetics) led to a significant increase in both the area under cultivation and production levels during the late 1980s. The main production areas are in harsh environments found in Morocco, the southeastern Iberian peninsula, Turkey, and the Italian islands of Pantelleria and Salina. This species has developed special mechanisms in order to survive in Mediterranean conditions, and introduction in semiarid lands may help to prevent the disruption of the equilibrium of those fragile ecosystems. Little information on this species is available despite the increasing worldwide demand for capers and the socioeconomic influence of the caper crop. In the context of the potential use of this species as an alternative for marginal lands, caper bush deserves further research and diffusion. A. History Capers (flower buds) and caperberries (caper fruits) have a long history of use by humans. A fragment of thick fruit skin of the caper type was obtained by archaeological excavations from an Old World Paleolithic site (Wadi Kubbaniya, west of Nile Valley, Upper Egypt) and provides direct evidence of the consumption of Capparis spp. from 18,000 to 17,000 years ago (Hillman 1989). Prehistoric remains of wild caperber-

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ries were also found in southwest Iran and in Iraq (Tigris) and dated to 6000 BCE (Renfrew 1973). Caper seeds were found in quantity at Tell esSawwan (Iraq) and dated to 5800 BCE (H. Helback, cited by Renfrew 1973). The earliest known mention of capers is in a Sumerian legend of 2000 or 3000 BCE (Stromme 1988; Trager 1995). The Greek term Capparis (καππαρις) has no convincing etymology (Carnoy 1959). Nevertheless, in ancient Greece, capers were used as a condiment and for other purposes. Hippocrates first wrote about the medicinal properties of different caper plant tissues, including use as a treatment for pneumonia (Diseases III, 7, 142, 7), pleurisy (Diseases III, 7, 150, 11), and fistulas (Fist. VI, 460, 3, 6). Theophrastus suggested that wild caper plants could have greater pungency and that caper bush could not grow well on cultivated lands (De Causis Plantarum III, 1, 4). Capers were also used in cosmetics. Phryne (4th century BCE), who was said to have modeled for both her lover the sculptor Praxiteles and the painter Apelles, used them regularly (Cerio 1983). A caper cream was utilized to help one stay young and to keep the skin free of wrinkles and with a healthy color (Castro Ramos and Nosti Vega 1987). Capers were known as an appetizer by ancient Hebrews (Duke 1983). They are mentioned in a nostalgic poem of the Hebrew Bible (Ecclesiastes 12, 5) probably written in the 3rd century BCE. Moreover, the Babylonian Talmud cites caper bush several times (Shabbath 30b and 150b; Bezah 25b; Baba Bathra 28b), as well as caper flower (Abodah Zarah 38b) and capers (Demai I, 1, 6; Abodah Zarah 40b). The Midrash Rabbah describes how a caper bush grew up and fenced a breach in the vineyard of a pious man (Leviticus -Behar- 34, 16). Dioscorides indicated the therapeutic properties of capers in his De Medica Materia (II, 192). In Latin literature, Plinius Secundus included several passages about the caper bush in his Historia Naturalis. He recommended that capers coming from foreign countries not be used, as they could be a health risk (XIII, 44, 1), but considered that the direct consumption of Italian capers or extracts from different parts of the plant could be beneficial for the organism (XX, 59, 1). He claimed that caper bushes should be sown in dry localities and sandy soils, “. . . the plot being hollowed out and surrounded with an embankment of stones erected around it: if this precaution is not taken, it will spread all over the adjoining land” (XIX, 48, 2). Apicius classified capers as a condiment in De Re Coquinaria (IV, 1) and the poet Marcus Martialis mentioned them in one of his famous Epigrams (III, 77). Claudius Galenus cited the caper bush among medicinal plants and Columella gave a description of this perennial bush in his agricultural treatise De Re Rustica (XI, 3).

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Athenaeus mentioned capers several times in his cookery book Deipnosophistae and cited comedians who included them in their plays: Aristophon (II, 63a), Nicostratus (IV, 133c), Alexis (IV, 170a), Timocles (XIII, 567e), and Antiphanes (II, 68a; IV, 161e). According to Plutarch’s Moralia (Quaestiones Convivales), pickled capers were used either alone to restore the appetite (VI, 2, 687d), or as an ingredient of a delicious meal (IV, 4, 668a–b). Remains of capers have been recovered from Mons Claudianus, a Roman quarry settlement (1st–2nd centuries; van der Veen 1999) and from the Roman port of Berenik (1st–4th centuries; Cappers 1999). During the Middle Ages, the School of Salerno documented caper bush as a medicinal plant in the Regimen Sanitatis (II -Materia Medica-, II, 24; VIII -Therapeutica-, VI). Platina (1475) included capers in the first dated cookery book, De Honesta Voluptate et Valetudine (IV, 15) and added new medical advice. Mattioli described caper bush and its qualities (De Plantis Epitome Utilissima) and pointed out some therapeutic properties of capers (Commentari al Dioscoride II, 169). During the Renaissance, Spanish writers and poets such as Lope de Vega, Juan de Zabaleta, and Alfonso Ortiz de Ovalle cited them. In France, Olivier de Serres reported different preparation methods in his Théâtre d’ Agriculture (XI, 6), and the famous surgeon Ambroise Paré wrote: “Capers are good, in that they sharpen the appetite and relieve bile” (ToussaintSamat 1992). Capers were used not only by common people but also at the tables of the higher classes; e.g., in the famous dinner to honor the Holy Roman Emperor Ferdinand III’s ambassador in Rome in 1638 (Trager 1995). Some documents reveal caper consumption in England during the 17th century (Allen 1994), which is corroborated by the cookery books of that time (Hackwood 1911). Pierre Belon du Mans (16th century) described caper plants growing in the regions of Alexandria, Suez, and Sinai (Darby et al. 1977). He found those bushes different from other European types, as they were thornless and kept their leaves in winter. Coles (1657) distinguished five kinds of caper plants without clear morphological differences and indicated places and seasons in which they grew. At the beginning of the 19th century, the Spanish poet Gaspar de Jovellanos mentioned the caper plant in his description of the castle of Bellver, Palma of Majorca (Font Quer 1962), where it grows on the ramparts. Alfred Kaiser found C. spinosa var. aegyptia in Sinai; it was used as fodder, and its buds and seeds were utilized as condiments (Darby et al. 1977). Alexandre Dumas (1873) pointed out the Asiatic origin of caper bush and marked caper qualities as an aperitif in his Le Grand Dictionnaire de Cuisine.

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The caper bush was grown in Provence during the 18th and 19th centuries. At the end of the 19th century French capers were replaced by the Spanish and Italian product. B. World Production Caper production and trade have become highly competitive. Spain, Morocco, Turkey, and Italy are the four major producers. Beginning in 1977/78, Spain had a rapid increase in caper growing areas and its production rose to first place in just a few years. Spain had the most important caper-producing area from 1978 through 1991, with an average production of 3,550 t/year and a maximum of 4,685 t in 1985 (Fig. 4.1). The ready availability of seeds and seedlings, the favorable soil and climate, the possibility of using low yielding soils, its ready sale and attractive profits, low initial investment, low production costs, and the crisis of traditional cultures such as that of the olive tree in Andalusia

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contributed to the success of caper bush growing in Spain. Different government research services, such as the Centro Regional de Levante, developed and transferred new technologies and helped to remove technical barriers (e.g., propagation) for caper bush culture (Luna Lorente and Massa Moreno 1979; Luna Lorente and Pérez Vicente 1979). During the 1980s, caper culture extended to different provinces: Almería, Murcia, Balearic Islands, Jaén, Granada, and Córdoba and, to a lesser extent, Ciudad Real, Cadiz, Málaga, Alicante, Barcelona, and Valencia. Most of the caper plantings (90%) had less than 5 ha, but fields with more than 5 ha represented 49% of the total area (Millan Campos 1987). The unchecked expansion of production, the competition of Morocco and Turkey, and the lack of promotion in Spain (Ruíz Avilés 1987; Villena 1988) brought about an oversupply and a 40% drop in the price between 1985/86 and 1994. This caused both the removal of many caper plantings and the decline in the average yield (Fig. 4.1), because the remaining plantings are located in marginal lands. Morocco is a major caper-exporting country. In Morocco, production is mainly based on prickly wild plants of different closely related species. Local consumption is negligible, as capers are not usually utilized in the traditional Moroccan cuisine. Most of the produce is exported to European countries (Anon. 1982). During 1986, exports exceeded 3,000 t. One third of the produce was sold to Italy but substantial amounts were also exported to France, the United States, Germany, and Switzerland (Barbera 1991). Three-fourths of the Moroccan production comes from the region of Fez, Taounate, and Boulemane; other areas of minor production are located in the surroundings of Safi and southwest of Marrakech. Traditionally, no cultural practices were carried out. Harvest began around mid June and lasted two months. The Moroccan product quality was not considered outstanding. It generally consisted of a mix of capers from different species that were not adequately processed. The creation of regional associations and the promotion of caper plantings during the past 15 years has resulted in the stabilization of profits and the improvement of caper production and quality. Turkey has come to be the leading caper-exporting country, although local consumption is trivial. Turkey began to export capers in 1982. The average annual production is estimated to be around 3,500–4,000 t per year. Turkey exported 1,095 t of capers in 1989, 5,072 t in 1994, and 3,500 t in 1998 (Turkey Embassy in Argentina, pers. commun.). In 1999, 3,226 t were exported in large packs and 1,025 t in small packages for retail sale (Aegean Exporters’ Unions, Izmir, Turkey, pers. commun.) but

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these figures may be overestimated, as they are based on gross weights that include the packaging materials. Most of Turkish production of capers comes from the provinces on the Aegean and Mediterranean coast (Çanakkale, Balikesir, Izmir, Aydin, Mugla, Antalya, Içel, Adana, Hatay). The remainder of production is obtained from warm places in the provinces of Us¸ak, Denizli, Konya, Ankara, Zonguldak, Tokat, Malatya, and from others in the northeast and southeast of the country (Rize, Artvin, Erzurum, Kars, Igdir, Van, Mardin, and Sanliurfa). Caper plants grow naturally in these areas. However, agricultural production in private arable lands has started in recent years. Italy is not only a traditional caper producer but also the country where the caper bush has been grown intensively for a long period. Nevertheless, the caper culture still shows an important territorial concentration in two Sicilian insular areas that produce 95% of the Italian capers: Pantelleria Island (36° 47′ N, 12° 00′ E), and the Aeolian Archipelago, especially in Salina Island (38° 34′ N, 14° 51′ E) and, to a lesser extent, in the islands of Lipari and Filicudi. The remaining plantings are located in Apulia, Sicily, and in other Italian islands (Ventotene, Ustica, Egadas) (Barbera 1991). Between 1960 and 1985, there was an increase in caper production and areas under cultivation in Pantelleria and Salina due to: (1) the decline of traditional cultures such as grape vine and olive tree; (2) the increasing internal demand for capers, which provided a better profit margin; (3) improvement in cultural techniques and early preservation procedures; (4) the creation of mutual associations; and (5) improvement in the socioeconomic levels (Caccetta 1985; Barbera and Butera 1992). Between 1973 and 1983, the area under cultivation increased by 67% and production levels by 90%. Caper production in 1983 was 1,900 t, 1,360 in Pantelleria and 440 in Salina; and the cultivated area was 1,000 ha, 770 in Pantelleria and 180 in Salina (Caccetta 1985), but these figures may have been overestimated (Barbera and Di Lorenzo 1984; Barbera 1991). After 1983, Italian caper production reached the historically lowest levels: 600 t in Pantelleria and 250 t in Salina (Barbera 1991), due to the open competition with Spain and Morocco. In the early 1990s, Italian production was supported by a better organized and dynamic marketing, and the improvement of Italian quality standards. Tunisia produces 300 t/year obtained from wild plants with or without stipular spines. Most of these wild specimens grow in the mountainous region situated at the north and northeast of Tunisia. Most of the spineless genotypes are situated around Beja (Barbera 1991). The main problem is the high cost of harvesting capers, which could be

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significantly reduced by increasing the density of spontaneous plants and initiating specialized plantings. Studies performed in the kibbutz Maagan Michael (south of Haifa) in 1973 discarded caper bush as a profitable intensive culture due to the spiny populations available in Israel and the high cost of harvesting (Putievsky 1977). In France, it is still grown in the departments of the Maritime Alps, Var, and Bouches-du-Rhône. In other countries around the Mediterranean basin such as Greece and Cyprus occasional harvests from wild plants are carried out. In the United States, Stromme (1988) considered caper bush to be invaluable for hillside planting in California but growers could only recently propagate it. The first caper farm was established in Gilroy, California, but it supplied only local restaurants. The United States imported more than $5 million worth of processed capers annually during the late 1980s and almost $10 million during the late 1990s (USDA Foreign Agricultural Service 1999). In Argentina, genus Capparis is represented by several species (Gómez 1953); some of them are an important supplement to the cattle diet during the dry season (Van den Bosch et al. 1997). Caper bush plantings were first established in the province of San Juan (Dimitri 1959). Nowadays, there are small (0.5–5 ha) plantings in the provinces of San Juan (Rawson) and Mendoza (San Martín, Maipú, Junín); some of them are nurseries of young plants. In 1990, the Instituto Nacional de Tecnología Agropecuaria (INTA) initiated a research program to determine the feasibility of caper production in the provinces of Catamarca (Andalgalá) and La Rioja (Arauco) (Paunero et al. 1996).

II. BOTANY A. Taxonomy and Distribution The caper bush is known as alcaparro, alcaparra, tapanera, tapenera, tapena, tápano, alcaparrera, or caparro in Spanish; câprier in French; cappero in Italian; Kappernstrauch in German; alcaparreira in Portuguese; ahveeyonah in Hebrew; al-kabbar in Arabic; kapari or kebere in Turkish; and dàmá in Chinese. Alkire (1998) has compiled the name of caper bush, capers, and caperberries in 16 different languages. The caper plant is a member of the Capparidaceae, which comprises 40 to 50 tropical, subtropical, and temperate genera with 700–930 species of trees, shrubs, and herbs (Zohary 1966; Heywood 1985; Zomlefer 1994), from

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which only Capparis, Cleome, Crateva, and Polanisia are cultivated (Bond 1990). Most of the members of this family are distributed in the low-altitude regions of the tropics and subtropics in both hemispheres, as well as in temperate climates of the Mediterranean basin (Heywood 1985). In Europe, only Cleome and Capparis have been found (Heywood 1964). Lately, Capparis and relatives have been proposed to form a basal paraphyletic complex within Brassicaceae (Zomlefer 1994; Judd et al. 1999) on the basis of molecular (Rodman et al. 1993) and morphological (Judd et al. 1994) cladistic analyses. In fact, taxonomists have long agreed that the caper family is very closely related to Cruciferae, based on some major shared characters, particularly the original bicarpellate ovary with parietal placentae, the vacuolar and utricular cysternae of the endoplasmic reticulum, the presence of myrosin cells and glucosinolate production (Rodman 1991a,b; Zomlefer 1994). Capparis is the largest genus of this family but has been taxonomically neglected (Jacobs 1965). The number of species in this genus varies according to different authors: 150 (Luna Lorente and Pérez Vicente 1985), 250 (Jacobs 1965), 300 (Bond 1990), or over 350 (Stocker 1974; Barbera et al. 1991; Judd et al. 1999). Only a few are economic plants: 9 (Uphof 1968) to 15 (Bond 1990). Species identification in this highly variable genus is very difficult and there are different opinions concerning the rank assigned to the different taxa and to their subordination (Zohary 1960; Jacobs 1965; Higton and Akeroyd 1991). The limits of the species are not always clear, because intraspecific crosses are relatively common in the overlapping areas. Capparis spinosa may be considered a species complex (Rao and Das 1978) and continuous flux of genes (Jiménez 1987) throughout its evolution has made relationship determination hard. The existence of morphological variations with many intergrading forms has led to the recognition of many varieties, resulting in an unsatisfactory working classification (Rao and Das 1978). According to Jacobs (1965), Capparis spinosa L. represents one polymorphic and variable species that embraces C. antanossarum Baill., C. cartilaginea Decne., C. cordiflora Lam., C. elliptica Hausskn. & Bornm., C. galeata Fres., C. hereroensis Schinz, C. himalayensis Jafri, C. leucophylla DC., C. mariana Jacq., C. mucronifolia Boiss., C. murrayana Grah., C. napaulensis DC., C. nummularia DC., C. obovata Royle, C. ovata Desf., C. sandwichiana DC., and C. uncinata Edgew. Thus, this polymorphic species is widespread in Europe (Mediterranean region), Africa (northern, northeastern and southwestern part, as well as the eastern coast and Madagascar), Asia (Turkey, the Caucasus, the Near East, Arabia,

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Iran, Afghanistan, West Pakistan to Kashmir and Nepal, India, East Malaysia, and the Pacific) and Australia. On the other hand, some of the taxa included in C. spinosa by Jacobs (1965) have been considered as independent species by other authors (Zohary 1960; Bokhari and Hedge 1975; Rao and Das 1978). Higton and Akeroyd (1991) recognized three entities in Europe, mainly based on vegetative characters: (1) C. spinosa subsp. rupestris (Sibth. & Sm.) Nyman, distributed in the coastal rocks and cliffs of the Mediterranean basin; (2) C. spinosa subsp. spinosa var. spinosa, predominantly found in Spain, France, and Italy and thought to be related to the cultivated caper bush; (3) C. spinosa subsp. spinosa var. canescens Cosson, widespread in Southern Europe and Anatolia and proposed to be the native variant of the cultivated plant. Nevertheless, Fici and Gianguzzi (1997) suggested that C. spinosa subsp. spinosa var. spinosa and var. canescens could represent two edaphic variants of the same entity. Both C. spinosa subsp. spinosa var. canescens and C. spinosa subsp. rupestris are present in Pantelleria Island. The first one is widespread on regosols, lithosols, and sedimentary rocks such as clay and marl; the second one is present along the coastal limestone and volcanic cliffs (Fici and Gianguzzi 1997). Caper bush is present in almost all the circum-Mediterranean countries (Greuter et al. 1984) and is included in the floristic composition of most of them (e.g., Willkomm and Lange 1880; Halácsy 1901; Post 1932; Rechinger 1943a,b; Colom Casanovas 1957; Garnier et al. 1961; Heywood 1964; Davis 1965; Maire 1965; Zohary 1966; Blatter 1978; Danin 1983; Barclay 1986), but whether it is indigenous to this region is uncertain (Zohary 1960; Pugnaire 1989). Although the flora of the Mediterranean region has considerable endemism, the caper bush could have a tropical origin and only been naturalized in the Mediterranean basin (Pugnaire 1989). B. Morphology and Anatomy The caper bush is a shrub 30–50 cm tall but old specimens can achieve 80 cm in height and occupy an area of 15 m2. It is a perennial deciduous plant that becomes woody at maturity. Its roots are deep. Plants have been reported with 6–10 m long roots (Reche Mármol 1967; González Soler 1973; Luna Lorente and Pérez Vicente 1985; Bounous and Barone 1989). The root system may account for 65% of the total biomass (Singh et al. 1992). Caper canopy is made up of 4–6 radial decumbent branches from which many secondary stems grow. In wild bushes, Singh et al.

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Stem of caper bush showing flowers and fruit.

(1992) observed up to 47 branches per plant. Branches (Fig. 4.2) are 2 to 3 m long. Stipular pale yellowish spines are often recurved and divaricate but sometimes weakly developed and setaceous. Leaves are alternate, 2–5 cm long, simple, glabrous to densely pubescent, chartaceous or coriaceous, obovate to elliptic or ovate to orbicular, with a rounded base and a mucronate, obtuse, or emarginate apex. Flowers are 5–7 cm across, axillary and solitary, with a sweet scent, scattered along the twigs on a sturdy pedicel. Their calyx, with 4 purplish sepals, is from almost symmetrical to strongly zygomorphic. The 4 petals, slightly exceeding the sepals, are white and imbricate. Stamens are numerous

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(50–190), slightly exceeding the petals, glabrous, with purplish filaments and small subbasifixed introrse anthers. The gynophore is approximately as long as the stamens. The ovary is superior, 1-locular, with 5–10 placentas. The fruit (caperberry) is ellipsoid, ovoid, or obovoid, with a thin pericarp. The fruit bursts when ripe, exposing seeds in a pale crimson flesh. Seeds are 3–4 mm across, reniform, with a circinnate embryo. Germination is epigaeal. A thousand seeds weigh 6–8 g (Gorini 1981; Akgül and Özcan 1999; Li Vigni and Melati 1999). Few studies have been performed on this plant’s internal structure. Ponzi et al. (1978) examined caper bush ovular tissues, while Jørgensen (1981a,b) investigated the presence of myrosin cells; some characteristics of caper endoplasmic reticulum were also found in the sister group Cruciferae. Bokhari and Hedge (1975) studied different anatomical characters and Rao and Das (1978) researched the idioblast typology, in an attempt to shed some light on the taxonomic status of this complex species. Seidemann (1970) described the anatomical characteristics of sepals and petals, while Leins and Metzenauer (1979) examined the ontogenetic sequence of flower buds. Investigations on ultrastructure of caper anther and pollen have been carried out (Gori and Lorito 1988a,b). Recently, Ronse Decraene and Smets (1997a,b) discussed taxonomic affinities and possible trends in floral evolution on the basis of the androecium configuration and the increase in carpel numbers from an original bicarpellate condition. Doaigey et al. (1989) and Psaras et al. (1996) analyzed various anatomical and histological features of caper leaf and stem, which will be reexamined in the next section in relation to the ecophysiology of this species. C. Floral Biology and Seed Dispersal The caper bush is known to be noctiflorous (Jacobs 1965). It blossoms for approximately 16 h, from ca. 18:00 h to ca. 10:00 h the next morning (Ivri 1985; Petanidou et al. 1996). Flowers of Capparis attract different insects, most of which have low pollination efficiency (Eisikowitch et al. 1986). In Israel, the main pollinators are hawkmoths and large bees (Kislev et al. 1972; Eisikowitch et al. 1986; Dafni et al. 1987; Dafni and Shmida 1996). In Greece, flowers are to a major extent pollinated by bees (Petanidou 1991). Flower rewards in genus Capparis vary according to location and year (Petanidou et al. 1996) and differ significantly among taxa. C. spinosa var. aegyptia has a higher pollen grain weight and its nectar is richer in total amino acids (Eisikowitch et al. 1986). On the other hand, higher

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nectar concentration and volume are found in C. ovata (Eisikowitch et al. 1986; Dafni et al. 1987). Most nectar secretion in C. spinosa is nocturnal. Amino acid content and concentration, as well as hexose concentration, increase with flower age, while sucrose concentration decreases (Petanidou et al. 1996). Although alternative nectariferous species are available in the Mediterranean environment, the genus Capparis is almost the sole nectar source for pollinators under desert conditions (Eisikowitch et al. 1986). The juicy fruits of caper bush are consumed by animals. Seeds surrounded by sweet pulp are eaten by birds (Seidemann 1970; Danin 1983) like Sylvia conspicillata, Oenanthe leucura (Hódar 1994), and Chlamydotis [undulata] macqueenii (van Heezik and Seddon 1999), which transport the undigested seeds. Caper plant is also considered a myrmecochorous and saurochorus species; harvester ants (Luna Lorente and Pérez Vicente 1985; Li Vigni and Melati 1999) and lizards like Lacerta lepida (Hódar et al. 1996) feed on the fruit and carry off fragments together with the hard-coated seeds, which are left to germinate afterwards. Wasps are attracted by mature caperberry scent and also act as dispersal agents (Li Vigni and Melati 1999).

III. ECOPHYSIOLOGY A. Environmental Requirements The caper bush requires a semiarid climate. Mean annual temperatures in areas under cultivation are over 14°C and rainfall varies from 200 mm/year in Spain to 460 in Pantelleria and 680 in Salina. In Pantelleria, it rains only 35 mm from May through August, and 84 mm in Salina. A rainy spring and a hot dry summer are considered advantageous (Barbera 1991). A harvest duration of at least 3 months is necessary for profitability. Intense daylight and a long growing period are necessary to secure high yields. The caper bush can withstand temperatures over 40°C in summer but it is sensitive to frost during its vegetative period. The potential exposure of caper hydraulic architecture to cavitations has recently been proposed as an explanation for its susceptibility to freezing conditions (Psaras and Sofroniou 1999). On the other side, caper bush seems to be able to survive low temperatures in the form of stump, as happens in the foothills of the Alps. Caper plants have been found even 1,000 m above sea level though they are usually grown at lower altitudes (Barbera et al.

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1991). Some Italian and Argentine plantings can withstand strong winds without problems, due to caper bush decumbent architecture and the coriaceous consistency of the leaves in some populations. The caper bush is a rupiculous species. It is widespread on rocky areas (Heywood 1964) and is grown on different soil associations, including alfisols (Fernández Pozo et al. 1993), regosols, and lithosols (Barbera 1991; Fici and Gianguzzi 1997). In different Himalayan locations, C. spinosa tolerates both silty clay and sandy, rocky or gravelly surface soils, with less than 1% organic matter (Ahmed 1986). It grows on bare rocks, crevices, cracks, and sand dunes in Pakistan (Ahmed and Qadir 1976), in dry calcareous escarpments of the Adriatic region (Lovric 1993), in dry coastal ecosystems of Egypt, Libya, and Tunisia (Ayyad and Ghabbour 1993), in transitional zones between the littoral salt marsh and the coastal deserts of the Asian Red Sea coast (Zahran 1993), in the rocky arid bottoms of the Jordan valley (Turrill 1953), in calcareous sandstone cliffs at Ramat Aviv, Israel (Randall 1993), and in central west and northwest coastal dunes of Australia (Specht 1993). It grows spontaneously in wall joints of antique Roman fortresses, on the Wailing Wall, and on the ramparts of the castle of Santa Bárbara (Alicante, Spain). Moreover, this bush happens to grow in the foothills of the southern Alps (Verona, Italy) and is a common species on city walls (Baccaro 1978) in Tuscany (Italy) and on bastions of Mdina and Valletta (Malta) (Brandes 1992a,b). Hruska (1982) included caper as part of the floristic diversity on the walls of Castiglione del Lago (Italy). Clinging caper plants are dominant on the medieval limestone-made ramparts of Alcudia and the bastions of Palma (Majorca, Spain) (Brandes 1992b). This aggressive pioneering has brought about serious problems for the protection of monuments (Fairushina 1974; Brandes 1992b). Deep and well-drained soils with sandy to sandy-loam textures are favorable for caper bush (Barbera and Di Lorenzo 1982, 1984; Ahmed 1986; Özdemir and Öztürk 1996), though this shrub adapts perfectly to calcareous accumulations or moderate percentages of clay (González Soler 1973; Fernández Pozo et al. 1993). It also shows a good response to volcanic (Barbera and Di Lorenzo 1982) or even gypseous soils (Font Quer 1962) but is sensitive to poorly drained soils. Soil pH between 7.5 and 8 are optimum (Gorini 1981), though pH values from 6.1 to 8.5 can be tolerated (Duke and Terrel 1974; Duke and Hurst 1975; Ahmed 1986). Caper bush is usually not considered to be a halophyte but Abbas and El-Oqlah (1992) found this plant in the loamy solonchacks of Bahrain coastal lowlands, where the conductivity may reach 54 mS/cm. On the other hand, aerosols from sea-water-fed cooling towers proved to pro-

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duce leaf chlorosis or necrosis, probably due to chloride toxicity (Polizzi et al. 1995). C. spinosa withstands chronic levels of some toxic gaseous pollutants. Krishnamurthy et al. (1994) reported an unusual 93% retention of leaves when caper bush was exposed to a mixture of sulfur dioxide, oxides of nitrogen, ammonia, and suspended particulate matter, although the photosynthetic area per leaf was reduced by 61% and the fresh weight by 67%. The simultaneous increase in the free amino acid pool was attributed to the production of sulfur-containing amino acids and the hydrolysis of proteins due to pollution. B. Growth and Flowering In winter, most of the biotypes remain in the form of stumps; when spring comes, new stems appear, elongate and bear flower buds. The total growing cycle lasts 5–6 months depending on temperature and early frosts. There is a positive correlation between temperature and productivity (Luna Lorente and Pérez Vicente 1985). Fertility of the nodes is maximum (close to 100%) during the hottest periods and lower at the beginning and end of the season (Barbera et al. 1991). Flower bud appearance is continuous so that all transitional stages of development, from buds to fruit, can be observed simultaneously on the plant throughout most of its ontogeny whenever buds are not harvested. The first ten nodes from the base are usually sterile and the following ten only partially fertile; the subsequent nodes have a caper each, almost to the tip of the stem (Barbera et al. 1991). C. Adaptation to Water Stress and Poor Soils Genus Capparis has a C3 photosynthesis (Alkire 1998). As caper bush grows in semiarid environments, it routinely encounters high radiation levels, high daily temperature, and insufficient soil water during its growing period. The caper bush has developed a series of mechanisms that reduce the impact of said conditions and ensure its survival. It is not only capable of resisting periods of drought but also of making active use of these periods, e.g., by bloom at that time. Some caper anatomical features are typical of xerophytes: small cell size (including guard cells), thick outer epidermal cell walls both in leaves and stems, strongly developed palisade in the mesophyll, and dense vein network (Stocker 1974; Doaigey et al. 1989; Fici et al. 1995).

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C. spinosa subsp. spinosa var. canescens seems to be better adapted to semiarid conditions than C. spinosa var. rupestris due to some characteristics of the cuticle, epidermal cells, and stomata (Fici et al. 1995). Caper photosynthesis is favored by its amphistomatic leaves (Psaras et al. 1996). Mesophyll cells are densely packed and chloroplasts are arranged adjacent to the intercellular air spaces (Psaras et al. 1996). This feature may be of ecological importance in intense daylight regions where CO2 could be the photosynthetic limiting factor. Rhizopoulou (1990) analyzed different caper bush physiological responses to drought. Plasticity of young expanding tissues increases under water deficit with a simultaneous lowering of water potential and the production of a large canopy within a short period. Under water deficit conditions, new leaves reach full size more rapidly. Response to drought also includes regulated wall properties and stomatal opening, as well as increased root density. The radical growth is not inhibited under water deficit but roots change their distribution in the different soil layers and make metabolic adjustments. As its root system also spreads horizontally in the soil, it makes use of rainfall however light it may be. The caper bush is a stenohydric plant (Rhizopoulou et al. 1997) with high photosynthetic rates. This characteristic is supported by a high hydraulic conductivity due to the presence of wide vessels with non-vestured intervessel pits, both in roots and stems (Psaras and Sofroniou 1999). The caper bush also shows characteristics of a plant adapted to poor soils. This shrub has a high root/shoot ratio and the presence of mycorrhizae serves to maximize the uptake of minerals in poor soils (Pugnaire and Esteban 1991). Four different N2-fixing bacterial strains have been isolated from the caper bush rhizosphere (Andrade et al. 1997) and may, at least partially, explain its capacity to acquire and maintain high reserves of that growth-limiting nutrient (Pugnaire and Esteban 1991). Fertilization of cultivated bushes probably leads to a luxury consumption of some nutrients, a typical response of wild plants from infertile environments (Chapin 1980).

IV. HORTICULTURE A. Biotypes The chromosome number (sporophytic count) of Capparis spinosa is 38 (Taylor 1925; Kuhn 1928; Darlington and Wylie 1955; Murín and Chaudhri 1970; Fici et al. 1995). Few, if any, breeding programs have

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been undertaken. Considering the existence of extensive variation (whether one species or several), the possibility of intercrossing and the absence of strict selection of progeny, it is still difficult to define the genetic material available in all the world (Barbera 1991). The main germplasm collections are located in Italy and Spain (Alkire 1998). In any case, there are biotypes that have been chosen by growers according to some advantageous characteristics. Features of interest that should represent the current scope in caper bush improvement programs are: (1) high productivity (long stems, short internodes, and high node fertility); (2) deep green spherical flower buds, with close non-pubescent bracts (to ensure a better commercial quality) and late opening; (3) absence of stipular spines and easy stalk separation to simplify harvest and postharvest operations; (4) processed product with an agreeable appearance; (5) capacity for agamic reproduction; (6) resistance to water stress, cold, and pests; (7) stem tip thick and tender for food use; (8) oval fruit with light green pericarp and few seeds for food use. The most attractive Italian commercial biotypes are ‘Nocellara’ and ‘Nocella’ (Barbera et al. 1991). Both are highly productive and yield high-quality capers (almost spherical shape, conserved integrity after brining). ‘Nocellara’ does not bear spines, and ‘Nocella’ has very small harmless ones. Seedlings are used for the propagation of ‘Nocellara’ in Pantelleria; ‘Nocella’ is propagated in Salina using cuttings. On the other hand, ‘Nocella’ does not resist drought. Also in Italy, the biotypes ‘Ciavulara’, ‘Testa di lucertola’, ‘Spinoso of Pantelleria’, and ‘Spinoso of Salina’ have different disadvantages. ‘Ciavulara’ is less productive and its buds tend to open precociously; capers are flatter and flake easily during postharvest treatments, giving a poor aspect to the final product. ‘Testa di lucertola’ (‘Lizard’s head’) produces capers with a lengthened pyramid shape. ‘Spinoso of Pantelleria’ and ‘Spinoso of Salina’ have conspicuous axillary spines. In ‘Spinoso of Pantelleria’, the leaf tips also bear a small thorn. ‘Spinoso of Salina’ is less productive; its capers are flattened pyramidal and tend to flake during postharvest curing. Other Italian biotypes are ‘Tondino’, grown in Pantelleria, and ‘Dolce di Filicudi-Alicudi’, cultivated in the Aeolian Archipelago. The most important Spanish biotypes are ‘Común’ or ‘del País’ and ‘Mallorquina’ (Luna Lorente and Pérez Vicente 1985). ‘del País’ is a heterogeneous population with spiny stems. This biotype branches frequently. Its capers are covered with a dense indumentum at the beginning of their growth. Stems dry out completely in winter. This biotype is difficult to propagate by cuttings (Jiménez Viudez and Guillamón Garrido 1986).

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‘Mallorquina’ is widely cultivated in Murcia and Almería. It has long spiny stems with bright green leaves. Its fruit is small and seedy. ‘Mallorquina’ is highly productive and shows a very important vegetative growth. In addition, it can be easily propagated by cuttings. This biotype has extraordinary yields under irrigation. ‘Italiana’ is a biotype with a lower vegetative and productive potential. When scions of ‘Italiana’ were bark-grafted using ‘del País’ as rootstock, yields increased significantly. ‘Italiana’ has the advantage of being easily harvested due to the absence of spines. Other biotypes are cultivated to a lesser extent in the Balearic Islands. ‘Rosa’, ‘Redona’, and ‘Fina’ are spiny but highly productive biotypes. ‘Redona’ and ‘Fina’ yield high-quality capers. On the other hand, ‘Fulla Redona’ and ‘Cavall’ are biotypes without spines. ‘Cavall’ has a low productivity but ‘Fulla Redona’ can be considered a promising biotype due to the quality and quantity of its produce. ‘Boscana’ is another spiny biotype, with low productivity and low product quality. B. Propagation 1. Seed. Caper seed germination performance is poor. Caper bush produces a high number of seeds per generative shoot, but low germination percentages under semidesert conditions create a great gap between seed yield and germination (Ziroyan 1980). However, caper bush propagation is usually carried out by seed owing to the serious rooting problems associated with cuttings. In Pantelleria, 5% germination was obtained within 2–3 months of seeding (Barbera and Di Lorenzo 1982, 1984). Cappelletti (1946) in Italy, and Luna Lorente and Pérez Vicente (1985) in Spain, also observed low germination percentages after direct sowing under field conditions. In the United States, using fresh seeds kept in pots above 18°C, Bond (1990) obtained germination percentages of about 10% within 10 days, and another 5–10% over the following month or two. Stromme (1988) described her difficulties when germinating caper seeds in California, although caper bush is fully adapted to the Mediterranean climate. Different pretreatments had been performed in order to improve the germination percentage, including scarification, stratification, soaking in 0.2% K2MnO4, concentrated H2SO4, H2O2, 0.2% KNO3, or gibberellins (GA4+7) and manipulation of the environmental conditions (light/dark, temperature) (Reche Mármol 1967; Ministerio de Agricultura 1980; Orphanos, 1983; Singh et al. 1992; Macchia and Casano 1993; Sozzi and Chiesa 1995; Yildirim 1998; Söyler and Arslan 1999; Tansi 1999). The best results were attained by partially removing

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the seed coats from non-germinated seeds (Sozzi and Chiesa 1995). With this procedure, the germination rate was greatly enhanced and seed dormancy was completely broken: 100% viable embryos germinated within 3–4 days. Caper seed germination shows a complete dependence on the covering structures. The seed of the genus Capparis is bitegmic (Corner 1976). The testa is 0.2–0.3 mm thick, with all its cell walls somewhat lignified, some of them with distinct thickening; its tegmen consists of an outer fibrous, lignified layer 4–10 cells thick, with a lignified endotegmen composed of contiguous cuboid cells, with strongly thickened radial walls. Only the mesophyll between exo- and endotegmen is unlignified (Guignard 1893a; Corner 1976). Since the integrity of the seed coating is very important to the persistence of dormancy in caper seeds, it is likely that the seed coats, with their successive lignified structures, are the most important cause for the problematic germination of this species (Sozzi and Chiesa 1995). The seed coats and the mucilage surrounding the seeds may be ecological adaptations to avoid the loss of water and germination during the dry season (Scialabba et al. 1995). Longitudinally opened mature fruits, with a violaceous dark green pericarp and dark red pulp, are adequate sources of dull brown mature seeds. Seeds obtained from small not-yet-opened fruit are generally light brown and immature. Seeds lie without order in the pericarp mass, each of them surrounded by an adherent layer of pulp. They can be obtained by rubbing and washing, followed by drying in the shade for a couple of days. Seeds are over 90% viable (Orphanos 1983; Sozzi and Chiesa 1995; Tansi 1999) for two years if held at 4°C and low relative humidity. In Spain, many methods to obtain young plants have been tested (Ministerio de Agricultura 1980; Luna Lorente and Pérez Vicente 1985). Commercial lots of seed are usually pre-germinated in February or March. Pre-germination consists of packing the seed in moist river sand, though other materials may also be used, such as compost of two parts turfy loam and one part leaf-mold and sand (Foster and Louden 1980) or mixtures with vermiculite or perlite (Kontaxis 1989). Small lots can be pregerminated in boxes; moderate- to large-sized lots are usually pregerminated in bins located in a greenhouse or another protected place. Two to four layers of seed are packed in each bin and the top one is covered with a 6 cm sand layer. Seeds are sprinkled with water and treated with captan or captafol. The moisture content requires a careful control; use of well-drained containers is essential to ensure thorough wetting as well as aeration (Luna Lorente and Pérez Vicente 1985). Seeds are usually

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submitted to these conditions for 25 to 50 days (Ministerio de Agricultura 1980) depending on temperature. Only sprouted seeds are planted. In Spain, nursery preparation begins in February using calcareous soils with loam to clay-loam textures and available irrigation. Proper cultivating and fumigation under a plastic tarpaulin kills perennial weeds, weed seeds, nematodes, soil insects, and fungi. After removal of the tarp, seeds (1.5–2 g/m) are planted about 1.5 cm deep, in rows 30 or 40 cm apart. Irrigation is needed on a 2-week or even more frequent basis. Most caper nurseries use furrow irrigation (Luna Lorente and Pérez Vicente 1985). Yields of 45–50 seedlings/m are obtained after 30 days. In Pantelleria, planting is performed in a rudimentary way, with few defined cultural practices (Barbera 1991). Transplants may also be produced under protected conditions using floating row covers. Some nurseries seed directly into pots, containers, or polyethylene bags where plants remain until outdoor transplanting. 2. Vegetative Cuttings. Caper bushes grown from cuttings have an advantage over seed-propagated bushes: they are genetically identical with their source. This practice avoids high variability in terms of production and quality. Nevertheless, plants grown from cuttings are more susceptible to drought during the first years after planting. Caper is a difficult-to-root woody species. Successful propagation requires careful consideration of biotypes as well as seasonal and environmental parameters. Propagation from cuttings is the standard method for growing ‘Mallorquina’ and ‘Italiana’ in Spain, and ‘Nocella’ in Salina. In Pantelleria, utilization of rooted cuttings did not yield good results (Barbera and Di Lorenzo 1984). Hardwood cuttings are generally used to propagate ‘Mallorquina’. Workers collect 1-year-old wood, 1.0 to 2.5 cm in diameter, when the plants are still dormant, and cut it into sections 20 to 30 cm long (15 cm minimum, 40 to 50 cm maximum). When cuttings are at least 1.5 cm in diameter, rooting percentages of 55% are possible (Istituto di Coltivazioni Arboree, Università degli Studi di Palermo, unpublished). The shoots are planted in individual rooting plugs or lined out in a nursery row with a humid environment. Rooting percentages depend on cutting harvest time and substrate utilized (Pilone 1990a). Another possibility is to collect stems during February through the beginning of March, pretreat them with captan or captafol, and stratify them outdoors or in a refrigerator at 3–4°C, covered with sand or plastic. Moisture content and drainage should be carefully monitored and maintained until planting (Luna Lorente and Pérez Vicente

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1985). Using semihard cuttings, collected and planted during August and September, low survival rates (under 30%) have been achieved. Softwood cuttings are prepared in April from 25- to 30-day shoots. Each cutting should contain at least 2 nodes and be 6 to 10 cm long. Basal or subterminal cuttings are more successful than terminal ones. Then, cuttings are planted in a greenhouse under a mist system with bottom heat; 150 to 200 cuttings m–2 may be planted. Dipping the cutting basal end into 1500–3000 ppm auxin solution may enhance rooting (Pilone 1990b) but results depend on the type of cutting. Hardwood cuttings do not seem to respond to indole-3-butyric acid (IBA) or a-naphthaleneacetic acid (NAA) pre-treatments. On the other hand, dipping the herbaceous cutting base in a 2000 ppm NAA yielded rooting percentages of 83% (Luna Lorente and Pérez Vicente 1985). Micropropagation. Successful micropropagation was achieved from nodal shoot segments. Four µM 6-benzylaminopurine stimulated proliferation and shoot development; when combined with 0.3 µM indoleacetic acid (IAA) and 0.3 µM GA3, formation of proliferating clusters was enhanced (Rodríguez et al. 1990). High rooting response was obtained by using 30 µM IAA (Rodríguez et al. 1990). The presence of abnormal vitrified shoots was observed in some cases and could be prevented by means of alternate culture in cytokinin-enriched and hormone-free media, or normalized by using sucrose-enriched medium (Safrazbekyan et al. 1990). Because of the difficulties of caper bush conventional propagation, in vitro culture may be a promising alternative technique. Grafting. Grafting is a less common method of propagation for caper bush. In Spain, acceptable results (60% scion take) were obtained using bark grafting in plantings. Nurseries generally whip graft with survival rates of 70–75% (Luna Lorente and Pérez Vicente 1985). C. Cultural Practices 1. Plant Establishment. Caper is a long-lived bush and plantings over 25–30 years old are still productive. Thus, site selection for the planting is an important step. Soil, water availability, and climate are the main aspects to be considered. Physical properties of the soil (texture and depth) are particularly important. Caper bush can develop an extensive root system. Because of that, it grows best on deep, non-stratified, medium-textured, loamy soils. Moldboard plowing and harrowing are usual practices prior to caper plant establishment (Luna Lorente and

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Pérez Vicente 1985). Soil-profile modification practices, such as slip plowing operating 0.6 to 1 m deep, can alleviate restrictions (Massa Moreno 1987). In Pantelleria, digging backhoe pits for each shrub was found to be the most effective means of cultivating caper in small plantings with rocky soils (Barbera 1991). Most caper bush plantings are planted in a square or hedgerow design, as these patterns are very easy to lay out and plant. Subsequently, cultivation and harvest are simplified. Spacing is determined by the vigor of the biotype, the fertility of the soil, the equipment to be used, and the irrigation method, if any. Bush spacing of 2.5 × 2.5 m (Barbera and Di Lorenzo 1982) or 2.5 × 2 m (Bounous and Barone 1989) is common in Pantelleria. In Salina, 3 × 3 m is satisfactory for ‘Nocella’. Even wider spacing is common in Spain: 4 × 4 or 5 × 5 m may be entirely satisfactory for planting on fertile loam soils. ‘Mallorquina’ may be crowded at 3 × 3 or 3 × 4 m in a 5-year planting (Centro de Capacitación Agraria de Lorca—Murcia, unpublished results). If caper bush is used to control soil erosion, especially on slopes, 2- to 2.5-m spacing is appropriate. Fertilization should begin 20–30 days before planting. At that time, 100 kg/ha ammonium sulfate, 400 kg/ha single superphosphate, and 150 kg/ha potassium chloride have been suggested in Spain (Massa Moreno 1987). Fertilizers may be broadcast on the surface and incorporated by tilling or cultivating, or surface band applied. In Pantelleria, plots are enriched with organic or inorganic fertilizers applied to the backhoe pits (Barbera 1991). Nursery plants, propagated as seedlings or rooted cuttings are dug in the nursery row during the dormant season. In the Aeolian Archipelago, transplanting is performed during January through February, but in zones of the Iberian Peninsula with prolonged winter, it takes place during February through early March, after the last frosts. In Argentina, transplanting is generally made in July through August. Transplanting is carried out by hand and caper shrubs may be transplanted either bare-root or containerized. Most plants are handled bareroot and replanted immediately in their permanent location or heeled-in in a convenient place with the roots well covered. Field beds should be well prepared and watered. Afterwards, a good amount of irrigation should be applied. Containerized plants are only used where irrigation is the chief factor limiting transplanting success. Direct field seeding or cutting planting may be alternative practices. Although costs and labor requirements are lower than for transplanting, a major difficulty is the very heavy losses of young plants resulting from drying. Eight to 10 pregerminated seeds or 3 to 4 cuttings are placed by hand into each 30-cm hole after carefully pulverizing the soil. On rocky

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slopes, caper bush can be directly seeded into a natural setting for landscape purposes. 2. Intercropping. Intercropping caper bush with other perennial crops is a practice commonly used by farmers both in Italy and Spain, as it provides a variety of returns from land and labor, increases resource efficiency (e.g., reduces labor peaks; Millan Campos 1987) and attenuates the risk of dependence upon a single crop. In Italy, the intercropping pattern varies from strip cropping to row intercropping. In the strip cropping pattern used in Pantelleria, there are one or two rows of caper bush (usually along the borders of the fields) to several rows of grape vine. In the row intercropping system used in the Aeolian Archipelago (especially in Salina), caper bush is grown in association with olive (Barbera 1991). In Almería, Spain, intercropping of caper bush and almond tree was devolved during the 1970s and 1980s (Lozano Puche 1977). Three different planting patterns were examined, providing more favorable conditions for this intercropping arrangement (Lozano Puche 1977): alternate-row, alternate-plant, and same-hole intercropping systems. No significant differences were detected between systems, and almond tree productivity was not affected significantly. Nevertheless, the alternate-row system is the more appropriate (Cano García 1987) to avoid competition between both species and to prevent erosion. Tillage of orchard middles or soil cultivation for weed control may be performed when caper plant is in the form of stump. 3. Pruning. Caper bush is usually dormant pruned. Where winter temperatures are very low, the pruning operation is delayed until the severest weather is over. After the removal of dead or dying tissue, caper bush must be pruned of weak, non-productive wood and water sprouts. Caper bush benefits from a short and heavy spur pruning that reduces branches to a length of 1 cm (Barbera and Di Lorenzo 1984) to 3 cm (Luna Lorente and Pérez Vicente 1985) or 5–10 cm when the plant is young and vigorous (Barbera and Di Lorenzo 1982). It is important to leave several buds on the spur, as only the 1-year-old stems will bear flower buds for the current season. Early summer pruning involves thinning out weak stems when caper bush is in active shoot growth, 30–40 days after budding. The number of stems to leave upon a vine should be based on its general vigor. A strong plant may have as many as six, strategically distributed to obtain an open canopy with uniform light penetration throughout. Summer pruning also involves heading back a few of the new shoots, to stimulate the formation of new flower buds.

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Table 4.1. Nutrient levels on a fresh weight basis of caper bush flower buds, stems, and leaves with no apparent visual deficiency symptoms. From Centro de Edafología y Biología Aplicada del Segura, Spain, cited by Luna Lorente and Pérez Vicente (1985). Nitrogen Phosphorus (%) (mg/100 g)

Plant tissue Flower buds Stems + Leaves

0.87 1.01

Potassium Calcium Magnesium (mg/100 g) (mg/100 g) (mg/100 g)

86 55

135 173

32 18

55 116

Water (%) 81.1 70.1

4. Plant Nutrition. In Spain, different analyses were performed to make an adequate diagnosis of nutrient deficiencies (Centro de Edafología y Biología Aplicada del Segura, cited by Luna Lorente and Pérez Vicente 1985). The types of fertilizer used and application rates should be related to plant age (Table 4.2) and soil nutrient content. Measurements of the total concentration of a nutrient in the plant (fresh weight basis, Table 4.1) and the extraction of different elements from soil (Table 4.2) can be used for diagnosing mineral deficiencies. Phosphate and potassium fertilizers are generally applied every two to three years (Table 4.3), as caper P-K requirements are lower than that for N. Ammonium fertilizers are incorporated into the soil at the end of the winter, before sprouting. In Pantelleria and Salina, N-P-K fertilizers (15-6.6-12.5, 10-4.4-8.3, 20-4.4-8.3, or 11-9.6-13.3) are applied during winter (December and January) at a rate of 200–300 g/plant (Barbera and Di Lorenzo 1982; Barbera 1991). Bounous and Barone (1989) suggested that fertilizations with

Table 4.2. Extraction of mineral elements during the first seven years after implantation. From Centro de Edafología y Biología Aplicada del Segura, Spain; cited by Luna Lorente and Pérez Vicente (1985). Flower buds, stems, and leaves are on a fresh weight basis. Planting age (years) 2 3 4 5 6 7

Flower buds (kg/ha)

Stems and leaves (kg/ha)

N (kg/ha)

P (kg/ha)

K (kg/ha)

Ca (kg/ha)

Mg (kg/ha)

125 600 1100 1250 1350 1350

500 1500 2000 2250 2500 2500

5.6 2.4 29.8 33.6 37.0 37.0

0.17 0.59 0.87 1.01 1.11 1.11

0.86 2.82 4.07 4.65 5.10 5.10

0.13 0.45 0.70 0.80 0.87 0.87

0.65 2.07 2.92 3.30 3.64 3.64

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Table 4.3. Fertilizer application program for caper bush. From Centro de Edafología y Biología Aplicada del Segura, Spain; cited by Luna Lorente and Pérez Vicente (1985). Planting age (years)

Ammonium sulfate (kg/ha)

Single superphosphate (kg/ha)

Potassium sulfate (kg/ha)

2 3 4 5 6 7

50 150 200 200 250 250

— 25 — 50 — 50

— 25 — 50 — 50

150–200 kg/ha of ammonium sulfate and additional P-K applications would be appropriate for mature plantings. 5. Irrigation. Caper bush is cultivated mostly in non-irrigated areas represented by poor lands generally receiving little attention from farmers. Though the caper plant typically grows in semiarid regions and tolerates water stress well, water is the most limiting production factor. During the first year, caper bush is particularly sensitive to water stress. In Pantelleria and Salina, irrigation is impossible due to the lack of hydrous resources (Barbera and Di Lorenzo 1984). Nevertheless, a type of mulching—which may include placing stones around the young plants—is utilized to protect them from the wind action and thus reduce evaporation. In Spain and Argentina, additional water is usually provided during the first year. The caper bush shows its productive potential under irrigation (longer vegetative cycle, larger bud production that begins earlier, and shorter intervals between harvests), though the plant tends to be more prone to diseases (Jiménez Viudez 1987). In Spain, irrigation begins in January when caper bush is grown with almond trees, but in February or March when grown alone, and it ends in August in either case (Jiménez Viudez 1987). Yields were doubled and even tripled when irrigation was used in Almería (it rains 96 mm from February through August), Jaén (284 mm), and Murcia (156 mm). In 1984, the average yield in Spain was 1365 kg/ha in irrigated plantings and 650 kg/ha in non-irrigated plantings (Ministerio de Agricultura, Pesca y Alimentación 1989). In 1988, 837 ha had been irrigated in Almería, Murcia, and Jaén (Ministerio de Agricultura, Pesca y Alimentación 1988). In 1995, only 41 ha (mainly in Murcia, Córdoba, and Valencia) were still under irrigation due to the

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increasing competition from caper grown in Turkey and Morocco (Ministerio de Agricultura, Pesca y Alimentación 1997). A point source sprinkler system may be utilized. Total volumes of 12–140 L/plantweek, depending on the climatic conditions, are supplied under irrigation (Jiménez Viudez 1987). 6. Weed Control. Competition of weeds may be particularly serious during the seedling establishment. During the first and second years of the planting, preemergence herbicide treatments in combination with mechanical weed removal (4–5 supplementary well-timed operations using discs or rotary tillers) yield satisfactory results whenever direct pulverization on caper stump or stems is avoided. In Pantelleria, paraquat and simazine are used (Barbera 1991). After establishment, most of the ground is rapidly covered by the caper bush canopy and weed development is almost suppressed. In Spain, different preemergence and postemergence herbicides were tested in a 5-year planting prior to outgrowth of the new caper shoots (Lozano Puche 1984). Linuron (50%) at 2 kg/ha, prometryne (50%) at 4 kg/ha, methabenzthiazuron (70%) at 3 kg/ha, and metribuzin (30%) at 3 kg/ha were compared. Prometrine and metribuzin gave the most satisfactory results under such conditions. On the other hand, simazine and prometrine were found to be effective in controlling the “weed” caper bushes (Fairushina 1974). 7. Pests and Diseases Pests. The caper bush is not very sensitive to pest damage when growing without cultivation and insects do not appear to be the limiting problem under field conditions. Nevertheless, some phytophagous species that attack caper in its main production areas have been detected. Insecticide treatments are restricted by the short interval between harvests (7–10 days); only low-persistence active principles can be used, so as to avoid the presence of toxic residues at harvest. Research should also be undertaken to determine the possible influence of caper processing on persistence of different types of residues. In Pantelleria, the caper moth (Capparimyia savastanoi Mart.; Trypetidae) is considered to be the most important pest (Longo and Siscaro 1989; Longo 1996). Its control relies on the removal of infested leaves, combined with the use of poisoned hydrolyzed protein baits in summer when populations are high. Another major pest is the caper bug (Bagrada hilaris Bm.; Pentatomidae), a polyphagous insect that feeds on species of many plant families

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such as Brassicaceae, Fabaceae, Poaceae, Amaranthaceae, Apiaceae, Solanaceae, Rutaceae, Malvaceae, and Asteraceae (Gunn 1913). In Pantelleria, it was first found on wild plants (Carapezza 1981) and, later on, attacking cultivated caper plantings (Genduso 1990). The pale creamy oval eggs, which turn to orange as the insect develops (Mineo and Lo Verde 1991), are laid singly on the ground, in the cracks of the bordering field walls and, more rarely, on the leaves. At the beginning of spring it attacks different wild plants, among them caper bush, which grows weak and rapidly yellows. Pyrethroid formulations are used to control this insect. The chemicals are applied either to the walls or to the plants after harvest is finished (Barbera 1991). The painted bug (Bagrada picta Fabr.; Pentatomidae) is a pest of cruciferous oilseed crops and has been reported to thrive on caper bush at Tandojam during summer (Mahar 1973). Also in Pantelleria, the larval form of a type of weevil, Acalles barbarus Lucas (Curculionidae), causes damage to the root system (Liotta 1977). In general, its targets are weak adult plants previously affected by other insects. The only effective control is the removal of the attacked plants. Other insect pests in Italy are Phyllotreta latevittata Kutsch (Chrysomelidae), which causes oval to round erosions in leaves, leaf yellowing and stem decay, and Asphondylia spp. (Cecidomyiidae) and Cydia capparidana Zeller (Tortricidae), which alter the morphology of buds (Harris 1975; Orphanides 1975, 1976). The braconid Chelonus elaeaphilus Silv., a promising parasite of Prays oleae (an olive pest), was also recovered from C. capparidana infesting caper bush (Fimiani 1978). Rapisarda (1984–85) reported the occurrence of Aleurolobus niloticus Priesner & Hosny (Aleyrodidae), a polyphagous species that feeds only on caper bush leaves in Sicily. In Southern Spain, caper bush is the only larval host plant available during the dry season for various Pieridae: cabbage small white (Pieris rapae L.) and large white (Pieris brassicae L.) butterflies, and desert orange tip (Colotis evagore Klug.). P. rapae has also been found feeding on caper bush in the Badkhyzskii Reserve, Turkmenistan (Murzin 1986), and in California (Kontaxis 1990). The larvae of P. rapae and P. brassicae usually use cruciferous plants in the rainy season and caper bush in summer when Cruciferae are dry (Fernández García 1988). Oviposition takes place preferentially on the ground or on dried material around the food plant. On the other hand, C. evagore larvae are unable to survive on alternative cruciferous hosts (Jordano Barbudo and Retamosa 1988; Jordano et al. 1991) but they complete their life cycle successfully in certain coastal enclaves where caper bush provides sufficient resources throughout the year. The adult lays red eggs singly, on young

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leaves, stems, and inert supports next to the food plant (Fernández et al. 1986; Fernández Haeger and Jordano Barbudo 1986). Caper bush and other related species are also the commonest food plants of other Pieridae in Saudi Arabia, such as Anaphaeis aurota F., Colotis fausta fausta Olivier, and Colotis liagore Klug. (Pittaway 1979, 1980, 1981, 1985). These species deposit the ova on isolated bushes in rocky scarps and cliffs. Eventually, caper plants may be completely stripped of foliage, the resulting bare branches carrying pupae and larvae. Pyrethroids and trichlorfon can be used to control all of these Pieridae pests (Massa Moreno and Luna Lorente 1985). Larvae of Lampides boeticus L. (Lycaenidae), which have anthophagous and carpophagous habits, have also been found to feed on the buds of caper bush (Jordano Barbudo et al. 1988). In Spain, the pentatomid bug Eurydema ornata L. (Fernández et al. 1986) attacks caper bush leaves and may cause serious damage to plants. Other pentatomid bugs (Holcostetus punctatus and Carpocoris lunula) are less frequent. The green stink bug Nezara viridula L. has caused some minor damage in the Iberian Peninsula and Argentina. All these Hemiptera can be controlled by using trichlorfon, endosulphan, dimethoate, or chlorpyriphos. Other insect pests detected in caper include Ceuthorhynchus sp. (Curculionidae) and Heliothis-Helicoverpa (Noctuidae). Many ant species (Camponotus spp., Plagiolepis pygmaea, Crematogaster auberti, Crematogaster sordidula, Formica subrufa, Tetramonium hispanica, Cataglyphis viaticoides) have been found feeding on caper plant (Fernández et al. 1986). In California, caper plants can be damaged by cabbageworm, black vine weevil, and flea beetle, as well as gophers, snails, and slugs (Kontaxis 1998). Mosquitoes (Culex pipiens molestus Forskal; Culicidae) and sandflies (Phlebotomus papatasi Scopoli; Psychodidae), both of which are bloodsucking insects, also feed on caper plants (Schlein and Muller 1995). Photosynthate levels in caper bush may affect sandfly feeding (Schlein and Jacobson 2000). Sandflies are known to be vectors of human pathogens, Leishmania tropica and L. major (Schlein and Warburg 1986; Schlein et al. 1986; Schlein and Yuval 1987), but Leishmania-infected sandflies showed a significant reduction in the number of parasites and 55% of impaired infections when feeding on caper bush. This suggests a role for the plant in the epidemiology of leishmaniasis (Schlein and Jacobson 1994a,b). In fact, extracts of C. spinosa caused extensive parasite agglutination, apparently due to caper plant lectins (Jacobson and Schlein 1999). Screening tests were performed in Spain (Servicio de Sanidad Vegetal, Murcia, data not published) to determine the efficiency of insecti-

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cides (parathion, malathion, chlorpyrifos, deltamethrin, trichlorfon, tetradifon plus dicofol, and dithiocarbamates). These pesticides were found not to be phytotoxic for caper bush. Biologically integrated pest management approaches through the use of biopesticides have not yet been tested. Diseases. Damping-off diseases may be severe. Frequently, caper seedlings are completely destroyed either when they are placed in seedbeds or after being transplanted. Seedlings are usually attacked at the roots or in the stems at or below the soil line, and the invaded areas soon collapse. Damping-off in caper bush is caused by several fungi (Pythium spp., Fusarium spp. Verticillium spp., etc.), which often cause quite similar symptoms. These diseases can be controlled through the use of sterilized soil and chemically treated seeds. A list of fungi that affect caper bush was given by Ciferri (1949). The most important is probably the white rust disease (Albugo capparidis De By.) which affects aboveground plant parts, particularly the leaves and flowers. Neoramularia capparidis spec. nov. produces small grayishwhite leaf spots with a narrow brown margin in India (Bagyanarayana et al. 1994). Besides, caper bush is a host of Leviellula taurica (Lev.) G. Arnaud, causal agent of the powdery mildew (Gupta and Bhardwaj 1998). In California, Botrytis spp. and Pythium spp. attack caper plants (Kontaxis 1990). A caper vein banding virus (CapVbV) was reported in Sicily and was tentatively assigned to the carlavirus group (Majorana 1970). Gallitelli and Di Franco (1987) isolated the same virus and showed that it infects caper plant symptomlessly. Therefore, they suggested the alternative name caper latent virus (CapLV). The real causal agent of vein banding may be a rhabdovirus, the caper vein yellowing virus (CapVYV) that may infect caper bush simultaneously with the CapLV (Di Franco and Gallitelli 1985). New serological tests have shown that CapVYV is indistinguishable from the Pittosporum vein yellowing nucleorhabdovirus (PVYV) (Nuzzaci et al. 1993). 8. Harvest and Yield. Harvest is the costliest operation of caper production. It may represent 2/3 of the total labor in the crop management process (Caccetta 1985; Martínez Capel 1987) because it is done manually. Harvest is difficult and time-consuming due to: (1) the decumbent character of the branches; (2) the presence of stipular spines in some biotypes; (3) high temperatures and insolation during summer in caperproducing areas; and (4) the small diameter of flower buds. Besides, harvest has to be performed several times. Flower buds are arranged along

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twigs that have an indeterminate growth habit. Since bud production is continuous, twigs should not be cut and mechanical harvesting is not a viable option at present. A harvester can collect around 12 kg of capers per day when working in a regularly designed planting (Lozano Puche 1983). Successive harvests are designed to maximize the yield of smaller (more valuable) capers. However, buds that are missed in one harvest will continue to develop and will be collected as caperberries in subsequent harvests. Temperature is the main environmental factor affecting caper harvest dates. In Italy, harvesting takes place from late May through late August. In Spain, caper production continues through September, but there are not always enough buds to justify collection. In Argentina, harvest operations last 75–90 (Mendoza) to 120 days (Catamarca) or even more, depending on the latitude. The genetic nature of the biotype also affects harvesting operations: in Spain, the production peak takes place in June–July when the biotype is ‘del País’, but in July–August if the biotype is ‘Mallorquina’. Harvest frequency has a direct bearing on the final size and quality of the product (Table 4.4). However, determining the optimum time interval is a difficult decision because there are different conflicting factors to consider. Shorter time intervals between successive harvests result in a high-quality product (Fig. 4.3); yet, on the other hand, the number of buds per kg increases when caper size decreases (Table 4.4) and harvesting costs increase when intervals between harvest dates are shorter.

Table 4.4.

Caper international grading system. Number of flower buds/kg

Diameter (mm)

International commercial denomination

<7 7–8 8–9 9–10 10–11 11–12 12–13 13–14 >14

Non Pareil Surfine Capucine Capote Capote Fine Fine Grosse Hors Calibre

According to Barbera (1991)

According to Luna Lorente and Pérez Vicente (1985)

5,500 4,000 3,250 2,600 2,200 1,900 1,600 — —

7,000 4,000 4,000 2,000 2,000 1,300 1,300 800 —

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30 10-12 days

25

Distribution (%)

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7 days

20

15

10

5

0 6

7

8

9

10 11 12 13 14 15 16

Diameter (mm) Fig. 4.3. Distribution of caper diameters in relation to time interval between successive harvests. Prepared with data from Barbera (1991).

In general, 7- to 10-day intervals between successive harvests are appropriate, but they are usually shortened to 3–5 days during the peakproduction periods, as happens in Spain. Usually, 8 to 10 recollections per year take place in Pantelleria and Salina, and 12–14 in the Iberian peninsula. Caper bush yields are highly variable depending on the growing environment, cultural practices, and biotype, but a maximum yield is expected in the 4th year. Bounous and Barone (1989) indicated average annual yields of 1–1.5 kg/plant and yields as high as 4 kg/plant in the 3rd and 4th years of cultivated growth. Barbera and Di Lorenzo (1982) reported average annual yields of 1–1.5 kg/plant in Pantelleria (maximum yields of 4–5 kg/plant) and 2–3 kg/plant in Salina in 3-year plantings (average annual yields of 3–4 t/ha). According to Barbera (1991), a mature caper plant may produce 4–5 kg/year. On the other hand, Caccetta (1985) estimated annual yields of 1.2–2.5 t/ha in Pantelleria and 1.8–2.6 in Salina. According to Lozano Puche (1977), a wild growing plant yields 2–3 kg/year in Spain, but the same caper bush has the potential to produce

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8 Mallorquina del País

7

Average Yield (kg plant -1)

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6 5 4 3 2 1 0 1

3

5

7

9

Planting Age (years) Fig. 4.4. Caper bush yields in the first years after implantation. Open symbols mark drought years. Prepared with data from Ezequiel Sánchez García (Centro de Capacitación Agraria de Lorca, Murcia), unpublished.

6–9 kg/year when cultivated in irrigated fertile soils (Jiménez Viudez 1987). Great differences in yield are attributed to genetic variations. A 3-year-old ‘del País’ planting yields 1–1.5 t/ha-year, but this production may be doubled and even tripled by using ‘Mallorquina’ (Fig. 4.4).

V. POSTHARVEST TECHNOLOGY A. Capers 1. Handling and Curing. Immediately after harvest, capers are placed in shallow vats. In Spain, postharvest conditioning is generally performed by local traders, cooperatives, or producer associations, and consists of a series of steps. After cleaning away the rest of the leaves and pedicels, a first selection of capers takes place and blemished and open buds are discarded. Then, capers are subjected to a first sieving, which generally

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size-grades them into two size groups, with diameters lower or higher than 8–9 mm. This first classification provides an incentive for recollection of smaller capers and makes the subsequent industrial steps easier. After aeration in a well-ventilated place, capers are packed in wooden or PVC barrels, fiberglass tanks, or large casks and pretreated with high salt brine (ca 16% NaCl w/v at the equilibrium, increasing to 20% after changing the first brine). After filling, the casks are hermetically closed and placed in the sun. In order to reach the equilibrium in salt concentration, barrels are rolled during the early stage of brining. Periodical checks should be performed, to ensure that the brine completely covers the material. The completion of this “wet” curing process lasts 20–30 days (Luna Lorente and Pérez Vicente 1985), but capers may be stored under such conditions for several months, until final industrial conditioning takes place. Thus, capers may be classified as fully brined vegetables (Ranken 1988) that may be regarded as a stable product during storage. Fresh capers have an intensely bitter flavor and one of the purposes of the pickling process, besides that of preservation, is to remove this unpleasant characteristic. This is due to the presence of the glucoside glucocapparin, which is readily hydrolyzed to by-products completely lacking the bitter taste. Although Spanish regulations still accept the use of brine concentrations up to 25°Baumé (Dirección de Comercio Exterior 1984), high salt brines are increasingly being objected to (Alvarruiz et al. 1990; Rodrigo et al. 1992). Organoleptic characteristics and preservation of the final product proved to be the same over at least 27 months when capers had been pretreated with 10, 15, or 20% NaCl at equilibrium (Alvarruiz et al. 1990). High salt concentrations inhibit both the growth of undesirable microorganisms and the activity of lactic acid bacteria. Lower NaCl brines (i.e., 5%) are more likely to permit growth of coliform bacteria, yeasts, and molds (Özcan and Akgül 1999a). Fermentation takes place at a higher rate when pickling small (≤ 8 mm) buds (Özcan and Akgül 1999a). Capers are also pickled in vinegar (at least 4% acidity as acetic acid) in a 1:1 (w/v) ratio (Reche Mármol 1967). Regular topping-up with vinegar ensures that all the capers remain covered. This pickling process lasts 30 days. Only 10% of vinegar is absorbed by the product, with the remainder being discarded at the end of the period. In Italy, growers arrange capers in cement tanks, PVC or wooden barrels, or open drums, between layers of solid salt (10–15% w/w). This promotes the extraction of water from the raw product by osmosis and generates a saturated brine. This treatment lasts 7–8 days. Then, the

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brine is removed and the capers are submitted to the same process once or twice more (Barbera 1991). 2. Industrial Treatment and Packaging. Following the completion of the curing period, the industrial processing is completed in three steps. First, capers are drained and rinsed with several changes of water to dislodge and remove all sediment. Second, damaged buds are disposed of, and then capers are carefully size-graded according to an international grading system (Table 4.4). Third, capers are prepared in a variety of ways and packed as a finished product. Pasteurization (80°C, 15 min) of the final product renders capers with good flavor and consumer acceptance and is recommended to prevent the development of microorganisms (Ranken 1988; Alvarruiz et al. 1990). Without pasteurization, 6–10% NaCl and 1% acidity as acetic acid (w/v) are required in the final product to avoid the risk of spoilage (Alvarruiz et al. 1990; Özcan and Akgül 1999b). In some cases, NaCl is avoided and covering capers with diluted acetic acid or distilled malt vinegar (4.3 to 5.9% acetic acid) serves as an alternative. In Italy, the final product is treated with dry salt. Such preparation decreases the cost of transportation and grants a more intensive flavor. In Spain, a similar treatment is carried out with capers of large diameter. Capers are drained and mixed with dry salt (20% maximum). The caper industry discontinued the use of olive oil in caper preparations due to its high cost. Other special preparations, including wine vinegar, with or without the addition of tarragon, Artemisia dracunculus L. (Vivancos Guerao 1948), are also expensive and exclusively utilized with capers of small diameter. Sweetening ingredients like sugar are added to those capers exported to Denmark or some northern European countries (González Soler 1973). Capers are generally packed in PVC or wooden barrels of 180–200 kg for the pickle industry but 40-kg barrels are used for packing “non pareil” and “surfine” capers, depending on the country importing them. For retail sale, capers are packed in various kinds of glass or plastic bottles containing 20 g to 5 kg, or translucent sachets of 0.1 to 1 kg. Fivekg flasks and sachets are usually sold to restaurants and coffee-shops. B. Caperberries Traditionally, caperberries are fermented by dipping them in water for 4–7 days. This immersion produces a strong fermentation accompanied by a color change (from green to yellowish) and loss of texture due to flesh breakdown and gas accumulation. This step affects the value of the

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product and has proven to be unnecessary (Sánchez et al. 1992). Lactic acid bacteria show faster growth rates at low NaCl concentrations (Sánchez et al. 1992) but, as for capers, undesirable microorganisms can grow in 5% NaCl brines (Özcan 1999a). In order to protect caperberries from spoilage during fermentation, 4–5% NaCl brines may be adequate (Sánchez et al. 1992), but fermentation must be continuously controlled (Özcan 1999a). Yields are increased by the use of 0.35% sodium acetate (Sánchez et al. 1992). Fermentation should last 20–25 days. Brines with 10% (Sánchez et al. 1992) to 15% (Özcan 1999a) NaCl at equilibrium create a favorable environment for pickled caperberry storage. Sorbic and benzoic acids, as well as their corresponding sodium and potassium salts, are used as preservatives during final packing. A method combining steam distillation (extraction) and HPLC determination has proven to be excellent for the analysis of both preservatives at low concentrations in caperberries (Montaño et al. 1995).

VI. COMPOSITION AND UTILIZATION A. Composition The chemical composition of caper plant, leaves, flower buds, fruits, and seeds is summarized in Table 4.5. As data were obtained using different genotypes, which were grown under various environmental conditions and analyzed using different experimental protocols, values can only be considered as approximations leading, at best, to the right orders of magnitude (Duke 1992). Important differences in lipid and mineral (P, Ca, Mg, Na, Fe, Zn) contents have been found in raw capers. In general, lower levels of water content, starch, and carotenoids and higher levels of ash, protein, and calcium were found in smaller capers (Rodrigo et al. 1992; Özcan and Akgül 1998). Significant differences in composition were also detected among genotypes and harvest dates (Rodrigo et al. 1992; Özcan and Akgül 1998). In caper plants, variations in organic acids (citric, tartaric, and oxalic acid), as well as in sugar components (glucose, glucuronic acid, arabinose, and xylose) and alkaloids have been considered to be evidence of taxonomic differences (Hammouda et al. 1975). High salt brine treatments greatly affect the composition of capers. Fiber and protein, as well as mineral (Mg, K, Mn) and vitamin (thiamine, riboflavine, ascorbic acid) contents drop during those preservation procedures, while ash increases due to the addition of NaCl (Nosti Vega and Castro Ramos 1987). A similar trend has been observed after

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Table 4.5. Phytochemical constituents and other variables in caper bush, capers, and caperberries. Data are on a fresh weight basis. Constituent

Organ

Content

Reference

Water

Leaf Flower bud

80.5% 69.6% 76.8–81.9%

Fruit Seed

88% 82.7% 6.48%

Katiyar et al. 1985 Duke 1992 Nosti Vega and Castro Ramos 1987; Rodrigo et al. 1992; Özcan and Akgül 1998 Gorini 1981 Özcan 1999b Akgül and Özcan 1999

Ash

Leaf Flower bud

4.2% 13.2% 2.1% 1.33–1.84%

1.16–1.76%

Fiber

Protein

Fruit Seed

1.09% 1.73%

Leaf Flower bud

7.9% 2.04%

Fruit Seed

4.5–5.92% 3.13% 25.71%

Leaf Flower bud

3.85% 13.8% 3.2% 4.59–6.79%

4.81–7.27%

Amino Acids Alanine

Fruit Seed

3.34% 19–22%

Flower bud

3740 ppm

Katiyar et al. 1985 Duke 1992 Gorini 1981 Nosti Vega and Castro Ramos 1987; Rodrigo et al. 1992 Özcan and Akgül 1998 Özcan 1999b Akgül and Özcan 1999 Duke 1992 Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan 1999b Akgül and Özcan 1999 Katiyar et al. 1985 Duke 1992 Gorini 1981 Nosti Vega and Castro Ramos 1987; Rodrigo et al. 1992 Özcan and Akgül 1998 Özcan 1999b Duke 1992; Akgül and Özcan 1999 Nosti Vega and Castro Ramos 1987

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(continued )

Constituent

Organ

Content

Reference

Amino Acids (cont.) Aspartic Acid

Flower bud

6660 ppm

Glutamic Acid

Flower bud

7460 ppm

Glycine

Flower bud

1770 ppm

Isoleucine

Flower bud

3680 ppm

Leucine

Flower bud

4140 ppm

Lysine

Flower bud

4310 ppm

Methionine

Flower bud

910 ppm

Phenylalanine

Flower bud

3930 ppm

Proline

Flower bud

2110 ppm

Serine

Flower bud

1180 ppm

Threonine

Flower bud

2640 ppm

Valine

Flower bud

5410 ppm

Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987

Plant

0.22–0.5%z

Leaf

0.96% 0.71% 0.5% 0.47%

Lipids

Flower bud

1.51–1.77% 0.28–0.44% Fruit

Fatty Acids Palmitic Acidy

Seed

3.75% 0.84% 31.6–36%

Plant

0.076–0.084%z

Plant Flower bud

5.4–17.8% 23.9%

Mukhamedova et al. 1969 Katiyar et al. 1985 Rakhimova et al. 1978 Gorini 1981 Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan and Akgül 1998 Rakhimova et al. 1978 Özcan 1999b Pernet 1972; Duke 1992; Akgül and Özcan 1999 Mukhamedova et al. 1969 Katiyar et al. 1985 Nosti Vega and Castro Ramos 1987 (continues)

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162 Table 4.5.

G. O. SOZZI (continued )

Constituent

Organ

Content

Reference

Fruit Seed

31.9–32.4% 16.44% 13.2%

Flower bud

1.4%

Fruit Seed

1.4–8.9% 4.6%

Plant Flower bud

5.69–20.29% 7.4%

Fruit Seed

4.1–4.9% 2.7% 3.2%

Plant Flower bud

5.24–16.57% 5.9%

Fruit Seed

8.1–10.2% 29.7% 42–46%

Rodrigo et al. 1992 Özcan 1999b Akgül and Özcan 1999 Nosti Vega and Castro Ramos 1987 Özcan 1999b Akgül and Özcan 1999 Ahmed et al. 1972c Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan 1999b Akgül and Özcan 1999 Ahmed et al. 1972c Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan 1999b Hilditch and Williams 1964 Akgül and Özcan 1999 Ahmed et al. 1972c Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan 1999b Hilditch and Williams 1964 Akgül and Özcan 1999 Ahmed et al. 1972c Nosti Vega and Castro Ramos 1987; Rodrigo et al. 1992 Özcan 1999b Akgül and Özcan 1999

Fatty Acids (cont.)

Palmitoleic Acidy

Stearic Acidy

Oleic Acidy

49.9% Linoleic Acidy

Plant Flower bud

4.69–19.03% 14.9%

Fruit Seed

17.9–18.2% 29.9% 45–51% 25.2%

Linolenic Acidy

Carbohydrates Starch

Plant Flower bud

16.15–71.92% 35–37.5%

Fruit Seed

12.9% 1%

Leaf Flower bud

3.6% 0.83–1.24%

Katiyar et al. 1985 Özcan and Akgül 1998

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(continued )

Constituent

Organ

Content

Reference

Total Sugars Reducing Sugars

Fruit Flower bud Flower bud

0.61% 5.4% 2.62–4.69%

Pentosan

Fruit Flower

5.53% 40,000 ppm

Özcan 1999b Gorini 1981 Özcan and Akgül 1998 Özcan 1999b Duke 1992

Leaf Flower bud

290 ppm 1036 ppm

Carbohydrates (cont.)

Minerals P

166.5–264.5 ppm 591–806.4 ppm

S Ca

Fruit Flower bud

1167.9 ppm 180 ppm

Leaf Flower bud

1180 ppm 490.5–1344 ppm 1830 ppm 43.2–225.9 ppm

Mg

Flower bud

469–810.5 ppm

1118–1774 ppm K

Flower bud

4303–6135 ppm

Na

Fruit Flower bud

3269.3 ppm 59 ppm 190.5–285 ppm 24.3–36.7 ppm

Fe

Fruit Leaf Flower bud

121.4 ppm 150 ppm 13.7 ppm 9.25–21.1 ppm 1.59–4.68 ppm

Katiyar et al. 1985 Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan and Akgül 1998 Özcan 1999b Nosti Vega and Castro Ramos 1987 Katiyar et al. 1985 Rodrigo et al. 1992 Nosti Vega and Castro Ramos 1987 Özcan and Akgül 1998 Nosti Vega and Castro Ramos 1987; Rodrigo et al. 1992 Özcan and Akgül 1998 Nosti Vega and Castro Ramos 1987; Rodrigo et al. 1992; Özcan and Akgül 1998 Özcan 1999b Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan and Akgül 1998 Özcan 1999b Katiyar et al. 1985 Nosti Vega and Castro Ramos 1987 Rodrigo et al. 1992 Özcan and Akgül 1998 (continues)

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164 Table 4.5.

G. O. SOZZI (continued )

Constituent

Organ

Content

Reference

Mn

Fruit Flower bud

5.45 ppm 2.9 ppm

Zn

Fruit Flower bud

7.18 ppm 7.6 ppm

Özcan 1999b Nosti Vega and Castro Ramos 1987 Özcan 1999b Nosti Vega and Castro Ramos 1987 Özcan and Akgül 1998 Nosti Vega and Castro Ramos 1987

Minerals (cont.)

1.17–2.59 ppm Cu Vitamins Thiamine (Vit B1)

Flower bud

3.4 ppm

Flower bud

0.72 ppm 0.698 ppm

Riboflavin (Vit B2)

Flower bud

0.89 ppm 2.16 ppm

Choline Ascorbic Acid (Vit C)

Leaf Flower bud

100 ppm 260 ppm 2300 ppm 156–324 ppm

Secondary Metabolites Total Alkaloids L-stachydrine Rutin

Pigments Carotenoids

Leaf Fruit Leaf Plant

200 ppm 740 ppm 100 ppm 0.02–0.026%z

Flower bud

0.28%

Flower bud

1.028 ppm 1.29–3.38 ppm

pH

z

Fruit

1.15 ppm

Flower bud

5.9–6.3 ppm

Fruit

4.32 ppm

Lemmi Cena and Rovesti 1979 Nosti Vega and Castro Ramos 1987 Lemmi Cena and Rovesti 1979 Nosti Vega and Castro Ramos 1987 Duke 1992 Lemmi Cena and Rovesti 1979 Nosti Vega and Castro Ramos 1987 Özcan and Akgül 1998 Ahmed et al. 1972c Ahmed et al. 1972c Duke 1992 Mukhamedova et al. 1969 Nosti Vega and Castro Ramos 1987 Nosti Vega and Castro Ramos 1987 Özcan and Akgül 1998 Özcan 1999b Özcan and Akgül 1998 Özcan 1999b

Converted to a fresh weight basis considering 80% moisture. Each fatty acid is reported as the percentage of the total fatty acid content.

y

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caperberry preservation treatment (Özcan 1999b). This exchange of different components, i.e. ionic constituents, is not particular for capers and caperberries but a general trend when soaking fruits, probably enhanced by the presence of NaCl. Fatty acid analyses have revealed that capers are rich in linoleic and linolenic acids (Table 4.5). Moreover, seeds yield higher levels of oleic and linoleic acid and have lower contents of saturated acids than other Capparis species (Hegnauer 1961; Sen Gupta and Chakrabarty 1964). The lipid complex of the aerial part of caper bush has been extracted with chloroform-methanol and analyzed (Tolibaev and Glushenkova 1995). Many lipid products were obtained, including neutral lipids (mainly free fatty acids, triacylglycerols, and sterol and triterpenol esters), glycolipids (mainly digalactosyldiglycerides and sterol glycosides), and phospholipids (mainly phosphatidylglycerols, phosphatidylethanolamines, and phosphatidylcholines). In almost all classes of lipids, palmitic, oleic, linoleic, and linolenic acids were prevailing. Different flavonoids were identified in caper bush and capers: rutin (quercetin 3-rutinoside), quercetin 7-rutinoside, quercetin 3-glucoside7-rhamnoside, kaempferol-3-rutinoside, kaempferol-3-glucoside, and kaempferol-3-rhamnorutinoside (Rochleder and Hlasiwetz 1852; Zwenger and Dronke 1862; Ahmed et al. 1972a; Tomás and Ferreres 1976a,b; Ferreres and Tomás 1978; Artemeva et al. 1981; Rodrigo et al. 1992; Sharaf et al. 1997). Rutin and kaempferol-3-rutinoside are probably the most abundant flavonoids, followed by kaempferol-3-rhamnorutinoside in significantly lower concentrations (Rodrigo et al. 1992; Sharaf et al. 1997). Recently, Sharaf et al. (2000) identified a quercetin triglycoside (quercetin 3-O-[6’”-a-L-rhamnosyl-6”-b-D-glucosyl]-b-Dglucoside) in methanolic extract of the aerial part of caper bush. Guignard (1893b) first reported the presence of the enzyme myrosinase in C. spinosa. Brassicaceae are a major source of glucosinolates (Kjœr 1963; Kjœr and Thomsen 1963) whose hydrolysis to glucose, sulfuric acid, and isothiocyanates can be catalyzed by the enzyme myrosinase. The presence of glucosinolates is synapomorphic for members of this family and lends additional support to the new phylogenetic classification (Judd et al. 1999). In fact, the conclusion that Capparidaceae and Cruciferae should remain together, based on the presence of glucosinolates, was drawn almost 40 years ago (Hegnauer 1961; Kjœr 1963). Glucosinolates, their biochemistry, biological variations, and roles have been recently reviewed in detail (Rosa et al. 1997). Methyl glucosinolate (glucocapparin) is the most common glucosinolate occurring in the Capparis genus (Ahmed et al. 1972b) but others have also been detected in and isolated from caper plants. Those include 2-propenyl glucosinolate

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(sinigrin), 3-methylsulfinylpropyl glucosinolate (glucoiberin), indol-3ylmethyl glucosinolate (glucobrassicin), and 1-methoxyindol-3-ylmethyl glucosinolate (neoglucobrassicin) (Ahmed et al. 1972a). In leaves and stems, aliphatic glucosinolates seem to be prevailing (Kjœr and Thomsen 1963), but methanolic extracts from roots were found to contain 4methoxyindol-3-yl-methyl glucosinolate (4-methoxy-glucobrassicin) (Schraudolf 1989). Thus, there are qualitative and quantitative differences in glucosinolate composition in different caper tissues, as happens with most glucosinolate-containing species (Rosa et al. 1997). Methyl glucosinolate was reported to be present at levels in the range of 38–268 ppm in capers treated with dry salt, brine, or oil (Sannino et al. 1991). An interference in the determination of dithiocarbamate residues in capers has been reported and seems to be due to the presence of methyl glucosinolate (Sannino et al. 1991). However, thiocyanates and isothiocyanates (odoriferous breakdown products of glucosinolates), as well as other volatile compounds, do not interfere in those pesticide tests (Brevard et al. 1992). Brevard et al. (1992) identified 160 components of pickled caper flavor, including elemental sulfur (S8) and more than 40 sulfur-containing compounds, among them thiocyanates and isothiocyanates. These authors also detected “raspberry-like” constituents: a- and b-ionine, frambinone, frambinyl alcohol, and zingerone. Two different 1H-indole-3-acetonitrile glycosides, 1H-indole-3acetonitrile 4-O-b-glucopyranoside and 1H-indole-3-acetonitrile 4-Ob-(6’-O-b-glucopyranosyl)-glucopyranoside (capparilosides A and B, respectively), have been isolated in methanolic extracts of caperberries (Çalis ¸ et al. 1999). B. Utilization 1. Food Use. Capers are recognized as a safe product when used as a spice for natural seasoning (Simon et al. 1984). A site in the Internet (http://food.epicurious.com) offers more than 250 recipes that include capers (CondéNet 2000), most of them compiled from specialized journals (Gourmet, Bon Appetit). Capers have a sharp piquant flavor and are mainly used as a seasoning to add pungency to: (1) sauces (e.g., tartare, remoulade, ravigote, vinaigrette, sauce gribiche, tarragon sauce, and caper sauce, for serving with lamb or mutton); (2) salads (e.g., caponata, a cold eggplant salad with olives and capers) and dressings; (3) cold dishes (vithel tohnné), or sauces served with salmon, herring, whiting, or turbot; (4) pizzas and canapés; (5) cheeses (e.g., liptauer cheese); and (6) lamb, mutton, pork or chicken preparations (Hayes 1961; Knëz 1970;

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Machanik 1973; Nilson 1974; Baccaro 1978; Stobart 1980). An unusual and complex organoleptic profile is responsible for caper flavor (Brevard et al. 1992). Caperberries and tender young shoots of the caper bush are also pickled for use as condiments, as previously described. The unripe seeds or pickled buds of other species (Tropaeolum majus L., Caltha palustris L., Cytisus scoparius (L.) Link., Zygophyllum fabago L., Euphorbia lathyrus L.) are sometimes suggested as substitutes for capers (Redgrove 1933; Vivancos Guerao 1948; Seidemann 1970; Mitchell and Rook 1979; Stobart 1980; Bond 1990). 2. Ornamental Use. Caper foliage is attractive but the sweet-scented flowers, with delicate white petals and long-projecting staments, give the caper plant most of its ornamental value (Bailey 1927; Baccaro 1978; Foster and Louden 1980). Thus, caper plant is most commonly used in ornamental plantings, for terraces exposed to the sun, borders, rocky gardens, and walls (Coutanceau 1957). Caper bush may be used as part of a strategy for reducing potential or actual erosion hazard (Lozano Puche 1977) along highways or pronounced rocky slopes, locations for which control is often more difficult than on farmland because many species used for erosion control do not survive the stressful conditions of the C horizon or without irrigation. Caper plant has low flammability (Neyis¸çi 1987) and thus may play a vital role in preventing forest fires. 3. Medicinal and Cosmetic Value. Most of the organs of the caper plant have been extensively used as folk remedies—sometimes as part of polyherbal formulations—for various diseases (Pernet 1972; Kirtikar and Basu 1975; Boulos 1983; Duke 1983; Jain and Puri 1984; Abbas et al. 1992; Husain et al. 1992; Al-Said 1993; Ghazanfar and Al-Sabahi 1993; Ghazanfar 1994; Bhattacharjee 1998). Recent reports appear to confirm some claims of these traditional formulations. Liv.52, an Indian traditional medicine that contains different plant extracts, among them 24% of C. spinosa, is a “liver stimulant” with some protective action against hepatotoxic substances, radiation sickness, and dermatitis. This herbal formulation brings about an hepatoprotective action by inhibiting lipid peroxidation and improving antioxidant levels (Suja et al. 1997; Vijaya Padma et al. 1998; Sandhir and Gill 1999). Liv.52 has an hepatoprotective effect against ethanol in that it reduces the hepatic binding of both ethanol and acetaldehyde (Dhawan and Goel 1994) and accelerates acetaldehyde elimination (Dhawan and Goel 1994; Chauhan and Kulkarni 1991a,b). Studies with rats have shown that it also prevents the deleterious effects of maternal ethanol ingestion on the fetus

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during gestation (Gopumadhavan et al. 1993). In a chronic model using rats, Liv.52 normalized the blood ethanol and acetaldehyde levels in a dose-dependent manner (Chauhan et al. 1994). Liv.52 has a beneficial effect on the activity of superoxide dismutase and glutathione levels (Sandhir and Gill 1999). Nevertheless, Fleig et al. (1997) found that Liv.52 does not improve survival of patients with alcoholic cirrhosis. Different fractions of an ethanolic extract of the root bark of caper plant significantly reduce the hepatotoxic activity of carbon tetrachloride (CCl4) in rats (Shirwaikar et al. 1996). Furthermore, p-methoxy benzoic acid isolated from the aerial parts of caper bush was found to prevent the hepatotoxic effects of both CCl4 and paracetamol in vivo, as well as the hepatotoxic activity of thioacetamide and galactosamine in isolated rat hepatocytes (Gadgoli and Mishra 1995, 1999). Similar effects in rats were found using Liv.52. It impairs the CCl4-mediated reduction in aniline hydrochloride and p-aminopyrine N-demethylase activity (Thabrew et al. 1982), as well as cathepsin-B, acid phosphatase, glucose-6phosphatase, and ribonuclease activity (Kataria and Singh 1997). On the other hand, it prevents the CCl4-mediated increase in different serum and liver hepatotoxicity markers (alkaline phosphatase, alanine transaminase, and aspartate transaminase) (Dhawan and Goel 1994) as well as CCl4- and H2O2-induced lipid peroxidation (Pandey et al. 1994; Suja et al. 1997). Liv.52 was found to down-regulate the tumor necrosis factor in CCl4-treated rats (Roy et al. 1994). Liv.52-treated rats also showed less marked toxic effects when beryllium (Mathur 1994) and mercuric chloride (Rathore and Varghese 1994) were administered. The ingestion of Liv.52 reduced the number and mass of DMBA- and croton oil-induced skin papillomas in male Swiss albino mice (Prashar and Kumar 1994). Liv.52 also showed in mice some antiviral activity against the Semlike forest encephalitis virus and enhanced the protective activity of 6MFA, an interferon-inducing antiviral substance (Singh et al. 1983). Nevertheless, many of the effects of Liv.52 may be due to non-caper constituents and the active compounds and precise mechanisms of action are still not clear. Caper root bark and leaves may have some anticarcinogenic activity (Hartwell 1968; Khan et al. 1992). In fact, the hydrolysis products of indol-3-ylmethyl glucosinolates have anticarcinogenic effects (Rosa et al. 1997). Although the consumption of capers is low in comparison with the intake of other major dietary sources of glucosinolates (white cabbage, broccoli, and cauliflower; Dragsted 1999) it may contribute to the daily dose of natural anticarcinogens that reduces cancer risk. Glucosinolates are also known to possess goitrogenic (anti-thyroid) activity

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(Rizk 1986; Rosa et al. 1997). Some non-nutrient components of capers are antioxidant compounds, e.g., quercetin, rutin, and kaempferol (Miller 1996; Pietta et al. 1996). Rutin and quercetin may contribute to cancer prevention (Committee on Comparative Toxicity et al. 1996). Selenium, present in capers at high concentrations in comparison with other vegetable products (Herrero Latorre et al. 1987), has also been associated with the prevention of some forms of cancer (Committee on Comparative Toxicity et al. 1996 and papers cited therein). On the other hand, linoleic acid has been reported to enhance carcinogenesis; but at high levels such as those found in capers (>16%), a reduction in cell proliferation has been reported in the mammary gland (Committee on Comparative Toxicity et al. 1996). The decoction of C. spinosa has hypoglycemic properties and may be useful in antidiabetic therapy (Ageel et al. 1985; Yaniv et al. 1987). The oral administration of a caper root decoction or tincture to guinea pigs revealed strong desensitizing effects against various plant and animal allergens (Khakberdyev et al. 1968). Cappaprenol-12, -13 and -14 in ethanol extracts of caper leaves are antiinflammatory compounds (AlSaid et al. 1988; Jain et al. 1993). Methanolic extracts of C. spinosa showed some antimalarial activity when assayed in vitro against a multi-drug resistant strain of Plasmodium falciparum (K1) (Marshall et al. 1995). Extracts of the whole plant or its aerial part also exhibited variable degrees of antimicrobial activity against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Proteus vulgaris, Bacillus cereus, and Bacillus subutilis, as well as antifungal activity against Candida albicans, Candida pseudotropicalis, and Fusarium oxysporum (Nadir et al. 1986; Mahasneh et al. 1996). Nevertheless, Ali-Shtayeh et al. (1998) found that aerial plant extracts only have antimicrobial activity against S. aureus and Proteus vulgaris, but fail to display antimicrobial activity against C. albicans, E. coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Many spices and their derivatives have antifungal properties but Capparis flower bud extracts did not show inhibitory effects on Aspergillus parasiticus mycelial growth (Özcan 1998). On the other hand, the aqueous extracts of the aerial part of caper plant prevented the growth of Microsporum canis, Trichophyton mentagrophytes, and Trichophyton violaceum (Ali-Shtayeh and Abu Ghdeib 1998). Thus, antidermatophytic activity in caper extracts is comparable with that of griseofulvin preparations (often used as a standard in evaluating antibiotic potential), suggesting a possible use against dermatophytic infections in humans.

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The green parts of caper plant have been considered to be potentially irritating to the skin because of its glucosinolates (Mitchell 1974; Mitchell and Rook 1979; Cronin 1980; Foussereau et al. 1982). Caper leaf and fruit extracts, applied as wet compresses to inflamed skin, may produce acute contact dermatitis (Angelini et al. 1991). Nevertheless, Lemmi Cena and Rovesti (1979) pointed out that caper extracts may be used for treating enlarged capillaries and dry skin. Barbera (1991) suggested that they could be utilized for cosmetic preparations (creams, shampoo, lotions, and gels), due to the presence of some active principles: rutin and quercetin (flavonoids that produce effects similar to those of vitamin P), pectins (moisturizing and protecting effects), glucocapparin (rubefacient action), phytohormones, and vitamins.

VII. INTERNATIONAL TRADE A major obstacle to a satisfactory analysis of the economics of capers— as is the case with many other spices—is the lack of statistical information. Accurate production figures are lacking for most of the exporting countries. Moreover, export-import statistics do not include those capers that are marketed in other processed products, with loss of identity (G. Chironi, in Barbera 1991; Sozzi 2000). On the other hand, trade statistics are the only source of information on consumption in many regions of Europe, where capers are often produced for local or even household use. Caper commercial exchange involves more than 60 countries. Nowadays, the average annual production may be estimated to be around 10,000 t: 3,500–4,500 t are produced in Turkey, 3,000 t in Morocco, 500–1,000 t in Spain, and 1,000–2,000 t in other countries. The most complete time series reflecting the international trade is that of United States imports (Figs. 4.5 and 4.6). The United States is one of the most important consumer countries. Based on United States statistics, it may be concluded that: 1. Increasing amounts of capers are being consumed (Fig. 4.5), and this trend appears to be sustained for the next few years, the expanding ethnic populations and the interest in new tastes presumably accounting for most of the increase in caper consumption. 2. The Spanish and Italian production has increasingly been exposed to the international competitive influence of Turkey and Morocco and current prices have been on a downward trend. The decline in prices is more dramatic if inflation is taken into account, and the

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2400 2200

Turkey

2000

Spain

Tonnes

1800 1600

Morocco

1400

Others

1200 1000 800 600 400 200

99 19

97 19

95

93

19

91

19

89

19

19

87

85

19

83

19

81

19

19

79

77

19

75

19

73

19

19

19

71

0

Year Fig. 4.5. United States imports of capers from major producing countries (1971–1999). Prepared with data from the USDA, Foreign Agricultural Service (1973/2000).

8 1,000 Dollars per Tonne

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Spain Morocco Turkey Average

6 5 4 3 2 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 Year

Fig. 4.6. United States import prices for capers during the last decade. Prices are on a current basis. Prepared with data from the USDA, Foreign Agricultural Service (1991/2000).

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current prices are adjusted (i.e., using the consumer price index). On the other hand, caper quality and presentation are recognized by traders and higher prices are paid for the product made in Spain (Fig. 4.6) or Italy. In fact, the French and Greek products are generally even more expensive. Very small amounts are marketed and they are bought by traders concerned with securing a high-quality supply. 3. The caper trade is very dynamic, with imports and subsequent exports often combined. Turkey’s main markets for caper exports are Spain, the United States, France, Italy, Germany, The Netherlands, Brazil, United Kingdom, Belgium, Venezuela, Japan, Denmark, and Israel. Denmark does not produce capers but exports larger amounts to the United States than Italy. And the United States, whose production is negligible, exports some of its imports to other countries, e.g., Venezuela. Morocco also exports capers to Spain and Italy, traditional producers that devote most of their own production to exportation.

VIII. CONCLUDING REMARKS Caper bush has a unique and interesting biology but few scientific reports have been published to unravel its mechanisms of growth and survival in harsh and stressful environments. Apart from environmental conditions, success in caper bush cultivation depends mainly on five fundamental points: (1) biotypes of high quality and production; (2) adequate propagation; (3) good control of cultivation practices, particularly harvest; (4) adequate postharvest processing and storage; and (5) efficient marketing systems and strategies. In low-input systems with both low land and labor costs, the caper plant can provide the diversity required for sustainability. On the other hand, caper yields are much higher in irrigated plantings, with NPK fertilization, although much more research is required to determine the optimal cultivation conditions for this species. Diseases and pests do not seem to be a great problem in general but also need to be researched. Two major expenses are expected: implantation and harvesting. The latter may be the stumbling block in high-input systems, and the possibility of a semi-mechanical operation should be considered in order to remove this limiting factor. Moreover, further improvement in caper quality may be obtained by regulating harvesting dates. There is an assortment of opportunities for plant breeders to contribute to domestication of caper bush for agricultural purposes. Determination of the genetic bases for productivity, ease of propagation,

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absence of stipular spines, and flower bud quality and conservation are high-priority research needs. Finally, marketing research remains an area of high concern. Marketing of capers without a pre-arranged contract with processing or exporting companies could be very risky. Market promotion and the ability of handlers to provide a high-quality product at times that will yield a competitive price have become essential factors. Producers and handlers will be challenged to develop new and expanded markets for capers. Many ethnic foods have filtered into the diets of Americans and other developed countries. A global cuisine is coming, for which the world’s food diversity should be available to everybody. Caper plant, a simple drought-tolerant perennial bush that grows in semiarid areas, has a favorable influence on the environment, stabilizes eroding slopes, helps to prevent forest fires, benefits some rural economies, provides medicinal and cosmetic compounds, and brings a unique flavor to our meals, may play a significant role in the international spice trade in the future. LITERATURE CITED Abbas, J. A., and A. A. El-Oqlah. 1992. Distribution and communities of halophytic plants in Bahrain. J. Arid. Environ. 22:205–218. Abbas, J. A., A. A. El-Oqlah, and A. M. Mahasneh. 1992. Herbal plants in the traditional medicine of Bahrain. Econ. Bot. 46:158–163. Ageel, A. M., M. Tariq, J. S. Mossa, M. S. Al-Saeed, and M. A. Al-Yahya. 1985. Studies on antidiabetic activity of Capparis spinosa. Federation Proc. 44:1649 (7243). Ahmed, M. 1986. Vegetation of some foothills of Himalayan range in Pakistan. Pak. J. Bot. 18:261–269. Ahmed, M., and S. A. Qadir. 1976. Phytosociological studies along the way of Gilgit to Gopies, Yasin and Shunder. Pak. J. Forestry 26:93–104. Ahmed, Z. F., A. M. Rizk, F. M. Hammouda, and M. M. Seif El-Nasr. 1972a. Glucosinolates of Egyptian Capparis species. Phytochemistry 11:251–256. Ahmed, Z. F., A. M. Rizk, F. M. Hammouda, and M. M. Seif El-Nasr. 1972b. Naturally occurring glucosinolates with special reference to those of family Capparidaceae. Planta Med. 21:35–60. Ahmed, Z. F., A. M. Rizk, F. M. Hammouda, and M. M. Seif El-Nasr. 1972c. Phytochemical investigation of Egyptian Capparis species. Planta Med. 21:156–160. Akgül, A., and M. Özcan. 1999. Some compositional characteristics of caper (Capparis spp.) seed and oil. Grasas Aceites 50:49–52. Ali-Shtayeh, M. S., and S. I. Abu Ghdeib. 1998. Antifungal activity of plant extracts against dermatophytes. Mycoses 42:665–672. Ali-Shtayeh, M. S., R. M. R. Yaghmour, Y. R. Faidi, K. Salem, and M. A. Al-Nuri. 1998. Antimicrobial activity of 20 plants used in folkloric medicine in the Palestinian area. J. Ethnopharmacol. 60:265–271. Alkire, B. 1998. Capers. (http://www.hort.purdue.edu/newcrop/CropFactSheets/caper.html). Allen, B. (ed.). 1994. Food: an Oxford anthology. Oxford University Press, Oxford-New York. p. 43, 48.

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5 Water Relations and Irrigation Scheduling in Grapevine* M. H. Behboudian Institute of Natural Resources College of Sciences Massey University Palmerston North, New Zealand Zora Singh Department of Horticulture Muresk Institute of Agriculture Curtin University of Technology GPO Box U 1987 Perth, WA 6845, Australia

I. INTRODUCTION II. PHENOLOGY III. ASPECTS OF WATER RELATIONS A. The Soil-Plant-Atmosphere Continuum B. Plant Roots and Water Absorption C. Transpiration 1. Energy Supply and Leaf Interception 2. Vapor Pressure Deficit 3. Wind Speed 4. Resistance to Transpiration D. Development of Water Deficit: Measurement and Recovery E. Plant Responses to Water Deficit 1. Plant Water Status 2. Stomatal Conductance *We are grateful to Dr. Tessa Mills (HortResearch, New Zealand) for constructive discussion and for commenting on the manuscript. We thank Dr. Stephen Lawes (Massey University, New Zealand) for critical comments on the manuscript. Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 189

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3. Photosynthesis 4. Plant Growth F. Acclimation of Plants to Water Deficit 1. Osmotic Adjustment 2. Stomatal Behavior IV. IRRIGATION OF VINEYARDS A. Regulated Deficit Irrigation (RDI) 1. Timing of RDI Application 2. Early RDI: Budburst to Flowering 3. Mid-season RDI: Fruit Set to Veraison 4. Late-season RDI: Veraison to Harvest 5. Postharvest RDI 6. Degree of Deficit B. Partial Rootzone Drying (PRD) V. QUALITY ATTRIBUTES FOR WINE, DRIED, TABLE, AND JUICE GRAPES A. Wine Grapes B. Dried Grapes—Raisins C. Table Grapes D. Juice Grapes VI. FUTURE PROSPECTS LITERATURE CITED

I. INTRODUCTION The grape, associated with humans since prehistory, spread throughout the world in antiquity both for fresh fruit and wine production (Smart and Coombe 1983). Grapevine, mainly Vitis vinifera but also V. labruska and other species, is now the most widely grown fruit plant in the northern and southern hemispheres (Grimes and Williams 1990). In 1999, grapes were grown on 7.5 million hectares worldwide, with production estimated at 58.7 million tonnes (Table 5.1). Generally Vitis is a temperate plant but is successfully grown in Mediterranean and subtropical climates and can be grown as an evergreen where the temperature is consistently in the range of 20°C to 30°C and where rainfall patterns have a high degree of reliability (Possingham 1994). There are approximately 35,000 ha of grapevines grown in the tropics and tropical viticulture is characterized by the production of two to three crops per vine each year (Araujo et al. 1999). At present, more than 80% of the world’s grapes are used for wine production (Mullins et al. 1992). The preferred wines are mainly cultivated in the areas with low annual precipitation. While the traditional areas of grape growing are non-irrigated, irrigation greatly increases yield. As an excess of moisture often decreases wine quality, many traditional wine growing areas in Europe have legally limited irrigation or even pro-

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Table 5.1. Area and production of grapes in different parts of the world in 1999. Source: FAO STAT 1999 (http://www.FAO.org/)

Continent Europe Asia South America North and Central America Africa Oceania Total (World)

Area (millions of ha)

Production (millions of tonnes)

4.5 1.7 0.5 0.4 0.3 0.1 7.5

29.7 13.2 5.2 6.5 3.0 1.1 58.7

hibited it both to insure wine quality and to reduce overproduction. At present, irrigation in many European areas may only be used in an emergency to save vines in times of drought. Nevertheless, the judicious use of irrigation in grape production is now an established practice in many non-European countries. Previous reviews related to water relations in grapevine and stress physiology include chapters in books on crop physiology or irrigation science. Plant and environmental factors affecting water relations in grapevine have been reviewed by Smart and Coombe (1983) and Williams and Matthews (1990), while stress physiology has been covered by Williams et al. (1994). The scope of this presentation is to review new information on basic and applied aspects of water relations in grapevine. It introduces some modern technologies used in studying water relations in general and specifically in grapevine, irrigation scheduling, and describes some of the known effects of deficit irrigation on fruit and wine quality. Except where specifically mentioned, all grapes refer to Vitis vinifera.

II. PHENOLOGY Phenology of the grapevine is similar to other deciduous fruit. In late summer or autumn the plant enters dormancy characterized by leaf senescence and abscission, and lack of visible bud growth, which allows the plants to survive cold winter temperatures. After exposure to sufficient chilling, growth is resumed the following spring (Lavee and May 1997). In the humid tropics grapevine behaves as an evergreen (Possingham 1994). In temperate and subtropical regions that have mild

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winters, budbreak is erratic and less uniform than in environments with lower winter temperature. Water uptake precedes rapid shoot growth and is followed by budbreak and by flower differentiation within the cluster primordia that continues up to anthesis. In California, anthesis occurs approximately eight weeks after budbreak (Williams et al. 1994). In contrast to many deciduous fruit trees, vegetative growth of grapevines precedes flowering and fruit growth. The degree of overlapping between vegetative growth and fruit growth varies among cultivars. Fig. 5.1 shows some growth parameters of ‘Thompson Seedless’ growing in the San Joaquin Valley of California as a function of growing degree days (GDD) (Williams and Matthews 1990; Williams 1987). Grape cultivars tend to develop at consistent rates relative to other cultivars regardless of seasonal conditions (Williams and Matthews 1990). Some irrigation scheduling is done according to the stages of fruit growth, a double sigmoid curve (Matthews et al. 1987a). Three growth stages are recognizable on the curve (Fig. 5.2). Stage I is the initial phase

Fig. 5.1. Changes in dry weight, leaf area, and soluble solids of ‘Thompson Seedless’ grapevines grown in the San Joaquin Valley of California as a function of growing degree days (GDD> 10°C). Average date of budbreak was March 9. Adapted from Williams and Matthews (1990) based on the data of Williams (1987).

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Fig. 5.2. Changes in fruit diameter for Vitis vinifera cv ‘Cardinal’ made on two berries (open and closed circles) for 70 days after anthesis. Stages I, II, and III are explained in the text. Adapted from Matthews et al. (1987a).

of rapid growth and stage II is the lag phase of slow or no growth. At the start of stage III, berries resume their final phase of growth and maturation. The transition from stage II to III (veraison) is characterized by many physiological changes, most of which could occur rapidly, i.e., within 24–48 hours.

III. ASPECTS OF WATER RELATIONS Grapevines are frequently grown in areas where water supply limits optimum growth and production. Thus, information on the physiology of water relations, the assessment of water status, and the basic plant responses to reduced water status is important to optimize water use. A. Soil-Plant-Atmosphere Continuum In the soil-plant-atmosphere continuum, water in liquid form moves through the soil to plant roots, is absorbed by the roots, and transported

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through the stem to the leaves, where it is lost to the atmosphere as vapor. Important processes in the pathway include: water absorption by plant roots, water movement within the plant, and loss of water vapor from the leaves to the atmosphere (transpiration). Along the pathway, water movement is driven by the gradients of water potential in that water moves from the phase of high to low potential and is retarded by a series of resistances present in the system (Cowan 1965). B. Plant Roots and Water Absorption Root factors that contribute to absorption efficiency are root density and distribution, maximum root depth, and rate of growth. The higher the root density, the larger the volume of soil water that can be utilized. The distribution of grapevine roots has been studied extensively for various grape-growing areas of the world (Richards 1983). The distribution depends both on species and on soil properties. For the majority of soils, most of the roots occur in the top 1000 mm, although individual roots might penetrate to depths of 6000 mm or more (Richards 1983). The fine lateral roots, which are thought to form the main absorptive area, occur in the top 100 to 600 mm of the soil. Barriers such as compacted layers, water tables, and saline and acid zones restrict root depth (Richards 1983). Mapfumo et al. (1994) found that in ‘Shiraz’ water flow into the main roots via the lateral roots is likely to be much smaller than that via the direct radial flow pathway through the main roots. Only about 1% of surface area of main roots is directly occupied by lateral roots, leaving the other 99% of main root surface area available for the direct radial flow pathway. Irrigation and mulching affect root distribution. In drip irrigated vines there is a confined soil wetted zone beneath the emitter that largely coincides with a confined and shallow root system, while furrow irrigated vines have a deeper and more widespread root system (Araujo et al. 1995). Black plastic mulch doubled the root:shoot ratio and berry weight of ‘Chenin Blanc’. The increased growth of mulched vines was attributed to improved weed control and to conservation of soil moisture, more uniform soil temperatures, and less soil compaction (Van der Westhuizen 1980). C. Transpiration Grape berries have stomata whose activity decreases with fruit age. On a surface area basis, leaf transpiration rate is 2.5–10 times higher than that of the grape berries (Blanke and Leyhe 1987). Various environ-

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mental and plant factors that control transpiration have resulted in different rates reported throughout the world. Over 100 days, between anthesis and harvest, the cumulative sap flow of 3-year-old ‘Chardonnay’ plants, which is presumed to be the same as the transpiration rate, was 461 ± 44 kg plant–1 or 124 ± 12 mm on a land area basis (Lascano et al. 1992). This was exclusive of soil evaporation, which was 77% of evapotranspiration (ET). Myburgh et al. (1996) found the same rate of transpiration for three-year-old ‘Pinot Noir’ growing in the coastal region of the Western Cape in South Africa. Schmid and Braun (1997) used the heat balance method for measuring the rate of transpiration of ‘Riesling’ on four different rootstocks and obtained higher values of 2 to 2.5 mm day–1, on a land area basis, in Germany, which is expected to have a lower evaporative demand than Texas and the Western Cape. Possibly the plants were older in Schmid and Brown’s experiment or daylengths were significantly longer. 1. Energy Supply and Leaf Interception. Solar radiation is the major source of the energy required to evaporate water from transpiring leaves and has a direct effect on stomatal aperture, photosynthesis, and leaf temperature, with the latter influencing vapor pressure deficit. In addition to direct solar radiation, other sources of energy include re-radiation from the soil and surrounding objects and sensible heat that flows between the leaf and the environment. In grapevines, seasonal changes in leaf area thus influence seasonal water loss through transpiration. Transpiration is low during the dormant period and becomes considerably higher during the active growth period, especially when leaves are fully developed and during fruit growth. Leaf area rapidly increases during the spring until reaching the maximum value, then becomes stable and later decreases (Fig. 5.1) as the leaves start to fall during autumn. The actual pattern of leaf area development in grape during the season, however, varies among cultivars and training systems used. 2. Vapor Pressure Deficit. Vapor pressure deficit (VPD), which is the driving force for water vapor to move from the leaf to the air, is the difference between the vapor pressure of the leaf and the vapor pressure of water in the bulk air (the humidity). Over the normal range of leaf water potentials, the vapor concentration of the air inside the leaf is nearly constant and very close to saturation (Wenkert 1983). A large change in cell water potential causes only a small change in its vapor pressure. For example, at 20°C, as the leaf water potential drops from 0 to –2.7 MPa, the vapor concentration drops from 100 to 98% of saturation (Wenkert

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1983). Thus VPD is largely determined by the water content of the outside air and the temperature difference between the leaf and the air. Temperature is important because a certain volume of the air can hold more water as its temperature increases. When other factors are equal, transpiration rate is proportional to the value of absolute humidity difference or vapor pressure difference between the leaf and the air (Kramer and Boyer 1995, page 210). In addition, VPD also has a direct effect on stomatal movement. Stomatal closure in response to the dryness of the air (high VPD) regardless of the leaf water potential has been observed in various species including V. vinifera. Williams et al. (1994) cited the literature covering the reactions of V. vinifera cultivars to VPD and state that these reactions are cultivar dependent and a high VPD seems to cause non-uniform stomatal closure in Vitis species. The effect of VPD on stomatal closure seems to be more severe if plants are undergoing water stress as shown in Fig. 5.3 (Williams et al. 1994). For ‘Thompson Seedless’ the higher stomatal conductance occurred when plants were watered at 100% of ET followed by those watered at 60% and 20% of ET (Fig. 5.3). For each watering regime stomatal conductance became

Fig. 5.3. The relationship between stomatal conductance of ‘Thompson Seedless’ grapevine and vapor pressure deficit for three irrigation treatments replacing 100% of ET (1.0), 60% of ET (0.6), and 20% of ET (0.2). Adapted from Williams et al. (1994).

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lower with increasing VPD. The lowering of stomatal conductance with increasing VPD might have implications for assimilation rates and the subsequent growth of the plants. However, there are not conclusive data in the literature to warrant such an assessment for grapevine. 3. Wind Speed. Wind seems to have opposite effects on transpiration. As wind speed increases, the thickness of the boundary layer surrounding the leaf or the canopy decreases, lowering the layer’s resistance and leading to a higher rate of transpiration. Wind may also increase transpiration rate by adding energy to evaporate water from the flow of sensible heat. However, wind also cools the leaves, resulting in a decrease in vapor pressure gradient from leaf to air. Studies carried out in the field and laboratory indicate that winds with a speed higher than 3 m s–1 will decrease stomatal conductance and transpiration (Williams et al. 1994). The effect on stomatal conductance could be both through increased production of abscisic acid (ABA) in the leaf and/or mechanical damage to the leaf. In addition to transpiration, wind influences assimilation, growth, and yield. The effect of a windbreak on ‘Chardonnay’ in California is shown in Fig. 5.4 (Williams et al. 1994). The vines in the control treatment, which were exposed to higher wind speeds, had lower stomatal conductance and lower assimilation rate than the vines protected by the windbreak. Dry and Botting (1993), in a study of the effect of windbreak on the performance of ‘Cabernet Franc’, found shoot length and pruning weight of sheltered vines averaged 59% more than exposed vines. The fruit yield increased by 15% in sheltered vines as a result of increased bunches per shoot and increased shoot and bunch number per row. 4. Resistance to Transpiration. The total resistance of a leaf to transpiration includes the resistance of a leaf’s air boundary layer and the leaf resistance, which is composed of stomatal and cuticular resistance. Air boundary layer resistance (ra) is the resistance to vapor diffusion across the laminar boundary layer. Water vapor from the leaf surface moves through the boundary layer mostly by diffusion and therefore it happens faster through a thinner layer (Jones et al. 1985). Boundary layer resistance of fruit orchards is usually lower than that of the field crops at a certain wind speed, due to the aerodynamically rough and nonhomogeneous surface of the fruit orchards (Jones et al. 1985). Stomatal resistance (rs), and its reciprocal stomatal conductance (gs), is regulated mainly by the size of stomatal aperture such that rs increases as the aperture decreases. Stomatal aperture is the major passage for

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Fig. 5.4. The effect of windbreak on the diurnal course of CO2 assimilation rate, stomatal conductance, and wind speed of ‘Chardonnay’ grapevine grown in the Salinas Valley of California. Adapted from Williams et al. (1994).

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water vapor movement out of the leaf and for CO2 movement from the air into the leaf. Because gs is affected by leaf water status, attempts have been made to correlate it with leaf water potential (ψ) for different species. For grapevine a linear relationship was observed between ψ and gs of ‘Thompson Seedless’ (Williams et al. 1994), although a clear relationship between ψ and gs could not be determined for ‘Colombard’ (Van Zyl 1987). For grapevine the relationship seems to depend on the cultivar, environmental conditions, and the rate at which water stress develops. This inconsistent relationship in grapevine might also be related to non-uniform stomatal closure over the leaf surface, i.e., stomatal patchiness, which is characteristic of grapevine in response to water stress (Downton et al. 1987). Düring and Loveys (1996) recommend that in measuring the gas exchange of grapevine leaf, areas that include both patches with open and closed stomata should be used to counteract the variability of gas exchange rates. The effect of chemical messages from roots on stomata during soil drying will also affect the relation between ψ and gs. The effectiveness of stomata in controlling canopy transpiration depends on the state of air boundary resistance. Stomatal movement affects transpiration only when the canopy is closely coupled to the atmosphere in terms of water vapor pressure (Hsiao 1990). A closely coupled canopy refers to the canopy in which the temperature and the water vapor in the boundary layer and the air stream outside the boundary layer are very similar, resulting in a very small gradient in temperature and water vapor pressure between the two locations. In contrast, a poorly coupled canopy has a relatively thick boundary layer; thus water vapor exchange between the canopy and the ambient air above the canopy does not occur rapidly and stomatal movements will not have an effective role in transpiration. Most fruit trees, including grape, are considered to have a closely coupled canopy. D. Development of Water Deficit: Measurement and Recovery The term water deficit implies that water status is less than the optimum value for plant growth and development. Plant water deficit occurs when water absorption lags behind transpiration. Thus excessive transpiration, slow absorption, or their combination can lead to plant water deficit. Plant water deficit is characterized by a decrease in plant water content, turgor, and total water potential resulting in wilting, partial or complete stomatal closure and a decrease in cell enlargement and plant growth (Hsiao 1973). Determination of plant water deficit can therefore

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be done through measurements of plant water content, plant water potential and its components, trunk diameter fluctuations, sap flow, and stomatal aperture. Generally, a plant routinely experiences water deficit both diurnally and seasonally. During the day, absorption often lags behind transpiration due to resistance to water flow in the system (Williams and Matthews 1990). The rate of replenishment at night is dependent upon water availability in the soil and the efficiency of water absorption and the water conducting system of the plant. In dry soils, the deficit persists and increases in magnitude with time as the plant is not recovered at night. Leaf water potential (ψ) and its components of turgor and osmotic potentials have been widely used as indicators of plant water status and as a measure of plant water deficit. A diurnal variation in ψ, which indicates the strong influence of evaporative demand on plant water status, has been observed in most plant species, including grapevine (Fig. 5.5). McCutchan and Shackel (1992) advocated the assessment of predawn ψ as the measure of water stress in plants due to the strong influence of environmental conditions on ψ during the day. However, the predawn ψ of the plant may not indicate the soil-water status over the entire root zone because it tends to be biased towards the water status of the wettest part (Jones 1990). In some cases leaf ψ has been found not to be sensitive enough as a measure of plant water status. Stem water potential (ψstem) has been suggested as an indicator of plant water status (Naor et al. 1995). Stem water potential can be measured by enclosing the leaf (inside the canopy) in a plastic bag and covering it with aluminum foil while it is attached to the plant. After the water status of the leaf and of the stem has reached an equilibrium, proposed to be 90 min, the leaf is removed and the water potential measured (Naor et al. 1995). Naor (1998) measured stomatal conductance, ψ, and ψstem on grapevine as well as apple and nectarine from early morning to midafternoon under several irrigation treatments and showed that ψstem was more sensitive to irrigation level than was ψ. Stem water potential correlated better with gs than did ψ. He proposed a model in which gs, ψ, ψroot, root signal intensity, and transpiration rate are linked in a feedback mechanism that leads to a higher correlation of gs with ψstem , and the correlation is higher than with ψ. Sipiora and Lissarrague (1999) reported that for ‘Tempranillo’ vines diurnal changes of gs and ψ were linearly related only when the midday data were analyzed separately from those obtained earlier and later in the day. They concluded that changes in ambient light and CO2 were involved in the observed stomatal responses. Due to the difficulty of relating ψ to metabolic processes, Sinclair and

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Fig. 5.5. Diurnal changes in water potential of leaves for fruiting (+F) and defruited (–F) ‘Riesling’ grapevine measured on two separate occasions: February 1, 1985 (a) and February 15, 1985 (b). Vertical bars represent average least significant differences between means at P = 0.05. Adapted from Downton et al. (1987).

Ludlow (1985) proposed that plant water status should be measured in terms of cell volume change, expressed as relative water content (RWC). The disadvantage of RWC is that it varies with plant species, age, and habitat; and it cannot be related to soil water status (Kramer 1988). Because measurements of both water potential and RWC are usually destructive and cannot be done continuously, other parameters that can

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be measured continuously and are non-destructive, such as stem diameter and sap flow, have been proposed as indicators of plant water status. Stem diameter, measured using a linear variable differential transducer (LVDT), changes rapidly in close correlation with plant water content (e.g., Simmonneau et al. 1993). Cohen et al. (1993) demonstrated that sap flow, measured by the heat-pulse method, is more sensitive to water deficit than is predawn ψ. Fluctuations in environmental conditions at the time of measurement may mask the effects of water stress on the above parameters. The measure of a plant’s discrimination against 13CO2 will be another tool to assess the extent of water stress. The discrimination is a measure of internal physiological and environmental parameters influencing the performance of the plant throughout the season and not subject to prevailing conditions at the time of sampling (Meinzer et al. 1991). This technique relies on the fact that due to discrimination against 13CO2 during photosynthesis, the ratio of 13C:12C in plants is lower than that found in the atmosphere. Organic matter is often depleted in 13C relative to the standard against which it is compared. Therefore values of δ13C (the notation used for expressing 13C discrimination) are negative because: δ13C = ((Rsample/Rstandard) – 1) × 1000,

[1]

where R is the ratio of 13C:12C. Rstandard is for Pee Dee belemnite (Farquhar et al. 1982). Assuming isotopic composition of atmospheric CO2 does not vary, a less negative figure represents less discrimination (higher 13C concentration in the tissue) against 13CO2 during photosynthesis. Water stress results in less discrimination against 13CO2, or less negative values of δ13C, as exemplified by apple leaves (Mills et al. 1998) and chickpea pods and seeds (Behboudian et al. 2000). For grapevine, values of δ13C have been reported only twice in the literature and those reported by Gaudillere et al. (1999) are related to water stress effects. They reported that for Angers and Bordeaux areas of France, the δ13C values in berry of non-stressed grapevines (cultivar not specified) were, respectively, –24.6 ± 0.8 and –25.0 ± 0.8. The corresponding values for the dry plots were –21.7 ± 0.6 and –23.3 ± 0.6. The authors found that different scion cultivars differed in δ13C values, as did different rootstocks. They concluded that 13C discrimination could be used for assessing water stress effects in grapevine, as has also been done for some other species. This technique could be of value when assessing long-term effects of different deficit irrigation regimes on grapevine. The effects of water deficit are enhanced with increased crop load as indicated for grapevine (Downton et al. 1987), ψ is lower in fruited

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grapevines compared with de-fruited vines, or in heavy crop load compared with light crop load (Fig. 5.5). The effects of crop level on tree water relations may be due to an increase in tree water use with the presence of fruit or with higher crop level. A concomitant increase in transpiration via increased gs also occurs when fruit is present. This fruit effect may also be due to decreased root growth in a heavily cropping vine. Roots will be receiving less assimilates because of competition by fruit leading to a reduction in plant water uptake capacity as suggested by Lenz (1986). If water deficit is not too long or too severe, plants generally are able to recover following full irrigation. The time and degree of recovery from water deficit upon re-watering varies among plant species and cultivars and depends on the degree and duration of deficit treatment. Plants with high hydraulic conductivity may be expected to recover faster than those with lower hydraulic conductivity (McAneney and Judd 1983). Gucci et al. (1996) found that recovery of gs, CO2 assimilation, and transpiration rate of a kiwifruit vine was complete within 36 h of re-watering after the first drought cycle, while the recovery was slower during the second drought cycle when the deficit was more severe. Net photosynthetic rate and ψ was found to recover only partially after re-watering of severely stressed olive trees (Angelopoulos et al. 1996). E. Plant Responses to Water Deficit Plant responses to water deficit are numerous and somewhat complex. A significant amount of research on such effects has been conducted over recent years, often in conjunction with the study of deficit irrigation (Behboudian and Mills 1997). Deficit irrigation may be defined as a system of managing soil water supply to impose specific periods of plant water deficit to elicit some desirable responses in plants. When reference to deficit irrigation is made throughout this section, it is assumed that plant water status was reduced during the deficit irrigation period. 1. Plant Water Status. As the rate of water loss often exceeds that of water uptake, especially during the middle of the day, the plant becomes increasingly exposed to water deficit leading to a decreased water status. Changes in evaporative demand that occur during the day may also cause short periods of water deficit. In cordon-trained ‘Roditis’ grape with north-south orientation of the rows, water potential was lower on the eastern leaves throughout the day, but the rate of photosynthesis and transpiration was higher than that of the western leaves (Patakas 1993).

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This was attributed to a maintenance of turgor potential by active accumulation of solutes in the eastern leaves through the process of osmoregulation. 2. Stomatal Conductance. It was initially thought that stomatal closure often observed with a decrease in ψ was caused by the effect of ψ on guard cell turgor. However, it now appears that the mechanism behind stomatal closure may be more complex. The involvement of hormones such as abscisic acid (ABA) (Tardieu and Davies 1993) and cytokinins (Golan et al. 1986) that are modified under conditions of water deficit may play a role. In ‘Concord’ grapevines (V. labrusca) leaf water potential in irrigated and non-irrigated plants was similar during the day (–1.0 to –1.6 MPa and –1.3 to –1.6 MPa, respectively) but assimilation rate and stomatal conductance of the non-irrigated vines were significantly lower than the irrigated controls (Naor and Wample 1994). Stomatal closure was attributed to root signals arising from the drier soil in the non-irrigated plants. 3. Photosynthesis. Some recent data indicate that a reduction in stomatal conductance does not always fully account for decreases in photosynthesis under water deficit (Flore and Lakso 1989). Additionally, the sensitivity of photosynthesis to reduced plant water status varies between and within species and appears dependent on the pre-treatment these plants have received. Quick et al. (1992) found that potted grapevines may show a non-stomatal response causing reduction in photosynthesis with the onset of water deficit. However, the detailed studies of Flexas et al. (1998) with field-grown ‘Tempranillo’ showed that stomatal conductance was the main reason for reduction in photosynthesis under water deficit. Based on their measurements of chlorophyll fluorescence and gas exchange rates, the authors concluded that photoinhibition and disruption of electron transport rate were not the main cause of reduction in photosynthesis of the vines undergoing mild water stress in the field. The discrepancy of results between these experiments might be due to the rate at which stress was developed in plants, which was slower in Flexas’s experiment than that of Quick. The cultivar difference could have also played a role, as exemplified by the study of Schultz (1996), who investigated the adaptive responses to water deficit of ‘Grenache’, of Mediterranean origin, and ‘Syrah’ of mesic origin. Stomatal conductance and photosynthesis were more sensitive to water stress in ‘Grenache’ than in ‘Syrah’. Chlorophyll fluorescence measurements showed higher sensitivity of the former cultivar to water stress than the latter.

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4. Plant Growth. Growth is the most sensitive plant parameter to water stress (Hsiao 1973). In grapevine the effects of water stress have been reviewed for vegetative and reproductive growth (Smart and Coombe 1983; Williams and Matthews 1990; Williams et al. 1994). The growth reactions to water stress may be modified by interaction with plant nutrient status. This was demonstrated by experiments with ‘Cabernet Sauvignon’ growing in pots under glasshouse conditions that received two irrigation schedules and two nutrient regimes (Ussahatanonta et al. 1996). The watering treatment involved adequate watering (WH) and intermittent water stress (WL) and nutrients were either adequate (NH) or insufficient (NL) involving one-fifth of the nutrients applied to the NH treatment. NL reduced major growth parameters such as shoot length, stem weight, and leaf weight more than did WL. Both WL and NL reduced node and leaf number, but only NL reduced internode length, leaf area, and weight. WL had a larger effect on growth reduction when combined with NH than when combined with NL. WL, especially when combined with NH, advanced maturity by 10 days. The authors concluded that advancement of maturity could be of potential value if intermittent water stress were combined with adequate nutrition in areas with a short season. Low nutrient soils might be desirable in areas where other factors are likely to induce strong growth. The effects of water stress may also be influenced by exogenously applied gibberellic acid (Williams et al. 1994). When GA3 was applied at berry set to ‘Thompson Seedless’, final yields were similar between the irrigated vines and the non-irrigated vines despite large differences in leaf area per vine at harvest. Water stressed vines that had reduced leaf area were able to mature a crop similar to that of irrigated vines, indicating that alterations in source/sink relationship may be able to overcome the detrimental effect of water stress. Berry growth rate of irrigated and non-irrigated vines was similar after Stage I despite differences in water status. The authors concluded that involvement of hormones, other than ABA, should be studied in plants under water stress situations. F. Acclimation of Plants to Water Deficit Because plants are often exposed to water deficits during growth and development, it is important that they be able to acclimate to the prevailing conditions so as to avoid permanent injury. Included in the mechanisms of adaptation to water deficit are drought escape, drought tolerance with low plant water potential, and drought tolerance with high plant water potential (Turner 1986). Drought escape requires that plants complete their life cycle before significant water deficit can

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develop. This is not applicable to deciduous trees. Drought tolerance on the other hand requires the plant to respond in some way as water deficit develops. Osmotic adjustment is an example of drought tolerance with low plant water potential. Drought tolerance with high plant water potential can be realized through the stomatal control of transpiration. A brief account of osmotic adjustment and stomatal control is given below. 1. Osmotic Adjustment. Osmotic adjustment is the lowering of osmotic potential by the net increase in intracellular solutes in response to a decreased plant water potential. Rodrigues et al. (1993) observed osmotic adjustment in leaves of grapevine and Matthews et al. (1987a) reported osmotic adjustment in grape berries. Schultz and Matthews (1993) showed that the occurrence of osmotic adjustment in leaves of ‘White Riesling’ is due to both accumulation of solutes and changes in cell wall elasticity. However, the results of Patakas and Noitsakis (1999) for ‘Victoria’ indicated that solute accumulation was the main mechanism of osmotic adjustment. The rate of stress development generally influences the ability of plants to demonstrate osmotic adjustment with the gradual imposition of water deficit favoring osmotic adjustment (Morgan 1984). There is also a difference in the ability of cultivars to undergo osmotic adjustment. Düring (1999) indicated that this difference did exist for wine grape cultivars and it was used as a basis for selection for drought tolerance in a breeding program in Germany, where irrigation of wine grapes is not generally permitted for quality wine production. 2. Stomatal Behavior. The maintenance of turgor potential via osmotic adjustment of the leaves may be expected to maintain stomatal opening despite a reduction in ψ (Turner and Jones 1980). However, stomatal conductance is not solely dependent upon leaf turgor potential. For example, Golan et al. (1986) demonstrated that stomatal response of sunflower and wheat was closely related to soil moisture due to the role of hormones produced in roots in dry soil. Additionally, Düring and Dry (1995) speculated that chemical signals such as ABA are synthesized in the roots and transported to the leaves, which act to induce stomatal closure. If roots osmotically adjust, this signal is suppressed and stomatal closure is reduced. Naor et al. (1995) showed a good correlation between stomatal conductance and ψstem and ψsoil but a poor correlation with ψleaf. A good correlation with ψsoil further indicates that root signals play an important role in the control of stomatal conductance. Such root:shoot communication highlights the complexity of stomatal response to water deficit. Tardieu and Davies (1993) emphasize that it is inadequate to dis-

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cuss the concept of stomatal control based only on chemical signals (via the roots) or solely on plant water relations. These influences are integrated within the plant under water deficit conditions. Irrespective of the mechanisms involved, the closure of stomata under water stress will result in maintenance of a high water potential providing adaptation to stress.

IV. IRRIGATION OF VINEYARDS Grapevines are often grown in dry areas where frequent irrigation may not be possible and water should be saved (Matthews et al. 1987b; Pire and Ojeda 1999). Grapes are drought tolerant (Van Zyl and Weber 1981) because of their low water consumption and an extensive root system that makes them able to withstand long periods without water (Saayman and Lambrechts 1995). Water use efficiency has been reported to be similar for well-watered and droughted vines, further indicating adaptation to dry conditions (Novello and de Palma 1997). Grapevine increases productivity under irrigation if water loss due to ET exceeds rainfall (Van Zyl and Weber 1981; McCarthy et al. 1983; McCarthy et al. 1997). Irrigation scheduling to avoid plant water deficit should therefore minimize periods of low water status in plants by replacing an adequate percentage of ET. Judicious irrigation therefore necessitates estimation or measurement of ET. Based on ET, the amount and timing of irrigation depend on both meteorological and crop factors (Williams and Matthews 1990). Meteorological factors influencing crop water use include radiation, temperature, VPD, and wind speed. Major crop factors influencing water use include stomatal response, leaf morphology, vine architecture, rootstock, crop load, and cultivar. The importance of meteorological factors affecting ET was first discussed by Penman (1948) and further developed by Monteith (1965), who included crop factors of importance in the estimation of ET. Several attempts to use the Penman-Monteith approach to calculate ET have been made in recent years for a number of crops (Green and McNaughton 1996; Mills et al. 1999), including the grapevine (Williams et al. 1992). The introduction of the FAO Penman-Monteith method for ET calculation is presented in Equation 2 adapted from Allen et al. (1998). This equation is an accurate and simple representation of the physical and physiological factors governing the ET process. It represents the reference evapotranspiration (ETo), and the crop coefficients (Kc) could be calculated by relating the measured crop evapotranspiration (ETc) with ETo because by definition Kc = ETc/ETo. Reference ET is

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defined as the transpiration from an extended surface of actively growing green grass cover of uniform height, completely shading the ground and not short of water. Crop coefficient is indicative of the effect of canopy size, canopy architecture and stomatal response on water use of a specific crop (Doorenbos and Pruitt 1975).

ETo =

900 u2 (es − ea ) T + 273 ∆ + γ (1 + 0.34u2 )

0.408∆(R n − G) + γ

[2]

where ETo Rn G T u2 es ea es-ea ∆ γ

reference evapotranspiration [mm day–1], net radiation at the crop surface [MJ m–2 day–1], soil heat flux density [MJ m–2 day–1], air temperature at 2 m height [°C], wind speed at 2 m height [m s–1], saturation vapor pressure [kPa], actual vapor pressure [kPa], saturation vapor pressure deficit [kPa], slope vapor pressure curve [kPa °C–1], psychrometric constant [kPa °C–1].

Values of Kc for grapevine given by Doorenbos and Pruitt (1975) range from 0.35 early in the season to 0.9 in mid season. Goodwin (1995) also showed crop coefficients are low early and late in the season but are maximum during the middle of the season. He indicated that for the hot Australian climate, Kc values were 0.1 at budburst, 0.25 at flowering, 0.5 at veraison to harvest, and 0.25 after harvest. Similar trends in changes of crop coefficient with time of season are reported in grapes by Grimes and Williams (1990). Evans et al. (1993) indicate that published crop coefficients may not be suitable when applied to local conditions. Ideally, crop coefficients should be calculated for each region. Oliver and Sene (1992) discuss the use of a constant Kc of 0.2 for a semi-arid region, well below the recommended values by Doorenbos and Pruitt (1975). Measurement of evaporation from an open water surface, such as a class A evaporation pan, has also been used as a guide for irrigation scheduling in many crops, including grape (Evans et al. 1993). Canopy models have been used for scheduling irrigation. Greenspan and Matthews (1996) used a micrometeorological approach employing on-site measurements and the use of a canopy energy budget model. Understanding the soil as the reservoir from which plants get their water is essential when developing a water management strategy

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(Wample 1997; Cass 1999). Soil moisture status may be used as an indicator of irrigation requirement. The conservation of water equation is a useful way to calculate expected water loss from the soil-plant system and thus to schedule irrigation (Mpelasoka et al. 1997). WU = I – ∆W – D

[3]

Where WU = crop water use, I = irrigation, ∆W = change in soil-water storage, and D = drainage volume. The use of the water balance equation for irrigation of grapes is illustrated by Caspari and Neal (1998). As soil water is depleted, plants become increasingly stressed and they are unable to extract all the water from the soil. The well-known terms “field capacity,” “stress point,” and “permanent wilting point” are used to describe critical points on the soil moisture release curve. Ideally, soil moisture would be kept within the range of field capacity and stress point. The amount of water present in the soil at these critical points varies depending on soil type. The size and nature of the soil particles dictate how tightly water is held by the soil. Therefore different soils have different amounts of water still held by the soil at the stress point. Saayman and Lambrechts (1995) illustrate the changes in available soil water with different soil types. Irrigation scheduling must take the storage capacity of a given soil into account (Wample 1996). Soil moisture measurement involves several techniques, including time domain reflectometry (TDR) (Green and Clothier 1995), Gypsum resistance blocks (Van Zyl and Weber 1981), neutron probes (Araujo et al. 1995), and tensiometers (Klein 1983; Saayman and Lambrechts 1995). Such sensors may be linked to an automatic irrigation system so that, once sensors measure a predetermined value of soil water, irrigation is supplied. Care must be taken when considering placement of such sensors and due consideration also given to variability of soil throughout the vineyard (Naor et al. 1993). Goodwin (1995) gives a practical guide to the use of soil moisture sensors. Periodic water deficit in plants may still occur even if soil is adequately moist. If evaporative demands from the atmosphere are higher than the rate of water uptake from the soil, plants will develop water deficit. Plant parameters may also be used as a basis for irrigation scheduling. Techniques such as measurement of plant water potential (Grimes and Williams 1990), diurnal changes in stem and/or fruit diameter using LVDT (Myburgh 1996), heat pulse method (Eastham and Gray 1998; Ginestar et al. 1998a), and heat balance method (Lascano et al. 1992) are commonly used by researchers to indicate irrigation requirements of vines. Such plant-based parameters are also useful as a cross check on

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the accuracy of models used to predict irrigation requirements. Lovisolo et al. (1999) used the Penman-Monteith model for predicting transpiration in ‘Nebbiolo’ grafted on ‘Kober 5BB’ and found that their prediction was accurate based on measurement of sap flow using the stem heat balance technique. Some studies recommend the use of plant-based indicators for irrigation scheduling because they have the potential to be more accurate (Ginestar et al. 1998b). However, some limitations to the use of these plant-based systems have been reported. For example, Braun and Schmidt (1999) reported that the heat balance method is not suitable for old vines with thick stems. Previous irrigation schedules may also influence the current one. For example, if irrigation water were always applied in small amounts frequently then roots are likely to be shallow. Such poor development of the root system means vines are unable to tap into water deeper in the profile and therefore require more frequent irrigation. Deep watering may encourage deep rooting but may also result in loss of irrigation water as it passes beyond the rootzone and is lost via leaching. A prolonged schedule of deficit irrigation will also limit total tree size and therefore water requirement. A. Regulated Deficit Irrigation Regulated deficit irrigation (RDI) is a technique of partial replacement of ET and therefore delivers less water than the actual plant requirement at selected times during the growing season, depending on the expected beneficial outcomes. It was initially developed to control vegetative growth of fruit trees in high-density plantings (Behboudian and Mills 1997). Control of vegetative growth is usually required in most deciduous fruit crops in order to maintain consistent production and to facilitate crop management. Excess vegetative growth of grapevines results in berry shading and reduced fruit quality (Dry and Loveys 1998). Additionally, disease problems, especially fungal disorders, are increased when the canopy is dense and air movement within the canopy is reduced. In 1997 McCarthy stated that RDI is becoming increasingly popular in Australian vineyards. The potential of RDI in reducing vegetative growth without detrimental effects on fruit growth and yield is based on at least two physiological principles. These are: differential sensitivities of tissues, organs, and processes to reduced plant water status, and phenological separation of shoot and fruit growth. Regulated deficit irrigation has been shown to effectively control vegetative vigor in grapevines (Caspari et al. 1997; Pire and Ojeda 1999).

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Reduced vegetative growth is often desirable to increase light and air movement through the canopy and to improve spray penetration. A decrease in vegetative growth allows greater exposure of fruit to solar radiation, which contributes to sugar accumulation and, for red varieties, color development (Calò et al. 1995). In addition to these indirect influences on fruit quality, deficit irrigation at specific phenological stages may also result in direct changes in fruit quality. However, the deficit level required to induce direct quality changes is generally quite severe. Goodwin (1995) states that the primary reason for the use of RDI in grapes is manipulation of fruit quality. Calò et al. (1995) also state that excessive watering can result in a dilution of fruit contents in grape and detrimentally alter quality, especially for wine grapes. With this in mind, Williams and Matthews (1990) state that grapevines in California are often over irrigated and therefore the potential to implement RDI and to improve irrigation practice is high. 1. Timing of RDI Application. As plants are only sensitive to water deficit during specific periods and because vegetative growth and fruit growth have somewhat separated periods of active growth (Fig. 5.1), there is the potential to manipulate targeted organs at specific times. Stevens et al. (1995) and Williams and Matthews (1990) report that vegetative growth is more sensitive to water deficit than fruit growth and similarly fruit composition is less sensitive than is overall fruit growth.. With moderate water deficit, some parameters may be affected but others remain essentially unchanged. Deficit irrigation may result in restricted root growth of fruit trees, as less root development was observed in dry soil (Behboudian and Mills 1997). However, Richards (1983) suggested that root growth is generally less sensitive to water deficit than vegetative growth. In contrast Dry and Loveys (1998) showed that deficit irrigation can result in a reduction in root growth of grapevines, which is coupled with reduced vegetative growth. The impact of any deficit irrigation on root growth must be considered when irrigation strategies are proposed. Fruit yield is often reduced under water stress either because fruit size is compromised or because flower development, fruit set, and berry number is reduced. A decrease in leaf growth allows greater exposure of fruit to the sun, contributing to sugar accumulation, color development (Calò et al. 1995), and a reduction in malate concentration (Stevens et al. 1995). These examples highlight the importance of the indirect consequences of reduced vine water status. In the following the impact of RDI is described for different stages of the growing season. Fig. 5.2 could be used as an approximate match for

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these stages. Although RDI at selected times may be beneficial, there are also phenological stages when it could be damaging. 2. Early RDI: Budding to Flowering. Although water requirements at this time are low, lack of water at this stage may lead to irregular bud burst, short shoots, and fewer flowers. Studies of deficit irrigation in grape indicate a reduction in vegetative growth if limited irrigation is applied between bud burst and flowering (Van Zyl 1984; Wample 1996). Bud break will be uneven during water deficit and flower development detrimentally affected. Water deficit during flowering and fruit set is generally associated with poor pollen and pistil viability and therefore poor fruit set. Goodwin (1995) reports yield losses of up to 50% if water deficit is induced at this time, mainly due to increased fruit abscission. Berry size is adversely affected if water deficit is induced during flowering (Van Zyl 1984). 3. Mid-season RDI: Fruit Set to Veraison. Following fruit set, it is ideal to avoid stress during cell division and initial cell enlargement of the fruit, as water stress during this time will reduce maximal berry size and thus yield (Smart and Coombe 1983; Wample 1996; McCarthy 1997). Creasy and Lombard (1993) showed that pre-veraison berries are very sensitive to water deficit and it appears as though the water requirements of the vine out-compete that of the fruit. This is supported by Goodwin (1995), who reports yield losses of up to 40% caused by water deficit at this time. Similarly, McCarthy (1997) found the largest reduction in yield if stress is imposed at this time, as compared to other fruit development stages. Trunk growth is reduced if water deficit is induced at mid-season (Myburgh 1996). In contrast, Caspari et al. (1997) reported that although water deficit was sufficient to reduce vegetative growth in ‘Sauvignon blanc’, fruit yield was not affected. Their study was carried out in a humid area in contrast to the above studies, which were done in dry regions. Fruit compositional changes due to water stress at this stage include increases in soluble solids (predominantly sugars) and titratable acidity. Fruit color may also be enhanced as anthocyanin development is encouraged with increased fruit sugar. 4. Late-season RDI: Veraison to Harvest. Grapevines are generally tolerant to reduced plant water status at this time (Van Zyl 1987). Deficit irrigation following veraison may cause senescence of lower leaves, leading to fruit exposure and sunburn resulting in reduced fruit quality (Wample 1996). Shoot growth is unlikely to be affected by water deficit following veraison, as minimal shoot growth occurs after this time,

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although there may be some abscission of shoot tips if the level of stress imposed is high (Wample 1996). Post-veraison water stress increases fruit pH (McCarthy et al. 1983; Ginestar et al. 1998b). Naor et al. (1993) indicate that a post-veraison water deficit reduced soluble solids in grape berries. Water deficit at this time appears to have a minimal impact on berry weight (McCarthy 1997). Some reports indicate that water deficit will accelerate fruit maturity, but reports are contradictory. Jackson and Lombard (1993) state excess water delays the onset of ripening, but water stress has not always been found to accelerate it (Matthews and Anderson 1988). Sipiora and Granda (1998) indicate a severe water stress will retard sugar accumulation and therefore maturity. Williams and Grimes (1987) also report that severe water deficit delays maturity. However, McCarthy et al. (1983) showed reduced irrigation did advance fruit maturity. Generally, drier soil during fruit ripening will improve fruit quality for wine grapes, but the level of stress imposed is important (Jackson and Lombard 1993). 5. Postharvest RDI. Water deficit following harvest and before vines become dormant for the winter may cause a reduction in root growth, which will further confound the effects of water stress in the future (Wample 1996). Increased low temperature hardiness has been reported in vines undergoing a post-harvest water deficit (Evans et al. 1993). This may provide an important management tool in areas where vines are exposed to low winter temperatures. Although grapevines do not transpire when dormant, roots still require damp soil. If drought is experienced late in the winter or in spring, bud burst may be detrimentally affected (Davidson 1998). Williams et al. (1991) report that a postharvest water deficit encourages earlier budbreak in the following season. The above information leads us to the conclusion that RDI could be applied for some benefits during the later stages of the growing season (veraison to harvest). These include minimal impact on berry weight (McCarthy 1997), acceleration of fruit maturity (McCarthy et al. 1983), and improvement of quality for wine grapes (Jackson and Lombard 1993). Care should be taken in the post-harvest application of RDI and for other parts of the growing season and only a mild measure of water deficit should be allowed if RDI were used. A promising approach to RDI whose timing spanned most of the growing season was taken by Hamman and Dami (2000) in Colorado. They irrigated ‘Cabernet Sauvignon’ at the rate of 192 (T1, control), 96 (T2), and 48 (T3) liters per vine per week from bud burst until veraison and then reduced all irrigation by 25% through to harvest. Soil water content was reduced in T2 and T3 compared with T1. The T2 treatment had the best canopy size, yield, and

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fruit and wine quality. Cold hardiness of buds and canes was unaffected by irrigation treatment. Canes matured earlier and were less susceptible to early fall frost in T2 and T3. The approach of Hamman and Dami (2000), which involved starting the RDI earlier in the season and then increasing the severity of deficit later in the season, seems feasible for repetition on other cultivars and localities combined with allowing various measures of water deficit to develop. Unfortunately the authors did not measure ψ for better comparison with other experiments. Some quantitative examples of deficit developed during RDI are given below. 6. Degree of Deficit. The benefits, or not, of manipulation of water at specific times for grapevines depends on the cultivar and the end use of the fruit. It also depends on the level of deficit that may be imposed. The level of deficit could be applied by replacing a certain percentage of ET or irrigating the vines after depletion of a measure of plant available water (PAW). Myburgh (1996) provides data on ‘Barlinka/Ramsey’ (scion/rootstock) that was irrigated at 10%, 40%, and 60% depletion of PAW in a field trial on sandy soil in South Africa. These treatments resulted in, respectively, midday ψ values of –0.93, –1.03, and –1.2 MPa. It was concluded that the most acceptable combination of growth, yield, berry size, and eating quality was obtained by irrigation at 40% PAW depletion. Van Zyl (1984) suggests that a rather severe deficit is required to bring about changes in fruit quality attributes in a direct way. Van Zyl and Van Huyssteen (1988) demonstrated how different types of irrigation influence the level of deficit achieved. Trickle irrigation may be very effective in some situations but it may cause severe water stress to develop when used on highly permeable soils. In these situations microjet, sprinkler, or furrow irrigation may be required. B. Partial Rootzone Drying Partial rootzone drying (PRD) is an irrigation protocol whereby at each turn of irrigation only a part of the root zone receives water and the other part would be allowed to dry. This has proven effective in inducing regulated deficit in several horticultural crops, including grapevine (Dry and Loveys 1998). This system works because roots are only active in moist soil (Elfving 1982; Behboudian and Mills 1997). Therefore, by inducing “dry spots” within the rootzone, the effective rooting volume is reduced. Additionally, chemical signaling from the dry roots to the vegetative portions of the vine brings about further physiological changes. Partial rootzone drying may be effective in limiting leaching

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and also reducing evaporation of irrigation water from the soil surface (Van Zyl and van Huyssteen 1988). Partial rootzone drying can be achieved via the use of trickle irrigation (Araujo et al. 1995) or by careful placement of other water emitters. Experiments by Van Zyl and Van Huyssteen (1988) indicate the use of trickle irrigation has the potential in improving fruit quality. A recent study by Dry and Loveys (1998) showed the effectiveness of PRD in the control of vegetative growth and the enhancement of berry quality. The theory behind PRD centers on the role that chemical signals, primarily ABA, originating in the roots, play in the control of shoot growth and transpiration. Stimulation of these signals through PRD can result in reduced vegetative growth and total vine water use, while maintaining crop yield and improving fruit quality indirectly (Loveys et al. 1997). Van Zyl (1987) provided interesting results from a trial in which only 50% of the rootzone was wet. The top 50% of the roots was irrigated rather than a split of the ground area occupied by the vine. Such a partial rootzone irrigation did not result in any significant changes in vine performance or vine water status. The lack of impact that shallow irrigation had was probably due to the exposure of a high proportion of absorptive roots to moist conditions that prevented the initiation of root signaling typical in PRD treatments. Poni et al. (1992) conducted split root experiments in pots using grapes and found that the above-ground portion of the plant equilibrates with the wet region of the root zone and very little change in vegetative growth, stomatal conductance, photosynthesis and overall water use was observed. However, the experiment of Poni et al. (1992) was short term and therefore there may have been insufficient time for differences to develop. Clearly PRD is a tool with great potential as a management strategy to save water, decrease leaching, reduce soil evaporation, decrease vegetative growth, and possibly improve berry quality.

V. QUALITY ATTRIBUTES FOR WINE, DRIED, TABLE, AND JUICE GRAPES A. Wine Grapes As a general rule, wine grapes require less water than table grapes because of quality concerns. Wine grape irrigation often comprises an RDI regime to control vegetative growth and to improve wine quality (Sipiora and Granda 1998). In wine grapes, one desirable attribute is a

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specific level of soluble solids (SS). If SS is too low, the wine will lack sugar and if it is too high the grapes are termed overripe. Both titratable acidity and pH of the wine are critical quality parameters. As berry SS increases, pH also increases, which may result in a wine that has bland flavor and is, therefore, less desirable (Mullins et al. 1992, page 138). Freeman (1983) reports an increase in titratable acidity with irrigation and a reduction in anthocyanin development. Although deficit irrigation is reported to increase grape SS levels, the results are somewhat contradictory (Jackson and Lombard 1993; Naor et al. 1993). Titratable acidity may decline with reduced irrigation (Van Zyl and Van Huyssteen 1988; Pire and Ojeda 1999). Grapes grown for wine have distinct quality requirements that differ with cultivar. The changes in fruit quality effected by modified plant water status can give wine some fundamental characteristics and distinct flavors that are associated with region and cultivar, as reviewed by Jackson and Lombard (1993), who emphasized that these characteristics give each region and wine its uniqueness. When reference is made to a vintage year for wine, it is a description of a season that has encouraged the optimization of fruit quality attributes for a specific wine. It is suggested that irrigation management is a major contributor to wine quality. Jackson and Lombard (1993) reported that ‘Müller-Thurgau’ grown in pots and deficitirrigated from veraison to harvest produced wine that was rated as “fruity, fragrant, and elegant.” Plants fully irrigated in this period produced wine that was “full-bodied and less elegant.” Preferred wines were from vines that were irrigated until veraison and then deficit-irrigated, and least preferred were from the vines deficit-irrigated until veraison and then fully irrigated. B. Dried Grapes—Raisins ‘Thompson Seedless’ is the most widely planted grape variety for raisin production in California (Williams and Matthews 1990). Seedlessness, large size, and high sugar content are important quality attributes for raisin production. Both size and sugar content are altered with irrigation management (Jackson and Lombard 1993). There are similarities between the irrigation requirements of grapes grown for raisin production and for wine grapes. During the grape-ripening period it has been recommended that the soil moisture supply be gradually lessened and the irrigation cut-off should be early enough to slow vine growth and to provide for adequate soil surface drying in areas where terraces are made under the vines for fruit drying (Williams and Matthews 1990).

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C. Table Grapes Quality attributes of table grapes differ from those required of wine grapes. Visual factors such as berry size, color, texture, and the condition of the waxy bloom on the fruit surface are all important. Table grapes are harvested at a lower SS level than those used for wine or raisin production (Williams and Matthews 1990). Texture is a particularly important attribute of table grapes, and a firm and crisp texture is essential (Jackson and Lombard 1993). Generally, the level of irrigation required for table grapes is higher than that required for raisin or wine production. Therefore, a higher percentage of ET should be replaced in deficit irrigation of table grapes as exemplified by the experiment of Srinivas et al. (1999), who replaced 75% of the ET for ‘Anabe-e-Shahi’ and reported increases in bunch weight and berry size. Irrigation of vines following harvest of table grapes is also important. Generally, fruit is harvested well before leaf fall. The vines, although not carrying a current crop, are at this stage laying the foundations for the next season. Adequate irrigation at this time is critical to ensure good flowering and fruit set in the coming season (Williams and Matthews 1990). These authors also indicate that many vineyards producing table grapes use grass between rows to control dust and to influence the light exposure of bunches. Pieri et al. (1999) reported that nitrogen was strongly taken up by the grass early in the season in two vineyards of ‘Cabernet franc’ in France, but vines did not suffer from such competition for water. The root system adjusted and also changed significantly according to soil characteristics. Grass therefore might not have a major influence on the irrigation requirement of vineyards.

D. Juice Grapes The grape juice industry, especially in the United States, is mainly based on the Concord grapevine (Vitis labruska) (Lakso and Dunst 1999). Information on the effects of irrigation on juice quality is scant for V. labruska, whereas more information, although not conclusive, is available for V. vinifera. For the latter, Hamman and Dami (2000) showed that total soluble solids (TSS) decreased with a late-season reduction of irrigation, while the results of Wample (1997) showed an increase. Balo et al. (1999) reported that irrigated and non-irrigated vines had the same aroma volatiles in the must for ‘Chardonnay’ and a sensory panel did not find any significant qualitative differences for the wine from the two treatments. For Concord vines, Poni et al. (1994) found that at pre-dawn

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leaf water potentials of lower than –1.1 MPa, juice Brix was reduced in berries and this was aggravated with heavier crop loads. Lakso and Dunst (1999) showed that irrigation increased berry juice Brix in both minimally pruned and balance-pruned Concord vines. It is therefore doubtful that deficit irrigation would be of any advantage to V. labruska. Wample (1997) states that Concord vine may require irrigation in many areas where it is growing and it requires a uniform level of soil moisture throughout the growing season. He further indicates problems that V. labruska faces if water-stressed at different times during the growing season in Washington State. One problem is the development of “black leaf” for mid-season stress, which could result in defoliation of vines. He concludes that maintaining a higher and more uniform soil moisture for American grapevine varieties may be more critical than for European varieties.

VI. FUTURE PROSPECTS Recent advances in vine management indicate that high-quality wines may be produced under irrigation. However, in many arid areas the increased use of irrigation brought about by the demands of rising populations has greatly increased the cost of water. Consequently, there has been increasing interest in scheduling irrigation to reduce usage, the rationale of regulated deficit irrigation. Although this technique can enhance fruit quality in grape, insufficient irrigation water can cause problems equally as damaging as an excess. Thus, the threshold whereby the detrimental effects of reduced water outweighs the advantages should be determined precisely for all cultivars. Given the different fruit quality requirement of wine, juice, table, and dried grapes, future research should concentrate on formulating irrigation schedules specifically targeted to each end product based on the cost of water. More research is needed on the physiological and product quality impacts of PRD, which seems to be a promising irrigation strategy based on the available information. Gaps remain in various aspects of grapevine water relations. Despite the fact that grapevine is extensively grown in dry areas, the plant reaction to VPD has not been researched to a satisfactory conclusion. The physiology of recovery from water stress such as occurs after re-watering of deficit-irrigated vines, has not been investigated as extensively as for other crops. Although phytohormones, such as ABA, have been studied as they relate to the water relations of grapevines, others, such as gibberellins, have been under-researched.

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Finally, there are other approaches to water management that need to be explored. One is the genetic solution, i.e., breeding rootstocks with improved drought tolerance. The discovery that water channel proteins (aquaporins) facilitate water transport across membranes, i.e., control water flux through tissues (Kjellbom et al. 1999), suggests that molecular approaches need to be explored in rootstock improvement. Breeding scion cultivars for increased drought tolerance is another solution. Düring (1999) has reviewed the important criteria for selecting drought tolerance, which includes higher water use efficiency based on higher rates of photosynthesis to transpiration, and increased ability to undergo osmotic adjustment. However, industry conservatism in changing wine grape cultivars is a limiting factor arrayed against this approach.

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Mapfumo, E., D. Aspinal, and T. W. Hancock. 1994. Growth and development of roots of grapevine (Vitis vinifera L.) in relation to water uptake from soil. Ann. Bot. 74:75–85. Matthews, M. A., and M. M. Anderson. 1988. Fruit ripening in Vitis vinifera L.: Responses to seasonal water deficits. Am. J. Enol. Vitic. 39:313–320. Matthews, M. A., M. M. Anderson, and H. R. Schultz. 1987b. Phenolic and growth responses to early and late season water deficits in Cabernet franc. Vitis 26:147–160. Matthews, M. A., G. Cheng, and S. A. Weinbaum. 1987a. Changes in water potential and dermal extensibility during grape berry development. J. Am. Soc. Hort. Sci. 112:314– 319. McAneney, K. J., and M. J. Judd. 1983. Observations on kiwifruit (Actinidia chinensis Planch.) root exploration, root pressure, hydraulic conductivity and water uptake. New Zealand J. Agr. Res. 26:507–510. McCarthy, M. G. 1997. The effect of transient water deficit on berry development of cv. Shiraz (Vitis vinifera L.). Austral. J. Grape Wine Res. 3:102–108. McCarthy, M. G., R. M. Cirami, and P. McCloud. 1983. Vine and fruit responses to supplementary irrigation and canopy management. S. African J. Enol. Vitic. 4:67–76. McCarthy, M. G., R. M. Cirami, and D. G. Furkaliev. 1997. Rootstock response of Shiraz (Vitis vinifera) grapevine to dry and drip-irrigated conditions. Austral. J. Grape Wine Res. 3:95–98. McCutchan, H., and K. A. Shackel. 1992. Stem-water potential as a sensitive indicator of water stress in prune trees (Prunus domestica L. cv. French). J. Am. Soc. Hort. Sci. 117:607–611. Meinzer, F. C., J. L. Ingamells, and C. Crisosto. 1991. Carbon isotope discrimination correlates with bean yield of diverse coffee seedlings. HortScience 26:1413–1414. Mills, T. M., M. H. Behboudian, and B. E. Clothier. 1998. Discrimination against 13CO2 in the leaves of water stressed ‘Braeburn’ apple. J. Plant Physiol. 153:237–239. Mills, T. M., K. M. Morgan, and L. R. Parsons. 1999. Canopy position and leaf age affect stomatal response and water use of citrus. J. Crop Prod. 2:169–184. Monteith, J. L. 1965. Evaporation and environment. Symp. Soc. Expt. Biol. 19:205–234. Morgan, J. M. 1984. Osmoregulation and water stress in higher plants. Ann. Rev. Plant Physiol. 35:299–319. Mpelasoka, B. S., T. M. Mills, and M. H. Behboudian. 1997. Soil-plant-water relations in deciduous orchards. Trends Soil Sci. 2:59–81. Mullins, G. M., A. Bouquet, and L. E. Williams. 1992. Biology of the grapevine. Cambridge Univ. Press, Cambridge, UK. Myburgh, P. A. 1996. Response of Vitis vinifera L. cv. Barlinka/Ramsey to soil water depletion levels with particular reference to trunk growth parameters. S. African J. Enol. Vitic. 17:3–14. Myburgh, P. A., J. L. Van Zyl, and W. J. Conradie. 1996. Effect of soil depth on growth and water consumption of young Vitis vinifera L. cv. Pinot noir. S. African J. Enol. Vitic. 17:53–62. Naor, A. 1998. Relations between leaf and stem water potentials and stomatal conductance in three field-grown woody species. J. Hort. Sci. Biotech. 73:431–436. Naor, A., B. Bravdo, and Y. Hepner. 1993. Effect of post-veraison irrigation level on Sauvignon blanc yield, juice quality and water relations. S. African J. Enol. Vitic. 14:19–25. Naor, A., I. Klein, and I. Doron. 1995. Stem water potential and apple size. J. Am. Soc. Hort. Sci. 120:577–582. Naor, A., and R. L. Wample. 1994. Gas exchange and water relations of field-grown Concord (Vitis labrusca Bailey) grapevines. Am. J. Enol. Vitic. 45:333–337.

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Novello, V., and L. de Palma. 1997. Genotype rootstock and irrigation influence on water relations, photosynthesis and water use efficiency in grapevine. Acta Hort. 449:467–473. Oliver, H. R., and K. J. Sene. 1992. Energy and water balances of developing vines. Agr. For. Meteorol. 61:167–185. Patakas, A. 1993. Diurnal changes in gas exchange and water potentials in field grown grape vines. Acta Hort. 335:251–256. Patakas, A., and B. Noitsakis. 1999. Osmotic adjustment and partitioning of turgor responses to drought in grapevine leaves. Am. J. Enol. Vitic. 50:76–80. Penman, H. L. (1948). Natural evaporation from open water, bare soil and grass. Proc. Royal Soc. A. 193:120–145. Pieri, P., C. F. Riou, and C. Dubois. 1999. Competitions for nitrogen and water in two vinegrass systems—application of a water balance model. Acta Hort. 493:89–96. Pire, R., and M. Ojeda. 1999. Vegetative growth and quality of grapevine ‘Chenin blanc’ irrigated under three pan evaporation coefficients. Fruits 54:135–139. Poni, S., A. N. Lakso, J. R. Turner, and R. E. Melious. 1994. Interactions of crop level and late season water stress on growth and physiology of field-grown Concord grapevines. Am. J. Enol. Vitic. 45:252–258. Poni, S., M. Tagliavini, D. Neri, D. Scudellari, and M. Toselli. 1992. Influence of root pruning and water stress on growth and physiological factors of potted apple, grape, peach and pear trees. Scientia Hort. 52:223–236. Possingham, J. V. 1994. Production of table grapes in south India. p. 38–42. In: J. M. Rantz and K. B. Lewis (eds.), Proceedings of the international symposium on table grape production. American Society of Enology and Viticulture, Davis, CA. Quick, W. P., M. M. Chaves, R. Wendler, M. David, M. L. Rodrigues, J. A. Passaharinho, J. S. Pereira, M. D. Adcock, R. C. Leegood, and M. Stitt. 1992. The effect of water stress on photosynthetic carbon metabolism in four species grown under field conditions. Plant Cell Environ. 15:25–35. Richards, D. 1983. The grape root system. Hort. Rev. 5:127–168. Rodrigues, M. L., M. M. Chaves, R. Wendler, M. David, W. P. Quick, R. C. Leegood, M. Stitt, and J. S. Pereira. 1993. Osmotic adjustment in water stressed grapevine leaves in relation to carbon assimilation. Aust. J. Plant Physiol. 20:309–321. Saayman, D., and J. J. N. Lambrechts. 1995. The effect of irrigation system and crop load on the vigour of Barlinka table grapes on a sandy soil, Hex River Valley. S. Afric. J. Enol. Vitic. 16:26–34. Schmid, J., and P. Braun. 1997. Transpiration of grapevines in the field. Acta Hort. 449 (2):475–480. Schultz, H. R. 1996. Water relations and photosynthetic responses of two grapevine cultivars of different geographic origin during water stress. Acta Hort. 427:251–266. Schultz, H. R., and M. A. Matthews. 1993. Growth, osmotic adjustment, and cellwall mechanics of expanding grape leaves during water deficits. Crop Sci. 33:287– 294. Simmonneau, T., R. Habib, J. P. Goutouly, and J. G. Huguet. 1993. Diurnal changes in stem diameter depend upon variations in water content: direct evidence in peach trees. J. Expt. Bot. 44:615–621. Sinclair, T. R., and M. M. Ludlow. 1985. Who taught plants thermodynamics? The unfulfilled potential of plant water potential. Aust. J. Plant Physiol. 12:213–217. Sipiora, M. J., and M. J. G. Granda. 1998. Effects of pre-veraison irrigation cutoff and skin contact time on the composition, color, and phenolic content of young Cabernet Sauvignon wines in Spain. Am. J. Enol. Vitic. 49:152–162.

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Sipiora, M. J., and J. R. Lissarrague. 1999. Diurnal changes in vine water status and gas exchange parameters of Vitis vinifera L. cv. ‘Tempranillo’ grapevines as influenced by irrigation and pruning level. Acta Hort. 493:109–116. Smart, R. E., and B. G. Coombe. 1983. Water relations of grapevines. p. 137–196. In: T. T. Kozlowski (ed.), Water deficits and plant growth. Volume VII. Academic Press, New York. Srinivas, K., S. D. Shikhamany, and N. N. Reddy. 1999. Yield and water-use of ‘Anabee.Shahi’ grape (Vitis vinifera L.) vines under drip and basin irrigation. Indian J. Agric. Sci. 69:21–23. Stevens, R. M., G. Harvey, and D. Aspinall. 1995. Grapevine growth of shoots and fruit linearly correlate with water stress indices based on root-weighted soil matric potential. Aust. J. Grape and Wine Res. 1:58–66. Tardieu, F., and W. J. Davies. 1993. Interpretation of hydraulic and chemical signalling in the control of stomatal conductance and water status of droughted plants. Plant Cell Environ. 16:341–349. Turner, N. C. 1986. Adaptation to water deficits: a changing perspective. Aust. J. Plant Physiol. 13:175–190. Turner, N. C., and M. M. Jones. 1980. Turgor maintenance by osmotic adjustment. In: N. C. Turner and P. J. Kramer (eds.), Adaptation of plants to water and high temperature stress. John Wiley, New York. Ussahatanonta, S., D. I. Jackson, and R. N. Rowe. 1996. Effects of nutrient and water stress on vegetative and reproductive growth in Vitis vinifera L. Aust. J. Grape and Wine Res. 2:64–69. Van der Westhuizen, R. H. 1980. The effect of black plastic mulch on growth, production, and root development of Chenin Blanc vines under dryland conditions. S. Afric. J. Enol. Vitic. 1:1–6. Van Zyl, J. L. 1984. Response of Colombar grapevine to irrigation regards quality aspects and growth. S. Afric. J. Enol. Vitic. 5:19–28. Van Zyl, J. L. 1987. Diurnal variation in grapevine water stress as a function of changing soil water status and meteorological conditions. S. Afric. J. Enol. Vitic. 8:45–52. Van Zyl, J. L., and L. Van Huyssteen. 1988. Irrigation systems—their role in water requirements and the performance of grapevines. S. Afric. J. Enol. Vitic. 9:3–8. Van Zyl, J. L., and H. W. Weber. 1981. The effect of various supplementary irrigation treatments on plant and soil moisture relationships in a vineyard (Vitis vinifera var. Chenin Blanc). S. Afric. J. Enol. Vitic. 2:83–99. Wample, R. L. 1996. Issues in vineyard irrigation. Wine East (July–Aug): 8–21. Wample, R. L. 1997. When understanding irrigation—many things are to be considered. Vineyard & Winery Mgmt (Nov–Dec) 72:1–7. Wenkert, W. 1983. Water transport and balance within the plant: an overview. p. 137-172. In: H. M. Taylor, W. R. Jordan, and T. R. Sinclair (eds.), Limitations to efficient water use in crop production. American Society of Agronomy, Madison, WI. Williams, L. E. 1987. Growth of ‘Thompson Seedless’ grapevines: I. Leaf area development and dry weight distribution. J. Am. Soc. Hort. Sci. 112:325–330. Williams, L. E., N. K. Dokoozlian, and R. Wample. 1994. Grape. p. 85–133. In: B. Schaffer and P. C. Andersen (eds.), Handbook of environmental physiology of fruit crops. Volume I, Temperate crops. CRC Press, Boca Raton, FL. Williams L. E., and D. W. Grimes. 1987. Modelling vine growth-development of a data set for a water balance subroutine. p. 169–174. In: T. Lee (ed.), Proc. 6th Aust. Wine Ind. Tech. Conf. Adelaide, Aust. 14–17 July 1986. Australian Industrial Publ., Adelaide.

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Williams, L. E., and M. A. Matthews. 1990. Grapevine. p. 1019–1055. In: B. A. Stewart and D. R. Nielsen (eds.), Irrigation of agricultural crops. American Society of Agronomy, Madison, WI. Williams, L. E., R. A. Neja, J. L. Meyer, L. A. Yates, and E. L. Walker. 1991. Postharvest irrigation influences budbreak of ‘Perlette’ grapevines. HortScience 26:1081. Williams, L. E., D. W. Williams, and C. J. Pheves. 1992. Modelling grapevine water use. Proc. 8th Aust. Wine Indust. Tech. Conf. (8):29–33.

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6 Physiology and Biochemistry of Superficial Scald of Apples and Pears Morris Ingle Division of Plant and Soil Sciences West Virginia University Morgantown, West Virginia 26506

I. INTRODUCTION II . SCALD SYMPTOMS AND CELL CHANGES III. BIOCHEMISTRY OF SCALD A. Volatiles 1. α-Farnesene 2. Conjugated Trienes B. Antioxidants 1. Lipid Soluble 2. Water Soluble C. Anthocyanins IV. PHYSIOLOGY OF SCALD A. Cultivar, Maturity, and Year B. Temperature C. Storage Atmosphere D. Ethylene E. Scald-inhibiting Materials V. A MODEL OF SCALD DEVELOPMENT A. Step 1 B. Step 2 C. Step 3 D. Step 4 VI. PROSPECTS A. Predicting Scald 1. Maturity 2. Chlorophyll Fluorescence 3. Preharvest Temperatures B. Future Research LITERATURE CITED

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I. INTRODUCTION Superficial scald, a serious postharvest storage disorder of apple and pear, has received a marked increase in research activity in the last decade. The accepted dogma regarding the physiology and control of scald was summarized by Ingle and D’Souza (1989) as follows: (1) scald results from damage to hypodermal cells by the accumulation of αfarnesene oxidation products; (2) scald incidence and severity are influenced (or regulated) by harvest dates, maturity, preharvest air temperatures, preharvest treatments, geographical location, cultivar, rootstock, ethylene in storage, storage periods, and storage conditions; (3) diphenylamine (DPA) and 6 ethoxy-1,2-dehydro-2,2,4-trimethyl quinoline (ethoxyquin) are commercially effective scald inhibitors. Since that was written, restrictions have been placed on the use of those compounds and alternates have been sought. In this review, evidence that has accumulated in the last 10 years affecting those statements will be examined and a model will be offered that integrates what is now known. Although the model will be incomplete, it will emphasize what needs to be learned. Emonger et al. (1994) have reviewed preharvest conditions that affect scald development.

II. SCALD SYMPTOMS AND CELL CHANGES Scald is recognized as discoloration of the fruit surface ranging from light tan to dark brown (Ingle and D’Souza 1989). Sometimes the discolored lesions or patches appear wrinkled and sunken and may be rough to the touch, which is the result of the collapse of the hypodermal cells (Bain and Mercer 1956, 1963). Epidermal cells in affected areas appear normal, while the hypodermal cells beneath the epidermis are usually collapsed and filled with dark materials that are usually said to be polyphenols (Bain and Mercer 1956, 1963). The lesions usually are not visible until fruit have been in refrigerated storage (RS) for over 100 days, depending on cultivar and harvest date (D’Souza 1991; Mir et al. 1998; Meir and Bramlage 1988). Lesions will appear after less RS if fruit is held at 20°C. During that holding, lesions develop linearly (Ju et al. 1996; Mir et al. 1998). The scald lesions on ‘Law Rome’, ‘Delicious’, ‘Gala’, and ‘Cortland’ increased in area and became darker during holding at 22°C. The chromaticity values L* and hue were not influenced (Mir et al. 1998b). Minimal and maximal chlorophyll fluorescence (Fo and Fm) were reduced as lesions developed; however, photochemical efficiency did not change. The reduction in Fo and Fm suggests a loss in hypodermal

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chloroplast structure and function. ‘Cortland’ apples that had been treated with DPA did not develop scald and there was no change in chlorophyll fluorescence during holding at 22°C. The epidermis of apple fruits is covered by a cuticle that is covered by a layer of wax that is mainly composed of C25-C30 alkanes and alcohols but also some ketones, aldehydes, and free fatty acids. Cutin forms the thickest layer of the cuticle and is made up of fatty acid chains linked by ester bonds to hydroxyl and epoxide groups. These fatty acid polymers are usually embedded in wax. In the lower or inner layer of the cuticle, cutin and waxes blend with the polysaccharide of the outer epidermal cell walls. This part of the cuticle is less hydrophobic (lipophilic) than the outer parts.

III. BIOCHEMISTRY OF SCALD A. Volatiles 1. α-Farnesene. Brooks et al. (1923) proposed that scald was initiated or regulated by volatile substances produced by the fruit. Scald was reduced by wrapping apples and pears in paper impregnated with mineral oil, which was believed to absorb volatiles. Scald was also reduced by increasing the rate of ventilation, which could remove or dilute volatiles. α-Farnesene (C15H24, M=204[3E,6E]-3,7,11-trimethyl-1,3,6,10dodecatetraene) was the first volatile identified as a constituent of the waxy coating or cuticle apple (Murray et al. 1964). Huelin and Murray (1966) then reported the alpha isomer that was present in several apple and pear cultivars and it was subsequently shown that α-farnesene was evaporated or volatilized from apples and accumulated in oil wraps during storage (Huelin and Coggiola 1968, 1970a). There seemed to be more α-farnesene in scald-susceptible cultivars and less mature fruit and no other volatiles were known to be produced by the peel of apples or pears. Though there were no statistical analyses, it seemed that there was a relation between α-farnesene concentrations and scald incidence, although there were many exceptions (Huelin and Coggiola 1968; Meigh and Filmer 1969); however, α-farnesene as the cause of scald became uncertain when nonsignificant correlations were found between concentration at removal from RS and scald after holding an additional 7 days at 20°C (Huelin and Coggiola 1970b). Until 1995, the concentrations of α-farnesene and conjugated trienes (CTs, oxidation products of α-farnesene) were determined by absorbance at selected wavelengths of hexane or pentane extracts made by immersing

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intact fruits. The concentration of α-farnesene was estimated from absorbance at 232 nm while the concentration of CTs were estimated from absorbance at 269 nm or 282 nm (Huelin and Coggiola 1969; Anet 1972). Since it is now known that several other compounds absorb at these wavelengths (Rowan et al. 1995; Rupasinghe et al. 1998; Whitaker et al. 1997), spectrophotometric measurements need to be treated with some reserve, but not disregarded. High performance liquid chromatography (HLPC) and gas chromatography-mass spectrometry (GC-MS) are undoubtedly more accurate and precise than spectrophotometry, but the few studies using the former methods conform with the earlier reports (Meigh and Filmer 1969; Rowan et al. 1995; Whitaker et al. 1997). Production of α-farnesene may be monitored by measuring the headspace concentration above fruits or tissue slices that have been enclosed in static or flow through chambers or vials by solid phase microextraction (Mir et al. 1999; Ju and Curry 2000). α-Farnesene and other volatiles are absorbed on fibers coated with polydimethylsiloxane, which are then inserted into the injection port of a gas chromatograph for separation and measurement. Since there is variability in thickness and composition of epicuticular waxes (Belding et al. 1998), volatile release into the gas phase may be incomplete and uncertain, and thus confirmatory extraction may be necessary (Ju and Curry 2000). It is the α-farnesene and CTs in the wax covering and cuticle and perhaps the epidermal and outer hypodermal cells that are extracted when whole fruit are immersed in hexane or pentane. Some researchers have used “peel” (or “skin,” depending on authors) that has been removed from fruit. Peel is taken to be cuticle, epidermis, and hypodermis. Most workers seem to have used a vegetable or fruit peeler or razor blade that removed the outer 1–3 mm of the fruit and included some cortical tissue attached to the peel (Barden and Bramlage 1994a; Whitaker et al. 1997). In some studies, researchers have tried to scrape off the cortical cells attached to the peel. The definition and method for removing peel is far from standardized. In some recent studies the enzymatically isolated cuticle has been assayed for several constituents (Ju and Bramlage 1999a,b). Concentrations are given in a number of dimensions, e.g., nmole cm–1, µgm cm–1, or some unit unique measurement system (Ju and Curry 2000). Most of the α-farnesene has been found in the fruit coating or cuticle. After extraction of whole ‘Granny Smith’ fruit with hexane, fruit were peeled and peel pieces extracted with hexane. At harvest, both extracts contained 0.2 µg. cm–2 (Huelin and Coggiola 1968). The maximum concentrations were reached after 12 weeks RS, 32 µg cm–2 in the coating

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and 7 µg cm–2 in the cortex. After the maximum, concentrations α-farnesene decreased at about the same rate in both extracts such that the ratio of content in the coating versus that in the cells ratio stayed about the same. There are differences in α-farnesene concentrations in fruit from different parts of trees. When whole fruit extracts of ‘Cortland’ apples were measured after 1 month RS at 0°C, fruit from the exterior of trees harvested on Sept. 18 and 30 contained 20% more α-farnesene than fruit from the interior of the same trees. The internal ethylene concentration increased between harvests but more in the exterior fruit so that they finally contained 4-fold more than the internal fruit (Meir and Bramlage 1988). The nonblushed side of ‘Rome’ contained 1.66-fold as much αfarnesene as the blushed side (D’Souza 1991). There is now evidence that α-farnesene is synthesized by the isoprenoid pathway, starting with acetate. Trace amounts of [2-14C]-acetic acid and [5-3H]-mevalonate are incorporated into α-farnesene by ‘Red Delicious’ apple peel pieces (Rupasinghe et al. 1998), but there is no record of incorporation by a cell-free system. The committed, regulated step is the conversion of hydroxymethylglutaryl coenzyme A (HMG) to isoprenoid pyrophosphates by HMG reductase (HMGR). These isoprenoids are synthesized to farnesyl pyrophosphate. HMGR can be inhibited in both animals and plants by a number of compounds, including Lovastatin (Chappell 1995). As nontreated preclimacteric ‘Golden Supreme’ apple accumulated ethylene and α-farnesene during storage at 20°C, prestorage treatment with 1.25 and 2.50 nmole L–1 Lovastatin nearly eliminated α-farnesene without affecting ethylene (Ju and Curry 2000). The addition of 0.25 nmole L–1 delayed α-farnesene accumulation for 12 days, again without affecting ethylene. The effects of Lovastatin was the same on fruit that had been stored at 0° before treatment and transferred to 20°C. While the application of ethephon to preclimacteric fruit stimulated α-farnesene and ethylene production, addition of Lovastatin suppressed α-farnesene production. The incorporation of labeled trans, trans-farnesyl pyrophosphate into α-farnesene by ‘Delicious’ peel pieces has also been reported (Rupasinghe et al. 1998). There was no conversion by cortex tissue. Low activity was observed at harvest, even though there was no α-farnesene present, and increased rapidly to a peak at 20 weeks of RS, sometime after the concentration of α-farnesene had begun to decline (Rupasinghe et al. 2000) A cell-free extract of ‘Delicious’ peel has been prepared that transfers 14C label from farnesyl pyrophosphate to α-farnesene (Rupasinghe et al. 2000). This activity is ascribed to α-farnesene synthase.

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Total enzyme activity in the cytosol fraction was 16-fold greater than in the microsome fraction. The enzyme was purified 70-fold and many of its characteristics described. The molecular weight is approximately 100,000 Kd and the enzyme is composed of 2 or 3 subunits. Maximum activity was at 10°–20°C, with half-maximal activity at 0°. There is an absolute requirement for a metal ion, Mg+2 or Mn+2, which is common to other sesquiterpene cyclases (Vogeli et al. 1990). The enzyme from apple shows allosteric kinetics. The incorporation of radioactivity into α-farnesene from acetate, mevalonate, and farnesyl pyrophosphate, the α-farnesene synthesis-suppressing effect of the HMGR inhibitor Lovastatin, and the presence of an α-farnesene synthase in apple peel combine to strongly support that α-farnesene is indeed synthesized via the isoprenoid pathway in apple peel. During the commercial harvest period in Massachusetts, ‘Cortland’ peel α-farnesene concentrations ranged from 5–37 nmole cm–2 in 1991 (Du and Bramlage 1993; Barden and Bramlage 1994c). Measurements made from the same orchard were not started until after 1 month RS (Meir and Bramlage 1988). ‘Delicious’ contained 4.0 nmole cm–2 (Barden and Bramlage 1994c). In experiments conducted at West Virginia, ‘Delicious’ contained an average of 83.9 nmole cm–2, with small variations around the fruit. Fruit peel of ‘Golden Delicious’ from the same orchard contained 51.4, ‘York Imperial’ 56.2, and ‘Rome’ 62.0 nmole cm–2 (D’Souza 1991). The blushed side in red cultivars contained more αfarnesene than the nonblush side. ‘Granny Smith’ contained 13.8 nmole cm–2 which appears to be much higher than concentrations found in New Zealand fruit of that cultivar (Watkins et al. 1995). Between 1988 and 1990, the concentration of α-farnesene ranged from 17.75 to 80.85 nmole cm–2 in the peel of ‘Rome’ apples harvested from the same block 167 days after full bloom (DAFB) (D’Souza, 1991). There was little difference in α-farnesene concentrations of ‘Cortland’ apples between 1989 and 1990 (Watkins et al. 1993), but two studies with ‘Cortland’ and ‘Delicious’ from the same University of Massachusetts orchard showed a 3-fold concentration range for both cultivars (Barden and Bramlage 1994c; Du and Bramlage 1993). As the effects of maturity and environment are reviewed, it will be realized that these comparisons of cultivars must be viewed with caution. A number of studies have established a pattern of α-farnesene concentration increase with time on the tree (maturity), which continues during storage at any temperature, with peaks being reached after 50–60 days RS, followed by a decline (Huelin and Murray 1966; Meigh and Filmer 1969; Huelin and Coggiola 1968a,b; Du and Bramlage 1993, 1994;

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Barden and Bramlage 1994a; Watkins et al. 1993; Whitaker et al. 1997), but exceptions can be found (Barden and Bramlage 1994b). In ‘Rome’ apples the peel α-farnesene concentration increased between 83 and 97 DAFB and then declined to 1.5 µg cm–2 at 139 DAFB, after which there was an increase to a maximum of 77 µg cm–2 on the blush side at 169 DAFB, which is about the commercial harvest time in West Virginia. No correlation or regression coefficients between fruit age and α-farnasene concentration have been found in the literature; however, a significant relation between hours of exposure to hours <10°C after August 1 before harvest and α-farnesene concentrations in ‘Cortland’ and ‘Delicious’ has been reported (Barden and Bramlage 1994a). Hours <10°C was significantly correlated with harvest date (Barden and Bramlage 1994a). Enclosing ‘Cortland’ fruit in brown paper bags in mid-August to exclude light had no significant effect on peel α-farnesene concentrations when fruit were harvested 6–8 weeks after bagging (Barden and Bramlage 1994b). Bagging did not affect fruit maturity (called ripeness by the authors) at harvest. The effects of ethylene, applied as preharvest ethephon sprays, has been studied most extensively in ‘Cortland’. Ethephon applied 10 days before each harvest between early Sept. and early Oct. increased peel αfarnesene concentrations at harvest from 6.25-fold in the earliest harvest to 1.92-fold in the last harvest (Du and Bramlage 1994). Barden and Bramlage (1994b) obtained essentially the same response when ethephon was applied 11 days before a Sept. 1 harvest. The increase in concentration of α-farnesene with time in storage depends on harvest time. The increase was linear in ‘Rome’ apples harvested 154 DAFB, quadratic at 161 DAFB, and neither linear nor quadratic at 168 DAFB (D’Souza 1991). Several studies have shown that the maximum α-farnesene concentration in either whole fruit extracts or peel of ‘Cortland’ is reached after 55–60 days of RS and then decreases (Meir and Bramlage 1988; Du and Bramlage, 1993, 1994; Barden and Bramlage 1994b,c), although a further increase was observed when the RS was extended to 30 weeks, an unusually long storage time for ‘Cortland’ (Du and Bramlage 1993). The concentration of α-farnesene declined during holding time at 20°C after 30–45 weeks RS. Maximum concentrations were in fruit harvested close to Sept. 24 in Massachusetts with amazing consistency between years. ‘Delicious’ followed the same pattern, although α-farnesene concentrations were about 25% of ‘Cortland’ (Du and Bramlage 1993; Barden and Bramlage 1994b). When harvest dates were combined, the relation was linear in ‘Cortland’ and ‘Delicious’ (Barden and Bramlage 1994b), but the increase over 20 weeks

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storage was only 1.13-fold for fruit harvested Oct. 11 (150 h <10°C) as compared to 13-fold for fruit harvested Sept. 17 (21 h <10°C). The slower increase and more rapid decline means that the concentration of αfarnesene is less after 4–5 months of RS in later harvested fruit. During the extension of storage from 16 to 30 weeks an increase in α-farnesene concentration was observed (Du and Bramlage 1993). The accumulation of α-farnesene by New Zealand ‘Granny Smith’ after harvest increased with temperature between 0° and 20°C and was positively correlated with ethylene production (Watkins et al. 1995). There was also a large increase of α-farnesene during storage of ‘Cortland’ at 20°C for 1 week; during the second week, there was a decrease (Du and Bramlage 1993). The maximum concentration at 20°C was 1.44 of the concentration at 0°C. During warming of ‘Granny Smith’ at 5°C after RS for 2 weeks, α-farnesene increased 17.2-fold and 1.3-fold after 12 weeks RS, although the greatest absolute concentrations increase occurred as RS was extended. Ethylene production followed the same pattern. During additional RS after warming until a total of 25 weeks after harvest, α-farnesene concentrations decreased from an average of 45.2 nmole cm–2 after warming to 4.6 nmole cm–2 after total storage. As the warming time after 2 weeks of RS was increased from 1 to 14 days, α-farnesene concentrations rose from 1.5 nmole cm–2 to 78.2 nmole cm–2. Concentrations after additional RS were not reported. Holding ‘Granny Smith’ and ‘Grand Alexander’ at 38° to 46°C for 24–96 h reduced the concentration of α-farnesene in the cuticle, even though ethylene production was nearly completely suppressed (Klein and Lurie 1990, 1992, 1994). In these experiments DPA did not affect αfarnesene. The high temperature treatments increased yellowing of ‘Anna’ but increased firmness after RS. During RS fruit that had been bagged before harvest contained about 1.15-fold greater α-farnesene than the controls, but the differences were significant only after 6 and 12 weeks of RS. In ‘Cortland’ and ‘Delicious’ internal ethylene concentration and whole fruit extract α-farnesene concentration at harvest both increased simultaneously from mid-Sept. to mid-Oct., suggesting that the beginning of the α-farnesene rise on the tree coincides with the onset of the ethylene climacteric or is perhaps regulated by ethylene (Watkins et al. 1993). Although the α-farnesene was not measured until a month after refrigerated storage, maximum concentrations of surface α-farnesene and internal ethylene concentration and percentages of fruit with greater than 0.5 µl L–1 of ethylene both increased in ‘Cortland’ apples with delay of harvest throughout Sept. (Meir and Bramlage 1988). The application of 0.5 g L–1 ethephon 10 days before each of 6 harvests from early Sept. through early Oct. increased the concentration of α-

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farnesene in whole fruit hexane extracts from 12-fold in the early harvest to 1.9-fold in the last harvest. During RS for 20 weeks α-farnesene increased characteristically in the controls (2- to 12-fold) while in the preharvest treated fruit there was no further accumulation (Du and Bramlage 1994). Small but statistically significant differences between harvest dates persisted. Also in ‘Cortland’ α-farnesene concentrations at harvest increased linearly in response to 0, 0.25, and 0.50 g L–1 ethephon sprays applied 16-28 days before harvest but during storage α-farnesene declined more rapidly in the ethephon-treated fruit (Barden and Bramlage 1994). The application of 0.2–0.4 g L–1 ethephon 3–6 weeks before harvest to ‘Fuji’ and ‘Granny Smith’ had no consistent effects on condition at harvest. In the one year that α-farnesene was measured, concentrations in peel discs were increased up to 26% after 8 months RS. Scald incidence was decreased as the ethephon concentration and application time before harvest were increased (Curry 1994). Application of ethephon immediately after harvest increased ethylene production and α-farnesene accumulation during 18 days at 20°C, mostly between days 9 and 15. DPA suppressed ethylene production and α-farnesene accumulation 20–30%, with most of the effect occurring between day 6 and day 15. The ethylene production of fruit treated with both ethephon and DPA was above the controls, while α-farnesene did not differ from that in the nontreated fruit; i.e., the compounds seem to have counteracted each other (Du and Bramlage 1993, 1994). Ethylene production increased and α-farnesene decreased while fruit were being held at 20°C after 10 weeks of RS. Application of ethephon after the 10 weeks of RS increased ethylene production but did not affect αfarnesene concentration. Diphenylamine applied at the same time suppressed ethylene production and lowered the α-farnesene concentration (Du and Bramlage 1994). One day after removing fruit from 20 weeks of RS, DPA applied either before or after RS did not affect ethylene production or α-farnesene concentration. During subsequent period of 8 days at 20°C, ethylene production increased greatly and α-farnesene content increased. Increasing the hourly ventilation rate from 2 to 20 L increased the rate of α-farnesene evaporation from Australian ‘Granny Smith’ by 35-fold, but after 25 weeks the amount of α-farnesene retained was the same in both treatments, indicating that there was considerable synthesis (Huelin and Coggiola 1970b). Balancing α-farnesene lost by evaporation and αfarnesene retained suggests that synthesis continues into the thirtieth month of RS. Increasing temperature from 5° to 15°C increased the rate of evaporation of α-farnesene only at the higher ventilation rate. With

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‘Delicious’, there was no difference in peel α-farnesene between room and flowing air (Whitaker et al. 1997). The effect of atmospheric composition on α-farnesene concentrations has not been studied much. Transfer of ‘Delicious’ after 2 months of RS to flow-through chambers with 1.5% O2 or air caused a subsequent 20–30% decrease in peel α-farnesene after 2–3 months (Whitaker et al. 1997). After a total of 6 months storage there were no differences between treatments. When flow-through storage of ‘Granny Smith’ was started at harvest, α-farnesene rose to a maximum after 7 weeks in room air or 100% O2 and then declined. In flowing 1.5% O2 the maximum was reached in 12 weeks and was about one-third the maximum amount in air and 100% O2. After 12 weeks in 100% O2 the entire peel was bronzed and a high level of ethanol had accumulated. Scald-resistant selections of ‘White Angel’ × ‘Rome Beauty’ crosses contained less α-farnesene at harvest than the less red or yellow scaldsusceptible selections (Rao et al. 1998). During 16 weeks of RS, αfarnesene increased 444% in the resistant selection as compared to 68%–145% in the susceptible strains, although after 4 weeks all selections were about the same. 2. Conjugated Trienes. α-Farnesene is autoxidized to at least 2 conjugated hydrocarbons and a hydroperoxide (Anet 1969; Rowan et al. 1995), which are referred to as conjugated trienes (CTs). No evidence has been presented that any enzymes are involved in the oxidation of α-farnesene to CTs. By 1972, evidence accumulated that these CTs were more directly related to scald development than α-farnesene and CTs were found along with α-farnesene in air that had passed over ‘Granny Smith’ apples and then through hexane (Huelin and Coggiola 1970a,b; Anet 1972). Little more attention was given to the trienes over the next 15 or so years as scald control research emphasized the efficient use of diphenylamine and ethoxyquin in multifomulation dip treatments (Ingle et al. 1990; Johnson et al. 1980; Little et al., 1980) and the effects of storage atmosphere composition and temperatures on scald development, about which more will be said later. The structures of the α-farnesene autoxidation products (E, E)-5dimethyl-2-)4-methylpentyl-3-enyl)hepta-2,4,6-trienyl hydroperoxide (I) and erythro and threo (E,E,)-4-(1-hydroperoxy-1-methylethyl(-1-methyl1-(4-methylhexa-1,3,5-trienyl)tetramethylene peroxide (II) were deduced from the infrared and ultraviolet spectra of the alcohols produced by reduction. These CTs have complex absorbance spectra with peaks at 259–260 nm, 269 nm, and 280–282 nm (Anet 1969). These three peaks are sometimes referred to as CT258, CT269, and CT281 (Du and Bram-

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lage 1993). It was later recommended that a single measurement at 282 nm be used to estimate the CTs (Anet 1972). Because of interference across the 258–282 range of the UV spectrum, a blank reading at OD292 is usually subtracted from the ODs at the lesser wavelengths. In addition to the CTs, there is some evidence that 6-methyl-5-hepten2-one (MHO) is an oxidation product of α-farnesene (Meigh and Filmer 1969). MHO has been found in headspace of vials where apple peel slices were being incubated as well as evolved from whole fruit (Mir et al. 1999b). Following the establishment of the structures of α-farnesene oxidation products by syntheses (Spicer et al. 1993; Brimble et al. 1994), the structure of α-farnesene and CTs extracted from apple surface was compared to the authentic compounds (Rowan et al. 1995). The CTs were separated and quantified by high performance liquid chromatography (HPLC) and GC-MS as well as UV absorption. The principal (89–95%) CT was the I of Anet’s and is now named 2,6,10-trimethydodeca-2,7(E), 9(E), 11tetraen-6-ol. The 9Z isomer of I constituted the remaining 5–11% of the CTs absorbing at 259–282 nm. Anet’s I measured by HPLC and GC-MS accounted for only 12–35% of the CTs measured by UV spectroscopy in the apple skin extracts (Rowan et al. 1995). The CT structures have been confirmed using HLPC with diode array detection and GC-MS and a C18HPLC method was devised for the simultaneous measurement of αfarnesene and its major oxidation product (Whitaker et al. 1997). It has not been demonstrated where the autoxidation of α-farnesene to CTs occurs. The synthesis of α-farnesene must occur in cells, assuming that it is derived through something similar to the mevalonateisoprenoid pathway (Rupasinghe et al. 1998). It would be guessed that the α-farnesene moves into the nonpolar cuticle by diffusion rather than by an active transport system because of the lipid solubility. α-Farnesene may be lost from the cuticle either by autoxidation or volatilization (Huelin and Coggiola 1970b), or possibly enzymatic degradation, which has not been described. If indeed CTs cause the damage in the hypodermal cells that is recognized as scald, the CTs must either move back into the hypodermis from the cuticle or be formed in the hypodermis to toxic concentrations. At present, nothing but speculation is available. No breakdown products of CTs have been described, except possibly MHO. CT281 concentrations were 18–51 nmole cm–2 in ‘Delicious’ apples at commercial harvest in West Virginia, 7–8 nmole cm–2 for ‘Golden Delicious’ and 6–7 nmole cm–2 for ‘York Imperial’ (D’Souza 1991). ‘Granny Smith’, commonly said to be the most scald susceptible of these cultivars, contained the lowest levels of CTs, 13–16 nmole cm–2, and were

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similar to levels observed in ‘New Zealand’. CT concentrations were higher on the blushed side of ‘Delicious’ but were evenly distributed around the other cultivars (D’Souza 1991). As with α-farnesene, there are no published correlations or regressions between CTs and fruit age, but there was a significant negative correlation between CT281 and hours <10°C in ‘Cortland’ and ‘Delicious’ for one year (Barden and Bramlage 1994b). The changes in CT281 during maturation are not as great as the changes in α-farnesene. ‘Cortland’ CT281 rose from 1 nmole cm–2 in mid-Sept. to 3 nmole cm–2 in mid-Oct. In ‘Delicious’ the change was from 0.1 to 0.4 (Barden and Bramlage 1994b; Meir and Bramlage 1988). In West Virginia ‘Rome’ apples, CT concentrations decreased between 83 and 153 (DAFB) and then increased during the next 30 days (D’Souza 1991). There was considerable difference between years, 6.40 nmole cm–2 167 DAFB in 1989, 0.01 nmole cm–2 in 1990, and 1.04 nmole cm–2 in 1991. The CT concentrations were a little higher on the blushed sides, but the differences were not significant. It will be noted that the concentration of α-farnesene is always several-fold greater than CTs (D’Souza 1991). The first detailed study of CT concentration changes after harvest showed that in ‘Cortland’ apples CT281 starting after 1 month RS increased from 1.64 nmole cm–2 at the first harvest (Sept. 12) to 2.46 nmole cm–2 in fruit harvested Sept. 30 and mostly increased during 4 months of RS to a final concentration of 10–16 nmole cm–2, although the pattern was not entirely consistent (Meir and Bramlage 1988). The increases with delayed harvest were considerably less than the increases during RS. Measurements made after 3 months of RS showed high correlations (0.884–0.980) between CT258, CT269, and CT281. There were no consistent correlations between α-farnesene and CT. α-Farnesene was significantly correlated with scald incidence or severity when the α-farnesene was measured at very near the time the scald evaluation was made. Correlations with CT281 were higher, even when made 3 months before the scald evaluation (Meir and Bramlage 1988). Another study with ‘Cortland’ apples from the same orchard done 6 years later showed similar patterns of change, although maximum concentrations were lower (Barden and Bramlage 1994a). At harvest, the CT281 concentrations in ‘Delicious’ peel was one-tenth that of ‘Cortland’ but increased much more rapidly so that both cultivars were about the same after 20 weeks RS (Barden and Bramlage 1994a). From a study of the changes in α-farnesene, CT258, CT269, and CT281 in whole fruit extracts of ‘Cortland’ and ‘Delicious’ during RS, several reasons were found to suspect that CT281 was not directly involved in the initiation and development of scald (Du and Bramlage 1993) as fol-

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lows: 1. After 30 weeks RS, early harvested ‘Cortland’ contained one-half as much CT281 as late harvested apples but developed three times more scald. 2. After 16 weeks RS, early harvested ‘Cortland’ contained 11.8 nmole cm–1 and developed 23% scald. During storage for an additional 14 weeks, the CT281 decreased to 2.9 nmole cm–1 but scald increased to 60%. 3. ‘Cortland’ apples with varying amounts of scald contained about the same concentrations of CT281. 4. ‘Cortland’ apples held continuously at 20°C had high concentrations of CT281 but developed no scald. In ‘Cortland’ at harvest, CT258 and CT269 concentrations were about 2-fold that of CT281. All 3 rose to a plateau at 12 weeks of storage. CT258 increased 1.08-fold, CT269 3.06-fold, and CT281 4.07-fold The unequal rates of increase caused decreasing ratios between the CTs with the CT258:CT281 decline being greater and significantly different from CT269:CT281 change. As was true for α-farnesene, the CTs were much lower in ‘Delicious’ than in ‘Cortland’ but increased proportionately much more (9 to 12-fold). CT258 and CT269 increased faster and to a higher level than CT281 so that the ratios were much higher in ‘Delicious’ and declined more and always linearly. The postharvest dip application of 2000 mg L–1 DPA reduced the accumulation of αfarnesene (53%) and CTs during RS of ‘Cortland’ but the reduction of the individual CTs was not proportional. The CT258:CT281 ratio was increased while CT269:CT281 was not changed. For ‘Delicious’, DPA reduced α-farnesene and CT281, but not enough to alter the ratios significantly. During the extension of storage from 16 to 30 weeks, αfarnesene and CTs declined, as did the CT258:CT281 and CT269:CT281 ratios, while scald incidence increased. Delaying harvesting of ‘Cortland’ increased α-farnesene and CT concentrations; CT258:CT281 increased but CT269:CT281 did not change. Scald was typically less in the later harvested apples. Rather than being dependent on OD281, scald was thought to be less likely when the CT258:CT281 was low and that this ratio was regulated by postharvest conditions (Du and Bramlage 1993). It was concluded that α-farnesene is oxidized to CT281, which is then converted to CT258, from which the toxic compound responsible for scald initiation is formed. It is uncertain what the absorbances at 258, 269, and 281 nm represent, since it does not appear that there are structurally distinct CTs with their own spectral characteristics (Rowan et al. 1995). MHO has been proposed as an oxidation product of α-farnesene and there is evidence that the evolution of MHO by peel discs at 20°C parallels the development of scald (Mir et al. 1999b). New Zealand ‘Granny Smith’ harvested Apr. 12 contained 0.11 nmole cm–2 CT and increased to 2.96 nmole cm–2 after 20 weeks RS (Watkins et al. 1995). There was a 2- to 3-fold increase during holding at 20°C.

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Warming after 2 weeks RS for 3–14 days resulted in increased CTs but reduced scald after another 25 weeks RS. There was a consistent increase in CTs during storage, but less in fruit collected 176 DAFB than 162 or 169 DAFB (D’Souza 1991). Holding ‘Granny Smith’ at 38°–46°C reduced CTs to about the same extent as postharvest application of DPA (Klein and Lurie 1992). Neither control nor bagged ‘Cortland’ fruit contained CT281 at harvest but the increase during RS was more rapid in the fruit that had been bagged. The concentration ratios increased from 1.27 at 6 weeks of RS to 1.64 at 18 weeks of RS (Barden and Bramlage 1994b). The effect on CTs of ethephon applied preharvest to ‘Cortland’ apples depended on fruit age/maturity/ripeness (Du and Bramlage 1994). When ethephon was applied 10 days before harvest on Sept. 3 the starch index was advanced from 2.7 to 4.8, CT258 at harvest increased from 6.2 to 26.1 nmole cm–2, and CT281 increased from 1.7 to 8.7 cm–3. Both CTs increased erratically in nontreated fruit as they matured on the trees (called ripening by the authors) but decreased linearly in treated fruit. The ethephon effect decreased from 4-fold to 5 to 1.45-fold as fruit matured (or ripened). During 20 weeks of RS CT258 increased 5-fold in nontreated fruit but there was no change in the control fruit. CT281 increased 4-fold in the controls but only 1.60-fold in the treated fruit. CT258/CT281 increased 3-fold in the untreated fruit but decreased 0.75 in the treated fruit (Du and Bramlage 1994). In experiments conducted in the same laboratory in the same year, the application of 0.25–0.50 g L–1 ethephon applied 11 days before a Sept. 1 harvest (starch index 1.0 or 4.8) raised the CT281 concentration 10- to 13-fold at harvest but the concentration hardly changed during RS so that treated and nontreated fruit were not different after 12 weeks (Barden and Bramlage 1994b). The CT281 concentration in ‘Fuji’ and ‘Granny Smith’ skin after 8 months of RS was not consistently affected by preharvest ethephon treatment (Curry 1994). At-harvest CT281 concentrations in whole-fruit extracts were 3-fold greater than CT258. Ethephon dips at that time increased the concentration of both CTs equally during holding at 20°C and decreased CT258/CT281 (Du and Bramlage 1994). DPA dips reduced both CTs to nearly the same extent and did not affect the ratio. No scald evaluation was made. During 16 days at 20°C after 10 weeks of RS, there was a small increase in CT258, a decrease in CT281, and an increase in the CT258/CT281 ratio. The 20% increase in CTs in fruit treated with ethephon poststorage was significant, but the ratio was unaffected. The 12% increase in CT258 in DPA-treated fruit was not significant, but enough to significantly increase the ratio, since CT281 was not changed. The rate

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of ethylene production increased 4-fold during 19–20 weeks RS and was not affected by DPA treatment before or after RS and both CT258 and CT281 were reduced. In the selections of ‘White Angel’ × ‘Rome Beauty’ crosses, both CT 258 and CT281 were higher in the scald-susceptible strains than in the resistant strain at harvest but like α-farnesene the increase during RS was greater in the resistant strain (Rao et al. 1998). After 4 weeks of RS, both CTs were slightly higher in the susceptible strains, but it is hard to tell if the differences were significant. CT258/CT281 was about the same in all selections, except after 4 of weeks of RS, when the resistant selection was higher. After transfer of ‘Delicious’ apples to flow-through chambers after 2 months RS in boxes, 1.5% O2 was more effective than air in reducing the accumulation of CTs (Whitaker et al. 1997). In the peel of ‘Granny Smith’ stored from harvest in flowing gasses, CTs accumulated more rapidly in 100% O2 than in air. The peak of 60 µg g–1 fresh weight was reached in 12 weeks in 100% O2 and in 22 weeks in air. The CT increase in 1.5% O2 followed a time course similar to air but were about 5-fold lower in low O2. CT levels were the same in flowing air and room air. B. Antioxidants 1. Lipid Soluble. Early in the study of the relation of α-farnesene and CTs to scald inception and development, it was proposed that endogenous antioxidants may contribute to the regulation of CT concentrations (Anet 1974). Although that work was previously reviewed, additional comments seem in order. It was assumed that effective α-farnesene antioxidants would be confined to the lipid phase. The concentration of the antioxidants in the whole fruit hexane extracts, as judged by size and intensity of spots on thin-layer chromatographs, was seen to increase during maturation and storage. The presence of tocopherols in apple cuticle and peel has been reported in later investigations (Meir and Bramlage 1988; Barden and Bramlage 1994b,c). It was suggested that scald did not occur if antioxidant content remained adequate to prevent or sufficiently limit α-farnasene autoxidation. Whole fruit hexane extracts of ‘Cortland’ apples showed a prominent UV absorption at 200 nm in addition to the customary 232 nm, 259 nm, 269 nm, and 281 nm peaks (Meir and Bramlage 1988). Hexane extracts of peel (depth, cell types not specified) inhibited the Fe+2 catalyzed oxidation of linoleic acid. Thin layer chromatographs separated that extract into about 14 spots (compounds?) that had linoleic acid antioxidant activity. The spots absorbed at 200 nm. Antioxidant activity and 200 nm absorbance

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increased during maturation on the trees and RS. The optical density at 200 nm was correlated (r=0.625) with antioxidant activity (Meir and Bramlage 1988). The strength of the correlations with scald after RS increased from α-farnesene < CT281 < antioxidants < OD200/nmole αfarnesene. The magnitude of the negative correlation between concentration of CT281 and OD200 increased between 2 and 4 months of RS. At that time it looked like OD 200 nm in whole fruit hexane extracts at harvest could be used to predict scald after RS, at least in ‘Cortland’. At commercial harvest in West Virginia, peel disc hexane extracts of ‘Golden Delicious’ had an OD200 of 2.15, ‘Delicious’ 1.99, ‘York Imperial’ 1.83, ‘Granny Smith’ 1.03, and ‘Nittany’, a ‘Golden Delicious’ ‘York Imperial’ cross, 2.15. There were no significant differences between fruit sides. The OD200 of ‘Rome’ declined between 83 and 153 DAFB and then rose abruptly to 176 DAFB (D’Souza 1991). The increase between 154 and 180 DAFB was linear and there was a nearly 3-fold annual variation over 3 years. Over 126 days of RS OD200 increased linearly and there was little difference between blushed and nonblushed fruit sides. Tocopherols and tocols were among the spots identified on the TLC plates. The largest spot, with greatest OD200 and linoleic acid oxidation inhibition, was at a much higher Rf than the tocopherols and was not identified (Meir and Bramlage 1988). It was attempted to quantify αtocopherol under a number of conditions (Barden and Bramlage 1994b). OD200, α-tocopherol inhibition of linoleic acid oxidation, and total watersoluble reducing compounds (RWRC) in peel slice extracts all increased linearly as a function of hours <10°C in both ‘Cortland’ and ‘Delicious’ of the 1990 University of Massachusetts crop. α-Tocopherol was higher in ‘Cortland’ than ‘Delicious’ (45%) and increased more (42% vs. 10%). During storage, α-tocopherol increased nearly the same in both cultivars so that after 20 weeks ‘Cortland’ contained 34% more than ‘Delicious’. Carotenoids, another group of lipid-soluble antioxidants, increased more in ‘Cortland’ than in ‘Delicious’ (50% vs. 11%) over the four week harvest period, although at harvest the concentrations were about the same. During 20 weeks of RS the increase was greater in ‘Cortland’ (67%) than in ‘Delicious’ (11%) (Barden and Bramlage 1994b). Ethephon-induced preharvest ripening increased inhibition of lipid oxidation by peel extracts by about 50%. It also increased α-tocopherol concentration, but not that of carotenoids. During storage, inhibition of lipid oxidation changed little, but concentrations of α-tocopherol and carotenoids increased greatly. At the end of storage, peel of ethephontreated ‘Cortland’ apples contained higher concentrations of both αtocopherol and carotenoids than that of non-treated fruit (Barden and Bramlage 1994b). Preharvest fruit bagging did not affect linoleic acid oxi-

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dation and reduced the concentrations of α-tocopherol and carotenoids (Barden and Bramlage 1994b). 2. Water Soluble. In addition to lipid-soluble antioxidants, ‘Cortland’ apple peel contains one or more water-soluble constituents that are capable of reducing iron, which is equivalent to antioxidant activity (Barden and Bramlage 1994b,c). This reducing capacity (TWRC) increased slightly but linearly as harvest was delayed; during storage, there was a small but inconsistent decrease with a harvest × storage duration interaction. Application of 0.25 and 0.50 g L–1 of ethephon led to a small increase in TWRC at harvest 10 days later but was not related to a change during storage. Bagged fruit contained 20% more TWRC at harvest; no measurements were made during storage. Apparently no attempt was made to resolve the constituents of this extract. The water-soluble natural antioxidants glutathione and ascorbic acid were found in other aqueous extracts. The ascorbic acid was identified and quantified by a colorometric method, while glutathione was assayed by HPLC (Barden and Bramlage 1994b,c). There was no consistent change in glutathione concentration as harvest was delayed, and during storage there was a significant decrease, which was influenced by the harvest date. Ethephon treatment did not affect glutathione concentration at harvest but apparently caused a large decrease during storage. Ascorbic acid concentration did not change significantly with harvest date and decreased during storage, again with a harvest × storage interaction. At harvest, ethephontreated fruit contained more ascorbic acid than the controls but there was a sharp decline during storage. Bagged fruit contained more ascorbic acid than unbagged fruit at harvest and as with glutathione there are no measurements during storage (Barden and Bramlage 1994b). The methanol-water-HCl extract of cuticle scraped from the surface or isolated enzymatically was found to react positively with FolinCiocalteu reagent, which is a standard method for identifying phenolic compounds, and was quantified spectrophotometrically using gallic acid as a standard (Ju and Bramlage 1999). When the residue was treated with potassium hydroxide, 4- to 7-fold additional reactive material appeared and the phenolics were designated as “free phenolics” and “bound phenolics.” More phenolics were found in the isolated than in the scraped cuticle. The amount of cuticle cm–1 collected by the two methods was not reported, but it was said that the scraped fruit did not brown, showing that cells were not damaged. It would seem that the scraped material was composed of the surface and the middle layer of the cuticle, while the enzyme-isolated cuticle also contained the lower layer, freed by hydrolysis from the cell wall carbohydrates with which

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it is usually associated. The flesh of ‘Delicious’ and ‘Ralls’ contained considerably less simple phenols (apparently the same as the free phenolics of Ju and Bramlage 1999) than the peel (Ju et al. 1996). The peel from the shaded side of the fruit contained more simple phenols than peel from the exposed side, and they declined as fruit matured from Aug. 1 to Oct. 1 (Ju et al. 1996). The simple phenol concentration in the peel of ‘Delicious’ decreased during holding at 20°C, at the same time that the visual symptoms of scald were appearing. Comparative free phenolic content between cultivars was ‘Golden Delicious’ > ‘Delicious’ > ‘Empire’ > ‘Cortland’ (Ju and Bramlage 1999), while ‘Delicious’ contained the most bound phenols. Ju et al. (1996) used a different method for measuring phenols than Ju and Bramlage (1999) and a little different terminology, so that comparing the two sets of findings must be done cautiously. The phenolic extracts inhibited the Fe+2 catalyzed oxidation of linoleic acid and on a molar basis to about the same extent as quercetin, one of the flavonoids, which also have been found in apple peel (Ju et al. 1996). The bound phenolics inhibited linoleic acid oxidation to about the same extent as gallic acid, which was considered to be a free phenolic. Free phenolics and gallic acid also inhibited the increased absorption at 281 nm in an α-farnesene-hexane system exposed to oxygen. They were more inhibitory than DPA; lipid-soluble antioxidant from peel was ineffective (Ju and Bramlage 1999). Other than reactivity with Folin-Ciocalteu reagent or absorption at 313 nm, no attempt was made to identify individual compounds. The skins of ‘Golden Delicious’, ‘Empire’, and ‘Rhode Island Greening’ have been shown to contain at least 10 phenolics, including open chain phenylpropanoids, chalcones, and flavenoids (Burda et al. 1990). Several complex, synthetic phenolic compounds were found to inhibit in vitro oxidation of α-farnesene but did not always affect scald development (Anet and Coggiola 1974). Those workers rejected lack of uptake for ineffectiveness as scald inhibitors since diphenylamine and t-butyl-4-methoxyphenol were present in whole fruit hexane extracts after dipping and after core injection. This does not mean that there are no barriers to the entrance and distribution of larger molecules with more functional groups. C. Anthocyanins Anthocyanins, specifically cyanidin and cyanadin-3-glucoside, inhibit the in vitro autoxidation of linoleic acid (Tsuda et al. 1994). It has been reported that anthocyanins are negatively correlated with CT281 and scald development in ‘Cortland’, but no data were shown (Barden and

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Bramlage 1994b). It was also reported that ‘Redcourt’ contained more anthocyanins than the standard strain, but both strains developed the same amount of scald. The outside or exposed sides of ‘Delicious’ and ‘Ralls’ apples were found to contain more anthocyanin than the shaded peel; the exposed side developed less scald (Ju et al. 1996). This relation between light exposure, red color, and scald is commonly found in the literature, but there is little quantitative supporting data. Peel of fruit bagged on July 15 contained no anthocyanin at harvest on Aug. 1–10, Sept. 1–10, or Oct. 1–10. Anthocyanin in unbagged fruit peel increased with harvest date. Harvest date was not associated with scald incidence on bagged fruit but declined on the unbagged fruit (Ju et al. 1996).

IV. PHYSIOLOGY OF SCALD A. Cultivar, Maturity, and Year Extensive data show that superficial scald incidence and severity differs greatly among cultivars, harvest dates (maturity), and years (Meir and Bramlage 1988; Ingle et al. 1990; Barden and Bramlage 1994a,b,c; Bramlage and Watkins 1994; Du and Bramlage 1994; and Emonger et al. 1994). It is commonly recognized throughout the literature that the cultivars ‘Granny Smith’, ‘Cortland’, and ‘McIntosh’ are scald susceptible, while scald is rarely seen on ‘Golden Delicious’ and ‘Empire’, which are therefore designated as scald resistant (Emonger et al. 1994). Because scald has been seen on those two cultivars, they cannot be called scald immune. An example of scald variations between years is ‘Cortland’ apples harvested Sept. 22–24 from the same Massachusetts orchard that had 36%, 99%, and 78% of the fruit scalded in 1988, 1989, and 1990, respectively (Barden and Bramlage 1994a). In the same experiments, in 1988 36% of ‘Cortland’ were scalded, while 2% of ‘Delicious’ of comparable maturity (starch index ~2.0) were scalded. In 1989 and 1990 there were much fewer differences between those two cultivars. These variations suggest that scald incidence and intensity are regulated by preharvest and postharvest external and internal (including genetic) conditions. Incidence usually means the percentage of fruit affected, regardless of the fraction of the fruit surface affected or the color, from tan to nearly black. Almost every investigator uses a unique system to rate apple fruit for scald severity. A numerical scale may be used to reflect the percentage of the fruit surface affected, such as 1 = 1–10%, 2 = 11–33%, 3 = 34–66%, and 4 = 67–100% (Barden and Bramlage 1994a).

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This is not quite a linear scale and it does not tell how badly the surface is discolored, sunken, or wrinkled, indications of the extent of cell collapse. Usually the percentage of the surface affected is estimated visually, rather than actually measured. There seems to have been only one experiment that used a chromameter to measure the color intensity of scald lesions (Mir et al. 1998a). To compare scald between harvest times or between years or cultivars, it would seem necessary to have a way to define fruit age at harvest. Calendar date is the most obvious, but nothing is told about fruit age or how long the fruit has been developing unless there is familiarity with the cultivar and the weather and climate under which the fruit is produced. That can be measured from some fairly easily recognizable developmental stage like full bloom, pollination, or petal fall, all of which vary annually. Some of the external conditions that change during development and from year to year are temperature, insolation, and water availability. The fruit variables that most commonly have been measured are firmness, sugar concentration as soluble solids, starch content based on semi-quantitative scales, internal ethylene concentration or production rate. Rarely are all these variables measured in the same experiment. There are traditional harvest windows for harvesting each cultivar in every district and many experiments are started several days before the window and continued beyond the end of the window. If harvest date is not a variable of the experiment, a single date within the window is chosen, perhaps refined by measurement of fruit condition for a week or two so that fruit of the desired condition can be obtained. B. Temperature The idea that preharvest temperatures influence or regulate scald induction and development dates back to about 1920, but the first quantitative studies were done with ‘Stayman’ apples (Merritt et al. 1960). Scald was reduced as apples were exposed to increasing time below some maximum (around 10°C). “High” temperature episodes reversed the “low” temperature effects. There was little or no scald on ‘Stayman’ fruit that had been exposed to 150–190 cumulative hours below 10°C after the onset of cool weather in Sept. A temperature of 12°C may have been a better dividing point than 10°C but 10°C has become a “magic number.” Scald severity (scald index) of ‘Starkrimson Delicious’ from 7 stations in Canada and the United States over 3 years starting in 1986 was better related to hours <10°C (r=–0.61) than starch index (r=0.30), although both r values were significant (Blanpied et al. 1991). On the 1–3 scald

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severity index scale used at all the stations, severity was less than 1 (slight) when the starch index was 5.3 or higher, indicating advanced maturity, regardless of the hours <10°C up to harvest. When the lots at advanced maturity were eliminated, the remaining lots could be placed in three categories: scald moderate to severe when there had been < 70 h <10°C; trace to slight scald with 70–80 h <10°C; trace to no scald with >90 h <10°C. The effect of preharvest temperatures was next studied at the University of Massachusetts during the 1988, 1989, and 1990 crop years (Barden and Bramlage 1994a). For ‘Cortland’, there was a very high correlation between scald incidence and hours <10°C (r=–0.87) and somewhat lower but still significant correlations with days since Jan. 1 (r=–0.71) and starch index (r=–0.65). The correlations for ‘Delicious’ from the same orchard for hours <10°C were lower (r=–0.78) but a little higher for days (r=–0.75). There was no correlation with starch index, in contrast to a previous study with ‘Delicious’ that found a small significant correlation between scald severity and starch index (Blanpied et al. 1991). It should be noted that there were high correlations between days, hours <10°C, and starch index, except for hours <10°C and starch index in ‘Delicious’. There was ripening even as hours <10°C accumulated. From five years of data, it was concluded that for ‘Cortland’ scald resistance began to appear after a minimum of 50–60 h (Barden and Bramlage 1994a) or 80–90 h (Bramlage and Watkins 1994) <10°C before harvest and that after 150 h <10°C there was little susceptibility to scald. For ‘Delicious’ scald resistance began to appear after 100 h <10°C had accumulated before harvest and after 200 h there was little scald susceptibility. In New Zealand ‘Delicious’ that had been exposed to 100 h <10°C were usually nearly scald-free, whereas, as noted above, Massachusetts ‘Delicious’ were just beginning to lose scald susceptibility. New Zealand can be divided into cool districts, such as Otago, which had over 200 h <10°C by March 1, and warm districts (Hawkes Bay, Nelson), which had <100 h <10°C by the same date. Neither ‘Delicious’ nor ‘Granny Smith’ fruit harvested in the cool districts after the earliest commercial picking dates in early March developed any scald, while incidence on fruit from the warmer districts declined nonlinearly as harvest was delayed. The trends in scald decline differed between districts, as indicated by the different constants and coefficients in regression equations relating scald incidence to hours <10°C, starch, and date to scald incidence in each district. High correlations were produced when all districts were combined in a multiple regression. When those equations were applied to selected

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dates from three districts, a wide range in differences between observed and calculated incidence was found. The correlations from multiple regressions adding either starch index or days hours <10°C were no larger than those for hours <10°C alone, which does not agree with Blanpied et al. (1991). Fruit from the warmer districts tended to behave rather like Massachusetts fruit, in which the onset of low temperatures occur about the same time in relation to commercial harvest. Starch and date may not be good scald predictors because they change linearly, while scald and hours <10°C usually do not. Unlike Massachusetts, it does not appear that large changes in scald susceptibility occur after a certain number of accumulated hours <10°C (Bramlage and Watkins 1994). Scald development can be influenced by postharvest temperature experiences. Although there are a few reports of scald on apples held continuously at 20°–30°C, scald is not seen unless fruit has been exposed to <15°C (Bauchot et al. 1995; Watkins et al. 1995). Traditionally, scald appears after transfer of fruit from RS or CA to “room temperature,” which usually means 20°C. Increasing low temperature storage and poststorage room temperature increase scald incidence and certainly severity. Since the time-temperature relations of scald symptom development is similar to chilling injury in tropical commodities, it has been proposed that scald is a chilling injury, even though apple is a temperate fruit (Meir and Bramlage 1988). Intermittent warmings usually reduce chilling injury in tropical plants (Wang 1994). Scald incidence, measured 25 weeks after harvest, decreased on Hawkes Bay, New Zealand ‘Granny Smith’ that were warmed for 5 days at 20°C after RS for 2–12 weeks and then returned to RS for the rest of the 25 weeks (Watkins et al. 1995). The reduction in scald incidence was less when RS was >10 weeks before warming. Severity increased continuously with time in RS before warming. Incidence and severity were about the same in warmed and DPAtreated fruit. The effects of the intermittent warming on fruit condition after the 25 weeks of RS was not measured in this experiment, although ethylene production was increased during the warming period. Hexaneextractable α-farnesene and CTs were increased by the warming treatments, and although the differences were significant, they were small. Scald decreased as the 20°C warming period after 2 weeks of RS was increased from 1 to 14 days. The greatest decrease was between 1 and 3 days of warming. After 25 weeks of RS, warming had increased ripening as measured by increased ethylene production, greasiness, and decreased firmness. While this effect on warming was significant, it was probably not commercially significant. α-Farnecene and CTs were

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increased (Watkins et al. 1995). Scald developed on fruit that had been stored at 0° or 4°C but none after 10°, 15°, or 20°C. Warming at 10° and 20°C after 1–4 weeks RS reduced scald equally without affecting firmness, although ethylene production was increased. Fruit conditioned by holding it at 10°C for 5–10 days before RS developed severe scald and there were no significant changes in α-farnesene or CTs. Interrupting the RS for 20 h of ‘Granny Smith’ from France reduced scald to the level of DPA-treated fruit after 4 months of RS and 1 day at 20°C; however, as the time at 20°C increased, so did scald incidence and severity (Bauchot et al. 1995). After 6 months of RS, there was no effect of warming on scald on Spanish-grown ‘Granny Smith’. The effects of intermittent warming on scald incidence on some other cultivars were inconsistent. Scald was reduced on warmed New Zealand ‘Granny Smith’ (1994) and on New Zealand ‘Delicious’ (1993) (Watkins et al. 2000). On Massachusetts ‘Cortland’ (1992) and ‘Delicious (1992), New Zealand ‘Delicious (1994) and ‘Pacific Rose’ (1994) there were small, but often significant, differences between warming treatments. Never was the level of scald acceptable. Increasing the length of the warming time from 0 to 9 days decreased scald on all cultivars except ‘Cortland’, where scald was increased. A prewarming RS of at least 9 days was required for the warming treatments to be effective. Since overall scald on the control ‘Cortland’ was 97% and 92%–98% on the ‘Delicious’, these fruit may have been too susceptible to test any control method. Softening of warmed fruit was excessive only for ‘Cortland’, all of which were unacceptably soft after 20 weeks of RS. Warming of ‘Granny Smith’ from the Aukland region of New Zealand reduced scald much less effectively than on fruit from the Hawkes Bay region. Because the response to intermittent warming and preconditioning differs so much between cultivars, regions, and years, the question of whether scald is a chilling injury cannot be answered clearly at this time. C. Storage Atmosphere Several studies in the last 10 years have confirmed that the effects of storage atmosphere on scald incidence are cultivar specific and probably related to preharvest environment because of annual variations (Ingle and D’Souza 1989). ‘Delicious’ apples from Oregon stored in 0.5% O2 developed ribbon-like depressions that may have been related to low temperature as well as low O2 (Chen et al. 1985). Washington and British Columbia fruit showed little of that injury. Scald was satisfactorily suppressed at 1.0% O2 except when harvest was “before commercial

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maturity.” These studies reinforce the influence of location and preharvest conditions on scald development. British Columbia ‘Delicious’ tolerated 0.7% O2, as judged by the absence of skin purpling and alcoholic taste (Lau 1997a). Scald incidence was significantly less at 0.7% O2 than at 1.5% O2, although in some early harvests scald incidence was unacceptable. Scald incidence fell to nearly zero as the O2 concentration was reduced from the conventional 12% O2, 9.0% CO2 to 1.0% O2 or 0.4% O2 plus 5.0% CO2. There was, however, CO2 injury that varied between years and was therefore ascribed to unidentified environmental and orchard conditions (Dover 1997). Scald on five strains of ‘Cortland’ grown in Nova Scotia was unrelated to O2 concentrations from 1.5 to 4.5% or to 0°C or 3.5°C (DeEll and Prange 1998). Initial low oxygen stress (ILOS) at 0.04% O2 for 10 days on ‘Delicious’ before 9 months of storage in 0.7 or 1.5% O2 reduced scald significantly in 1990 but not in 1991 (Lau 1997b). The reduction in 1990 was judged not to be commercially significant. There were small but sometimes significant increases of scald in fruit treated with ILOS. Other cultivars have shown reduced scald after ILOS (Ingle and D’Souza 1989). D. Ethylene Since scald occurrence is usually less in later harvested, more mature, riper fruit, it might be logically assumed that there would be less scald on ethephon-treated fruit. The findings from experiments reported before 1989 (Ingle and D’Souza 1989) and after are inconsistent and contradictory, which is not surprising since there is little in common in the methods used. Concentrations and intervals between application and harvest have differed considerably. Application of 0.5 g/L–1 ethephon to ‘Cortland’ apples from the 1989 crop year at Belchertown, Massachusetts reduced scald incidence from 87% to 81% when fruit was harvested on Sept. 6, 21 days after spraying, and from 96% to 67% when fruit was harvested 7 days later when 52 and 62 hours <10°C had accumulated at the two harvests. In 1990, harvests were 16 and 21 days after application and the reductions were from 97% to 92% and from 99% to 96%, respectively. No hours <10°C had accumulated before either harvest. Concentrations of 0.25 and 0.50 g L–1 significantly increased the starch index and internal ethylene concentration at harvest, indications that ripening had been induced. The ethephon effects were significant at P<0.01, even though the differences were usually small (Barden and Bramlage 1994a). The scald reduction in the second 1989 harvest was attributed to the combination of increased ripening and the marginally greater

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number of hours <10°C, whereas there were no hours <10°C before either of the harvests in 1990, when the differences in scald between treated and nontreated were small although statistically significant (Barden and Bramlage 1994a). Greater scald reductions were observed when lower concentrations of ethephon was applied to ‘Fuji’ and ‘Granny Smith’ in Washington State (Curry 1994). The greatest effect was a reduction to 35–45% of control following application of 0.40 g L–1 5–6 weeks before harvest. 0.20 g L–1 was nearly as effective, but 50 mg L–1 and 100 mg L–1 only reduced scald to 75%–80% of control. Fruit condition at harvest was not affected, with only sporadic, inconsistent, statistically significant changes in firmness, soluble solids, starch, or acidity. Nothing was said of preharvest temperature history. Ethylene concentration in reduced oxygen (controlled atmosphere) storage have been observed to influence scald. When the ethylene concentration was reduced from 500 to 1 µL–1 in flow-through systems, the scald severity on ‘Delicious’ was decreased substantially (Liu 1977, 1986). With ‘Granny Smith’ there was no significant reduction in scald with the removal of ethylene to maintain concentrations between 0.4 and 1 µl L–1 compared to 25 and 71 µl L–1 (Chellew and Little 1995). The effect of ethylene was independent of O2 concentrations from 1.3 to 3.1%. A regression equation fitting scald incidence to O2 partial pressure was not made more accurate when ethylene was added. When storage atmosphere ethylene was reduced to 0.05 µl L-1 with a catalytic converter, scald was nearly absent from ‘Bramley’ apples after 5.5 months storage in 12% O2 and 9% CO2 (Dover 1997). The reduction was not as great as with DPA treatment. Above 1 µl L–1 there was little scald control. The effect of near complete ethylene or other volatiles (αfarnesene, CTs) removal may be greater in ‘Bramley’ than in other cultivars because they are harvested before internal ethylene has begun to accumulate so that the fruit will remain bright green throughout storage. Low oxygen (0.4–1.0%) and 5.0% CO2 reduced scald to <10% and maintained acceptable color and condition, but caused CO2 injury. E. Scald-inhibiting Materials Diphenylamine is the only remaining antioxidant scald inhibitor for apples, since ethoxyquin is now registered only for pears. In a study of the fate of 14C DPA in ‘Delicious’ apples, 41% of the applied radioactivity was found in intact DPA after 40 weeks RS (Kim-Kang et al. 1998). Most of that activity was in the peel and methanol wash of whole fruit,

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although there was a small amount in the pulp. The principal metabolites (37% of that applied radioactivity) were the glucose conjugate of 4-hydroxydiphenylamine and oligosaccharide conjugates of 2-, 3-, and 4-hydroxydiphenylamines. These were largely confined to the pulp. There were relatively small concentrations of the 2-, 3-, and 4-hydroxydiphenylamines. It is probable that the DPA is hydroxylated, then glycolsylated. There has been a continuing search for alternative scald inhibitors (Ingle and D’Souza 1989). The effects of alcohol vapors on ‘Granny Smith’ apples have been investigated (Ghahramani and Scott 1998a,b; Ghaharani et al. 1999). Scald was almost completely suppressed by ethanol, propan-1-ol, butan-1-ol, and pentan-1-ol at >0.03 mole kg–1. Hexan-1-ol was less effective, while α-terpineol was least effective. Volatilization into the bag atmosphere and uptake by fruit of the lower alcohols was demonstrated. Generally, as scald decreased, internal browning increased. In addition to that injury, problems with legalization, particularly of ethanol, and application methods need to be solved. α-Farnesene and CTs were reduced in fruit exposed to ethanol, propan1-ol, butan-1-ol, and hexan-1-ol. No explanation for these reductions was found, but they may account at least in part for the scald reductions. Ethanol dips did reduce scald to acceptable levels (Chellew and Little 1995). The addition of ascorbyl palmitate had no effect. Semperfresh, the trade name for a coating that is a mixture of sucrose carboxymethylcellulose ester and glycerides of fatty acids (Chellew and Little 1995) has affected scald development erratically. Scald was reduced on ‘Bartlett’ and ‘d’Anjou’ pears by as much as 80% after 4 months storage and 40% after 6 months, but this may not be commercially acceptable (Meheriuk and Lau 1988). Semperfresh alone reduced scald from 61% to 21% on Australian ‘Granny Smith’ after 43 weeks in CA but had no significant effect after 29 weeks of RS (Chellew and Little 1995). Semperfresh did improve the scald-reducing effects of low concentrations of DPA. Treatment of ‘Delicious’ and ‘Golden Delicious’ from Italy and ‘Granny Smith’ from France and Spain with a formulation of ascobyl palmitate or n-propyl gallate, alone or together with Semperfresh, delayed scald development, reducing scald after 3–4 months of RS but not after 6 months of RS or CA (Bauchot et al. 1995). Scald developed rapidly after removal from RS and the reductions were not comparable to those on DPA-treated fruit. None of the scald levels in any of the Semperfresh treatments could be considered acceptable control.

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V. A MODEL OF SCALD DEVELOPMENT The proposition stated at the beginning of this review and generally accepted in the literature is that scald is initiated as injury to hypodermal cells by the oxidation products of α-farnesene. A useful model of scald initiation and development should place most of the facts summarized so far in a cause-and-effect sequence. As this has been attempted, contradictions and missing facts have appeared. A few citations have been included to remind the reader of key findings. It has been mentioned that as early as 1970 the correlation between α-farnesene and scald was found not to be significant (P<0.05), while there was a significant (P< 0.01) with CT269 (Huelin and Coggiola 1970b). For ‘Cortland’ apples, the correlations between α-farnesene and scald incidence did not become significant until fruit had been in storage for 4 months, and then only if the α-farnesene had been measured at least after 3 months of storage (Meir and Bramlage 1988).The r was less than 0.526, which means that only 28% of the variation in scald was explained by α-farnesene. The correlations with CT scald incidence were consistently significant from 2 to 5.5 months of storage. The highest r was 0.755, accounting for 55% or less of the scald incidence variation. Correlations between OD200, which appear to be the absorbance of lipophilic natural antioxidants, were significant throughout storage and were particularly high when measured at harvest and increased as storage time was extended. In the peel of West Virginia ‘Rome’ apples harvested 162 DAFB there was no correlation of α-farnesene, CT281, or OD200 and scald severity after RS (D’Souza 1991). α-Farnesene may not be the compound that causes cellular damage, but its concentrations are still important because it is the precursor of the CTs, which are more closely related to scald incidence and severity. If the natural antioxidants are important in the regulation of scald by inhibiting the conversion of α-farnesene to CTs, they should increase during maturation on the tree as scald susceptibility decreases and decline during storage when scald is induced and as it develops. It has been seen that antioxidant activity can be measured in several ways. OD200 is a non-specific estimate that has been reported to increase from 1.14- to 9-fold over 4 weeks of maturation in both ‘Cortland’ and ‘Delicious’ (Meir and Bramlage 1988; Barden and Bramlage 1994c). Lipidsoluble antioxidants increase considerably with maturity, while the increase in water-soluble antioxidants is more modest (Barden and Bramlage 1994b,c). During storage, all of the antioxidant activities except

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α-tocopherol and carotenoids in ‘Cortland’ decline, while in ‘Delicious’ all increase except water-soluble reducing capacity and glutathione, which decrease (Barden and Bramlage 1994b,c). High correlations between OD200 and scald have been noted (Meir and Bramlage 1988). Similarly, it was found that OD200 and lipid oxidation accounted for large percentages of the nonreplicate variance of scald (Barden and Bramlage 1994c). Total water reducing capacity accounted for about one-half as much of the variance, while the specific antioxidants α-tocopherol, carotenoids, ascorbic acid, and glutathione each accounted for small percentages. Perhaps it is the total antioxidant capacity that influences αfarnesene oxidation. Even if α-farnesene oxidation were confined to the cuticle, which is certainly not established, it still wouldn’t be known whether lipophilic or hydrophilic antioxidants would be more effective because the cuticle grades from a very lipophilic exterior to a somewhat hydrophilic layer adjacent to the cell wall where cutin and wax blends with lamellar and cell wall carbohydrate. At present it is difficult to assess the relation of phenolics to scald development. When peel was extracted with acidic methanol and the constituents measured spectrophotometrically, simple phenolics were positively correlated, anthocyanins negatively correlated, and flavenoids not correlated with scald (Ju et al. 1996). It was suggested that phenolics and flavenoids were somehow involved in the initiation and development of scald, either as toxic compounds or as substrates for the browning reactions. When phenolics were extracted from apple fruit cuticle and it was shown that at least some of those phenolics could inhibit linoleic acid oxidation and the oxidation of α-farnesene in a hexane solution, it was suggested that the phenolics might contribute to the total antioxidant capacity of the peel, including the cuticle. The phenolic ρ-coumaric acid has been found bound to the cutin of apple cutin (Riley and Kolattukudy 1975). Antioxidant activity of any compound may depend on its behavior at water-oil or air-oil interfaces (Frankel et al. 1994). Phenolics may inhibit α-farnesene oxidation in the cuticle and in cells, which have most of the lipid-soluble antioxidant activity (Ju and Bramlage 1999) as well as substrates for the browning reactions. A model for the regulation of scald initiation and development should place at least most of the facts summarized so far into a cause-and-effect sequence. As that has been done, contradictions and missing facts have appeared. These oblige the reviewer to supply unique speculations that are sometimes hard to separate from fact. The steps or elements of the following sequence could be arranged in other ways and most probably

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additions could be made. Just a few citations are included to remind the reader of key findings. A. Step 1 α-Farnesene is synthesized from acetyl CoA in hypodermal and perhaps epidermal cells (Rupasinghe et al. 1998, 2000). Concentrations of αfarnesene are low at the time of commercial harvest but increase through the harvest window; however, a low amount of α-farnesene has been recovered from the surface of ‘Rome’ apples at least as early as 83 DAFB (D’Souza 1991). The increase in α-farnesene late in maturation or early in ripening may be influenced by endogenous ethylene since the rise of each roughly coincide and α-farnesene concentrations are higher when storage atmospheres are high in ethylene (Du and Bramlage 1993; Barden and Bramlage 1994a,b). Also, ethephon applications before or after harvest are followed by increased cuticle and peel α-farnesene concentrations without consistent associations with scald development. It is not clear why α-farnesene accumulation ends after 50–60 days of RS. There are several reasons to doubt that α-farnesene concentrations are directly related to scald development. The poor correlations between αfarnesene and scald have been cited (Huelin and Coggiola 1970b; Meir and Bramlage 1988). Rupasinghe et al. (1998) found lower levels of αfarnesene in nonscalded or scald-developing peel tissue than in scaldfree peel of ‘Delicious’. There were no significant differences in α-farnesene concentrations between the two types of tissue from ‘Rome’, ‘Stayman’, ‘York’, ‘Granny Smith’, and several budsports of ‘Delicious’ (D’Souza 1991). Generally, there seems to be no relation among cultivars between α-farnesene and scald susceptibility (D’Souza 1991; Rao et al. 1998; Rupasinghe et al. 2000). There was the same lack of relationship for in vivo α-farnesene synthase activity (Rupasinghe et al. 2000). B. Step 2 α-Farnesene is autoxidized to conjugated triene(s) (Anet 1969). It is unclear how many CTs there are or the sequence of their formation. It was once proposed that the three absorbance peaks at OD258, OD269, and OD281 represented three CTs (CT258, CT269, and CT281) with different physiological or metabolic activities (Du and Bramlage 1993). It was proposed that CT281 promoted scald, while CT258 retarded scald, and that the CT258/CT281 ratio was more indicative of scald susceptibility than the concentration of an individual CT. In the ‘White Angel’ × ‘Rome

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Beauty’ selections, poor relationships were found between scald and αfarnesene, CTs, or the ratios of CTs. The most recent and rigorous structural studies suggest that there is one principal CT with one minor (Rowan et al. 1995; Whitaker et al. 1997). MHO as well can be released from apple fruit surfaces and may be a CT oxidation product (Mir et al. 1999). Cell membranes, primarily the tonoplasts, may be damaged by αfarnesene oxidation products to the extent that they are no longer selectively permeable and vacuolar contents are released into the cytoplast. This is speculation. That the CTs or MHO are responsible for the membrane damage and the commencement of scald symptom development is based entirely on statistical correlations or deduced from sequences of changes, although one attempt has been made to determine whether applied α-farnesene or CTs initiate scald development (Huelin and Coggiola 1970b). α-Farnesene was prepared by immersing immature ‘Granny Smith’ apples in hexane. These extracts showed no absorbance for CT. The α-farnesene was transferred to ethanol and the solution was applied to other fruits. Fruit treated with freshly prepared α-farnesene did not scald earlier than untreated fruit; however, scald did develop more rapidly on fruit treated with the solution that had been exposed to oxygen until one-half of the α-farnesene had been oxidized, presumably determined by decreased absorbance at 232 nm. More experiments are needed on the influence of CT treatments on apple peel in addition to that one done 30 years ago. Phenols could also be factors in scald initiation (Ju and Bramlage 1999). When peel was extracted with acidic methanol and the constituents measured spectophotometrically, simple phenols were positively correlated, anthocyanins negatively correlated, and flavonoids not correlated with scald (Ju et al. 1996). It has been suggested that phenolics might be involved in scald development, either as toxic compounds or as substrates for the browning reactions. When phenolics were extracted from apple fruit cuticle, some of those phenolics inhibited linoleic acid oxidation and the oxidation of α-farnesene in hexane solution. It was suggested that the phenolics contribute to the total antioxidant activity of the peel, including the cuticle. The phenolic ρcoumaric acid has been found bound to the cutin of apple (Riley and Kolattukudy 1975). Antioxidant activity of any compound may depend on its behavior at water-oil or air-oil interfaces (Frankel et al. 1994). Phenolics may inhibit α-farnesene oxidation in the cuticle and in the cells, which have most of the lipid-soluble antioxidant activity (Ju and Bramlage 1999) as well as substrates for the browning reactions. Membranes apparently do not become susceptible to injury by CTs, MHO, or something else until after at least 60 days of RS, which could

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qualify scald as a senescence disorder. Apples and pears have not been observed to develop scald unless they have been exposed to <5°C, although high levels of α-farnesene and CTs are accumulated at high temperatures (Watkins et al. 1995; Du and Bramlage 1994). Work on the effects of interrupted RS on scald development on ‘Granny Smith’ lead to the proposition that scald is a form of chilling injury (Watkins et al. 1995). That may not be generally true since not all cultivars respond to intermittent warming, as does ‘Granny Smith (Watkins et al. 2000). Changes in membrane composition have been studied in tropical crops, in which chilling injury is more widely recognized. The chlorophyll fluorescence parameters, minimal and maximal fluorescence, photosynthetic efficiency, and photosynthetic fluorescence quenching declined with time in RS in scald susceptible ‘Cortland’ and ‘Delicious’, while the changes in scald resistant ‘Empire’ were slight. These changes seemed not to be directly related to scald susceptibility but rather are coincidental (Mir et al. 1998a,b). It is not known why CTs do not begin to accumulate until after harvest and storage. The site of α-farnesene oxidization or the required oxygen tension are not known. Oxidation may be confined to the cuticle, requiring the outward transport of αfarnesene and inward transport of CT. Since the concentration of α-farnesene always greatly exceeds CT concentrations, for some reason it seems that only a small fraction of α-farnesene is oxidized to CTs. It is difficult to estimate the conversion since synthesis, oxidation, and volatilization are continuous. Since the relation of scald to α-farnesene and the CTs have been found to be poor in some studies (Meir and Bramlage 1988; Rao et al. 1998), an alternate method of membrane damage may be considered. Polyunsaturated fatty acids (PUFA), either free or as glycerides, may be peroxidized nonenzymatically by the active oxygen species (AOS), superoxide, hydrogen peroxide, hydroxyl radical, or singlet oxygen or enzymatically by lipoxygenase (Anderson 1995). The AOS have glycerides as substrates and produce fatty acid hydroperoxide radicals that can propagate the reactions to oxidize several (hundreds) of additional PUFA molecules (Shewfelt and Purvis 1995). Antioxidants, such as α-tocopherol, ascorbate, and glutathione, serve as protectants, stopping peroxidation by scavenging free radicals. During the development of bean hypocotyls and carnation petals, microsomal membranes release lipoprotein particles (“bleebs”) that contain peroxidized PUFAs (Hudak et al. 1995). A mechanism of peroxidation is suggested. As these tissues senesce, bleeb production decreases, presumably allowing the hydroperoxides to accumulate in the membranes, which they can damage. Superoxide is degraded to hydrogen peroxide by superoxide dismutase (SOD), which

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in turn is degraded to water and oxygen by catalase or peroxidase. SOD activity could be regulated by compartmentation (Shewfelt and Purvis 1995). At harvest and through RS, the scald susceptible cultivar ‘Cortland’ had 2- to 3-fold higher total SOD activity than the resistant ‘Empire’ (Du and Bramlage 1994). ‘Delicious’, which is also susceptible, contained considerably less SOD activity than the other cultivars. What happened to the hydrogen peroxide in these apple peels was not indicated. There was an inconsistent downward trend in SOD activity with time at 0°C. Scalded and nonscalded peel areas had equivalent SOD activities, and activity was not affected by temperature or DPA. Higher hydrogen peroxide concentrations were found in the scald-susceptible selections of ‘White Angel’ × ‘Rome Beauty’ crosses than in the resistant selections (Rao et al. 1998). The resistant types had more guaiacol peroxidase and catalase activity than the susceptible types. Both groups of apples had about the same concentrations of carbonyl groups, indicating equal protein degradation. From these data it was concluded that resistance is a function of a genotype’s ability to lower AOS. It is not clear whether superoxide or hydrogen peroxide is responsible for peroxidation so that it is difficult a priori to associate scald susceptibility with high or low SOD activity. Lipoxygenase, which hydroperoxidizes unsaturated fatty acids, may be a factor in scald initiation (Feys et al. 1980). Activity has been extracted from the outer portion of the fruit, including the peel, of ‘Schone van Boskoop’ apples. There was slow but steady increase during storage at 3.5°C. Membrane injury would have to be from lipid hydroperoxides since lipoxygenase does not form free radicals and uses only free fatty acids as substrates (Shewfelt and Purvis 1995); examples of that type of injury have not been found. If membrane damage is a critical step in scald development, it is probably from the combined effects of CTs, MHO, AOS, and lipoxidase. C. Step 3 Released vacuolar contents are converted to colored materials. It is guessed that oxidation of simple phenols is catalyzed by polyphenoloxidase, which seems to be localized in the grana of apple peel chloroplasts (Murata et al. 1997). Histochemical assays showed the formation of brown pigments when cell suspensions were supplied with ortho-diphenol. The speculations about cell discoloration during scald development now has some experimental support and fits rather well

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with the changes in chlorophyll fluorescence, suggesting chloroplast structural changes accompanying scald development. D. Step 4 Cells collapse in an irregular pattern and the scald lesions are formed. It is not known why not all the fruit in a sample, box, bin, or indeed a block of trees are affected. Further, it is also not known why not all of the surface of the fruit is affected. Unequal distribution around fruit of the compound(s) that initiate and perhaps expand the lesions and of the naturally occurring antioxidants might be suspected but not much data has been found to support or refute that proposal. It has been cited that the blushed side of the red cultivars ‘Delicious’ and ‘Nittany’ contained more α-farnesene than the nonblushed side but there were no differences in CTs or antioxidants as measured by OD200 (D’Souza 1991).There were no differences in the red cultivars ‘Criterion’ and ‘York’ or the non-reds ‘Granny Smith’ or ‘Golden Delicious’. If anthocyanins were effective antioxidants, it would seem that there would be less CTs on the blushed side. Cyanidins may inhibit the oxidation of linoleic acid in vitro (Tsudsa et al. 1994) but may be separated from α-farnesene in apple peel. It seems that no one has determined the range in concentration in αfarnesene, CTs, antioxidants, phenolics, and pigments between individual fruit from a tree or block. VI. PROSPECTS A. Predicting Scald The goal of much research on scald has been to devise a method for predicting scald incidence and severity at harvest so that the minimum concentration of DPA could be selected or that fruit could be harvested at a time when scald after storage would be at a minimum. Minimizing the DPA rate would reduce treatment costs and largely avoid the risk of residues above the legal tolerances. Delaying harvest until the risk of scald is very low is fairly easy, but by that time fruit ethylene production and softening may leave fruit with insufficient shelf life after storage. Prediction methods are based on relating some fruit or environmental variable at harvest or early in storage to storage and poststorage scald development. Some of the variables that have been suggested and studied include fruit maturity, preharvest temperatures, natural antioxidant levels, fluorescence, and α-farnesene and CT concentrations.

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1. Maturity. Scald susceptibility usually decreases as harvest is delayed. As fruit develops or matures on the tree, ethylene production and internal ethylene concentration increase, starch disappears, soluble solids (mostly sugars) concentration increases, firmness decreases, chlorophyll decreases, and red pigments increase in some cultivars. The environment is also changing. Temperature decreases, measured as daily means, maxima, and minima. Daylength and therefore light exposure decreases. All of these changes are correlated but there are yearly magnitude differences. Internal ethylene concentration or production rates and starch content are the only maturity or ripeness measures that have received significant attention. Starch measurement is probably the only method for commercial use, because ethylene measurements are slower and require sophisticated apparatus. Neither of the methods account for enough scald variability to be used as the sole variable in a model, although it can add precision when used in conjunction with other variables (Barden and Bramlage 1994a; Bramlage and Watkins 1994; Bramlage and Weis 1997). 2. Chlorophyll Fluorescence. The changes in chlorophyll fluorescence were seen to be related to fruit senescence and only indirectly to scald, which give these measurements low potential for predicting scald after storage (Mir et al. 1998a,b); however, if scald indeed is a senescence injury, perhaps combined with chilling injury, further studies with this method may be fruitful. 3. Preharvest Temperatures. Preharvest temperatures have worked well for some cultivars in some climates. In western Massachusetts, days (rather than hours) with at least one temperature <10°C accounted for 31% of ‘Delicious’ scald variability (Bramlage and Weis 1997). Average temperature after Aug. 3 accounted for 16% and starch index only 4%. At a higher minimum temperature, starch made a little greater contribution. Using five variables accounted for no more than 64% of the scald variability. By contrast, at Elgin, Republic of South Africa, average temperature and days <10°C accounted for 22% of the scald variability, while adding starch scores raised the total to 83%. In neither case did rain or sun add more than 5%, and usually less. In Nelson, New Zealand and West Virginia in the United States, average temperatures alone accounted for 70% and low temperatures added another 15%.

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Considering temperatures <10°C or less increased the variability accounted for to 85–87%. Combined data from 10 locations in the northern and southern hemispheres showed correlation coefficients of 0.49 between scald and days after Jan. 10 or Aug. 12 when the coefficients and days were allowed to vary by location; however, the correlation coefficients differed significantly between locations. It seems that scald can be predicted well enough to help make scald inhibitor and storage management decisions from easily measured variables, but formulas will have to be developed for each region (Bramlage and Weis 1997). Using days after Aug. 31, days with at least one temperature <10°C and harvest starch score, formulas have been derived to classify New England ‘Delicious’ as of high scald susceptibility, of intermediate susceptibility, and of low susceptibility (Weis et al. 1998). DPA at 2000 mg L–1 is recommended for the high susceptibility group, 500 mg L–1 DPA for the intermediate susceptibility group, and no DPA for the low susceptibility group. The same group has been unable to develop a similar system for New England ‘Cortland’ (Weis et al. 1998). Formulas will have to be developed for each region (Bramlage and Weis 1997). Preliminary studies in West Virginia strongly suggest that two formulas would be required for the two major apple growing areas in that state, which differ by about 1,000 feet in elevation and topography. The utilization of the observed effects of temperature will be improved when it is known how these conditions work on the molecular basis. The increasing α-farnesene concentrations with accumulating low temperature exposures is probably associated with maturation/ripening. It would seem likely that rather than being involved in α-farnesene oxidation, preharvest low temperature exposures retard membrane senescence or alleviate chilling injury. It would be interesting to see scald development on fruit that had been stored at 5°–10°C for 100 h before RS. If preharvest low temperature exposure acclimates or adapts membranes to longer and lower temperature stress of RS, why does intermittent high temperature exposure reduce scald severity? It may be that the fluidity of membranes is increased so that they are less susceptible to chilling injury or resistant to CT or MHO injury. There is some evidence that preharvest temperatures >25°C or 30°C sometimes contribute to scald resistance (Bramlage and Weis 1997). There is clearly a need for studies of membrane composition and permeability after several temperature, ethylene, and antioxidant treatments similar to those done on tissues like senescing carnation petals (Fobel et al. 1987).

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B. Future Research Scald initiation and development seem to be regulated by several internal and external variables. The principal internal variables are genetic. Thus, the objective of research must be to understand scald at the molecular level. Any prediction model that accounts for at least 70% of scald variability will have to include more environmental variables than have been used so far. Hopefully, this review has suggested where research efforts should be focused. Prediction models will surely be unique for cultivars and climatic regimes. These prediction systems are important because pressure is increasing to reduce chemical and genomic manipulation of foods. A series of question remain to be answered. Why do some cultivars develop so little scald even though they produce αfarnesene and conjugated trienes? What are the pathways and enzymes that produce α-farnesene and conjugated trienes? How does the environment regulate the production, metabolism, and cellular effects of these compounds as well as the endogenous antioxidants? What is the molecular basis of scald reduction by pre-RS high temperature exposure and low-oxygen storage? Enhanced knowledge should improve development of control methods, including prediction, that could greatly reduce or eliminate the use of chemical additives.

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Merritt, R. H., W. C. Stiles, A. V. Havens, and L. A. Mitterling. 1960. Effects of preharvest air temperatures on storage scald of Stayman apples. Proc. Am. Soc. Hort. Sci. 78:24–35. Mir, N. A., and R. Beaudry. 1999a. Effects of superficial scald suppression by diphenyamine application on volatile evolution by stored Cortland apple fruit. J. Agr. Food Chem. 47:7–11. Mir, N. A., R. Perez, and R. M. Beaudry. 1999a. Chlorophyll fluorescence and whole fruit senescence in ‘Golden Delicious’ apple. Acta Hort. 464:121–126. Mir, N., R. Perez, and R. M. Beaudry. 1999b. A poststorage burst of 6-methyl-5-hepten-one (MHO) may be related to superficial scald development in ‘Cortland’ apples. J. Am. Soc. Hort. Sci. 124:173–176. Mir, N., M. Wendorf, R. Perez, and R. M. Beaudry. 1997. Variable fluorescence quenching in apple storage as a function of O2. In: E. Mitchum (ed.), Proc. 7th Int. Controlledatmosphere Res. Conf. Vol. 2. Apples and pears. Davis, CA. Mir, N., M. Wendorf, R. Perez, and R. M. Beaudry. 1998a. Chlorophyll fluorescence in relation to superficial scald development in apple. J. Am. Soc. Hort. Sci. 123:887–892. Mir, N. A., M. Wendorf, R. Perez, and R. M. Beaudry. 1998b. Chlorophyll fluorescence as affected by some superficial defects in stored apples. J. Hort. Sci. Biotechnol. 73:846–850. Murata, M., M. Tsurutani, S. Hagiwara, and S. Homma. 1997. Subcellular location of polyphenol oxidase in apples. Biosci. Biotech. Biochem. 61:1495–1498. Murray, K. E., F. E. Huelin, and J. B. Davenport. 1964. Occurrence of farnesene in the natural coating of apples. Nature 204:80. Poritt, S. W., and P. D. Lister. 1978. The effect of prestorage heating on ripening and senescence of apples during cold storage. J. Am. Soc. Hort. Sci. 103:584–587. Rao, M. V., C. B. Watkins, S. K. Brown, and N. F. Weeden. 1998. Active oxygen species in ‘White Angel’ × ‘Rome Beauty’ apple selections resistant and susceptible to superficial scald. J. Am. Soc. Hort. Sci. 123:299–304. Riley, R. G., and P. E. Kolattukudy. 1975. Evidence for covalently bound ρ-coumaric and ferulic acid in cutins and suberins. Plant Physiol. 56:650–654. Rowan, D. D., J. M. Allen, S. Fiedler, J. A. Spicer, and M. A. Brimble. 1995. Identification of conjugated triene oxidation products of α-farnesene in apple skin. J. Agr. Food Chem. 43:2040–2045. Rupasinghe, H. P. V., G. Paliyath, and D. P. Murr. 1998. Biosynthesis of α-farnesene and its relation to superficial scald development in ‘Delicious’ apples. J. Am. Soc. Hort. Sci. 123:882–886. Rupasinghe, H. P. V., G. Paliyath, and D. P. Murr. 2000. Sesquiterpene α-farnesene synthase: Partial purification, characterization, and activity in relation to superficial scald development in apples. J. Am. Soc. Hort. Sci. 125:111–119. Shewfelt, R. L., and A. C. Purvis. 1995. Toward a comprehensive model for lipid peroxidation in plant tissue disorders. HortScience 30:213–218. Spicer, J. A., M. A. Brimble, and D. D. Rowan. 1993. Oxidation of α-farnesene. Austral. J. Chem. 46:1929–1939. Trutter, A. R., J. C. Combrink, and S. A. Burger. 1994. Control of superficial scald in ‘Granny Smith’ apples by ultra-low and stress levels of oxygen as an alternative to diphenylamine. J. Hort. Sci. 69:581–587. Tsuda, T., M. Watanabe, K. Ohshima, S. Norinobu, S-W. Choi, S. Kawakishi, and T. Osawa. 1994. Antioxidative activity of the anthocyanin 3-O-B-Dglucoside and cyanidin. J. Agr. Food Chem. 42:2407–2410.

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Vogeli, U., J. W. Freeman, and J. Chappell. 1990. Purification and characterization of an inducible sesquiterpene cyclase from elicitor-treated tobacco cell suspension cultures. Plant Physiol. 93:182–187. Wang, C. Y. 1994. Chilling injury of tropical horticultural commodities. HortScience 29:986–988. Wang, H., G. Cao, and R. L. Prior. 1996. Total antioxidant capacity of fruit. J. Agr. Food Chem. 44:701–705. Watkins, C. B., C. L. Barden, and W. J. Bramlage. 1993. Relationships between alphafarnesene, ethylene production and superficial scald development of apples. Acta Hort. 343:155–160. Watkins, C. B., W. J. Bramlage, P. L. Brookfield, S. J. Reid, S. A. Weis, and T. F. Alwan. 2000. Cultivar and growing region influence efficacy of warming treatments for the amelioration of superficial scald development on apples after storage. Postharvest Biol. Technol. 19:33–45. Watkins, C. B., W. J. Bramlage, and B. A. Cregoe. 1995. Superficial scald of ‘Granny Smith’ apples is expressed as a typical chilling injury. J. Am. Soc. Hort. Sci. 120:88–94. Weis, S. A., W. J. Bramlage, and W. J. Lord. 1998. An easy and reliable procedure for predicting scald and DPA requirements for New England Delicious apples. Fruit Notes 63:1–8. Whitaker, B. D., T. Solomos, and D. J. Harrison. 1997. Quantification of α-farnesene and its conjugated trienol oxidation products from apple peel by C18 HPLC with UV detection. J. Agr. Food Chem. 45:760–765

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7 Health Functional Phytochemicals of Fruit Wilhelmina Kalt* Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, Kentville, Nova Scotia, Canada B4N 1J5

I. INTRODUCTION A. Oxidative Damage of Biomolecules B. Cancer C. Cardiovascular Disease D. Major Fruit Phytochemicals 1. Phenolics 2. Carotenoids II. CITRUS A. Phenolics 1. Composition and Localization 2. Anti-cancer Effects 3. Cardiovascular Protection 4. Anti-inflammatory Effects 5. Other Effects B. Carotenoids C. Other Components 1. Liminoids 2. Vitamin C 3. Fiber 4. Limonene 5. Folic Acid

*Contribution No. 2215. The author is grateful for the careful review of this manuscript by A.-M. Connor, M.D., University of Minnesota, and C. McRae, M.Sc.

Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 269

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III. GRAPES AND WINE A. Phenolics B. Evidence of Health Benefits 1. Anti-cancer Effects 2. Cardiovascular Protection 3. Antioxidant Activity IV. VACCINIUM A. Phenolic Components B. Health Properties Attributed to Bilberry C. Anti-cancer Activity D. Neurological Function E. Urinary Tract Health F. Antioxidant Activity V. OTHER FRUITS A. Strawberries B. Apples C. Melons D. Other Fruits VI. CONCLUSIONS LITERATURE CITED

I. INTRODUCTION The notion that dietary choices play a role in human health is not new. In the 5th century B.C. Hippocrates, the father of modern medicine, suggested “Let your food be your medicine and your medicine be your food.” In spite of this early and insightful recommendation, and our appreciation for the complex multi-component nature of foods, most of the research in human nutrition during the 20th century has focused primarily on a relatively small group of traditional food nutrients. This interest in “traditional nutrients” is justified since these food components are essential to the diet to prevent deficiency-related diseases (e.g., scurvy). Each of the essential nutrients has an established Recommended Daily Allowance (RDA) (Beecher 1999a). Substantial epidemiological evidence links fruit and vegetable consumption to a decreased risk of various degenerative diseases (Block et al. 1992; Steinmetz and Potter 1996), but this cannot be accounted for by the consumption of the essential nutrients contained in these foods (Beecher 1999a). Significant effort is now being devoted to investigating fruit and vegetable phytochemicals, and the responses that these food phytochemicals elicit in human metabolism, leading to improvements in human health. The term “phytonutrients” has been used to describe health-promoting fruit and vegetable phytochemicals (Beecher 1999a), while “chemoprotec-

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tion” and “chemoprevention” describe the action of dietary components in protecting against or preventing disease. Block et al. (1992) show that fruit and vegetable consumption had a protective effect against various cancers in 129 of 172 epidemiological studies. In a similar review of 206 human epidemiological studies, Steinmetz and Potter (1996) document an association between the consumption of fruits and vegetables and a reduced risk of several forms of cancer. With respect to cardiovascular disease, the “French paradox” is a widely cited example of the protective effects of specific dietary phytochemicals. France has a lower incidence of cardiovascular disease (CVD) than other populations with similar diet and lifestyle risk factors (e.g., high fat diets, smoking). Extensive research has led to broad agreement that it is the consumption of red wine by the French that is responsible for their lower incidence of CVD (Renaud and DeLorgeril 1992). Evidence from in vitro and in vivo studies indicate that phenolic phytochemicals of grapes possess several properties that are responsible for the cardio-protective quality of wines (Frankel et al. 1993; Kanner et al. 1994). Among the various components found in fruit, phenolic components have probably received the greatest attention with respect to possible health-promoting properties, although carotenoids, folate, fiber, and essential nutrients continue to be investigated. It is widely accepted that fruits play an important role in human nutrition. While they contribute relatively little to the protein and dry matter requirements of the human diet (Allard 1960), they are rich in both essential nutrients and phytonutrients. This article will address the chemistry and bioactivity of fruit phytonutrients. While the essential nutrients of fruit will only be discussed in cases where their role is relevant to the action of phytochemicals, it is understood that many types of fruit possess health-promoting properties due to their content and profile of particular phytochemicals (Tables 7.1 and 7.2). Information on the phytonutrient content of fruits will continue to be updated, as new and improved techniques for their analysis are developed (Song et al. 2000; Merken and Beecher 2000; Holden et al. 1999; Beecher 1999b). Two significant results of recent research are: (1) the overwhelming evidence that oxidative damage to biomolecules is a fundamental element in the development of the degenerative diseases of aging, and (2) that the antioxidant properties of certain plant phytochemicals are of paramount importance to their health-promoting properties (Ames et al. 1993; Halliwell 1996; German et al. 1997). Since these themes occur

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272 Table 7.1.

W. KALT Composition of fruits per 100 g FW edible portion (Nicholson 1997). Vitamins

Fruit Apple ‘Cox’s Pippin’ ‘Golden Delicious’ Apricot Banana Bilberry Blackberry Black currant Cherry Cranberries Damson Gooseberry Grapes Grapefruit Kiwifruit Lemon Lime Melon, cantaloupe Melon, ‘Honeydew’ Orange, assorted Peach Pear Plum Prune Quince Raisin Raspberry Red currant Strawberry Tangerine

Total fiber (g)

{2.2} {1.9} 1.9 3.1 {2.5} 2.0 7.8 1.5 3.8 3.7 2.9 0.8 {1.6} N 4.7 N 0.9 0.8 1.8 2.3 N 2.3 14.5 5.8 6.1 6.7 {4.5} 2.0 1.7

Carotene (µg)

E (mg)

B1 (mg)

B2 (mg)

C (mg)

Folate (mg)

18 15 405 21 30 80 100 25 22 {295} 110 17 17 37 18 12 1000 4.8 28 58 18 295 155 Tr 12 6 25 8 97

0.6 0.6 N 0.3 N 2.4 1.0 0.1 N 0.7 0.4 Tr {0.2} N N N 0.1 0.1 0.2 N 0.5 0.6 N N N 0.5 0.1 0.2 N

0.03 0.03 0.04 0.04 0.03 0.02 0.03 0.03 0.03 0.10 0.03 0.05 0.05 0.01 0.05 0.03 0.04 0.03 0.11 0.02 0.02 0.05 0.10 0.02 0.12 0.03 0.04 0.03 0.07

0.03 0.03 0.05 0.06 0.03 0.05 0.06 0.03 0.02 0.03 0.03 0.01 0.02 0.03 0.04 0.02 0.02 0.01 0.04 0.04 0.03 0.03 0.20 0.02 0.05 0.05 {0.06} 0.03 0.02

9 4 6 11 17 15 200 11 13 {5} 14 3 36 59 58 46 26 9 54 31 6 4 Tr 15 1 32 40 77 30

4 1 5 14 6 34 N 5 2 {3} {8} 2 26 N N 8 5 2 31 3 2 3 4 N 10 33 N 6 21

{ } = estimate; N = significant quantities, but data not reliable; Tr = trace.

repeatedly in the following discussions on specific fruit crops, a brief overview of oxidative damage in human disease, as well as an outline of the etiology of cancer and cardiovascular disease, is presented. A. Oxidative Damage of Biomolecules The role of oxidative damage in the development of disease and the phenomenon of aging is under intensive investigation. It is now widely

y

x

14–33

2.4–2.9

1.3–87 (peel)

0

167–200

Plum

170

85

1.4–3.1

28–70

2.1–17

2.4–9.6

1.8–6.1

0.1–6.2

5.3–14

220–370

v

1.4–52.7 (skin)

110–150

76–150

53

90–120y

32–78 (peel)

17 (peel)

100y 6–20

+y,v y

+y,v

400–3500 ppm cider

Tannins

5–20y

2.0–2.8

0.2–16

Flavan-3-ols

Kalt et al. 1999; Kühnau 1976; Hakkinen et al. 1999; oxoflavonoids (inc. flavones, flavonols, flavanones, flavanonols); + = present, but quantity not reported.

z

Strawberry

2.9x

z

Red Raspberry

2.0–5.2

4.2–160 (peel) <4.0–6.0x

1.9–5.3

5–10 (peel)

0

Red Currant

12–94

w

123–400

Pear

8.1–75

0.4–1.0

28–180

0

Peach

14–16

3.4x

1.9–10

0.81–8.20

50–100y,w

8–388

831

10–109 (peel)

Lingonberry

900–950

Grape, red

Orange

350 (peel)

Grape, white

50y,w

0

x

8.8–13.3

Grapefruit

46–172

160–503

15–23

18–41 (peel)

<0.01x 12–22

188–211

460

10–2160 (peel)

Flavonols

Gooseberry, green

Cranberry

Blueberry

250

6.2–134

Anthocyanins

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Blackberry

381–4651z

50–1100

Apple

Hydroxycinnamic acid derivatives

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Bilberry

Total phenolics

Fruits

Table 7.2. Total and individual phenolic content of various fruit crops. Adapted from Macheix 1990; values in mg/100 g FW, unless otherwise indicated.

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accepted that accumulated damage to DNA, proteins, and lipids by reactive oxygen species (ROS) is a precursor for the development of numerous degenerative conditions, including various cancers, cardiovascular disease, certain neurological diseases, immune system decline, cataracts, and the aging process itself (Ames et al. 1993; Halliwell 1996; German et al. 1997; Cantuti-Castelvetri 2000; Meydani 2000). Despite an extensive array of biochemical mechanisms for defense against ROS, and the repair and replacement of oxidatively damaged molecules, oxidative lesions to biomolecules accumulate. Oxidative damage sustained by the antioxidant metabolic machinery itself will compromise its protective and reparative functions, and degenerative processes may occur as a result of cumulative cellular damage. This, in large part, characterizes the phenomenon of aging (Ames et al. 1993). ROS, including hydrogen peroxide, superoxide anion, hydroxyl radical, as well as various peroxyl radicals, are ubiquitous. Normal metabolic processes give rise to ROS in several ways (e.g., mitochondrial electron transport, peroxisome function, phagocytic activity). It is estimated that in humans the DNA in each cell sustains 10,000 oxidative “hits” per day. External sources such as UV radiation, and xenobiotic agents, such as tobacco smoke and other air pollutants, also result in ROS within cells. Avoiding the external sources of oxidative assault will reduce some of its deleterious effects. Further protection against cellular oxidative damage may be provided by consuming dietary antioxidants. This is an exciting area of research that links epidemiological evidence for health benefits with the consumption of antioxidant-rich fruits and vegetables (Ames et al. 1993; Frei 1994; Halliwell and Gutteridge 1995). B. Cancer The transformation of benign cells to malignant ones may be induced by a number of endogenous and exogenous agents. Examples of exogenous factors contributing to cancer incidence include radiation and certain chemicals, while endogenous factors include particular viruses (oncogenic viruses), steroid hormones, and oxidative damage to biomolecules. Oxidative damage to biomolecules may originate from either endogenous or exogenous sources. Much of our understanding of the stages of cancer development is based on models of chemical carcinogenesis, although cancer induced by other agents shares certain features with the chemical carcinogenesis model. In this model, there are three major stages of cancer development (Fig. 7.1). The first stage, initiation,

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Initiation Fig. 7.1.

Promotion

275

Progression

Three stages in carcinogenesis (Wargovich 2000).

is characterized by damage to DNA. The second stage, promotion, involves tumor proliferation and altered cell differentiation. Initiation and promotion together are responsible for what is termed cancer induction. The third stage, progression, refers to the accelerated growth and increasing aggressiveness that results in the invasion of cancerous cells into surrounding tissues and its spread to other parts of the body (metastasis) (Fig. 7.1). Inhibition of cell division and enhanced cell differentiation are desirable effects against the promotion and progression stages of cancer. The protective role(s) fruit phytochemicals may perform in these three stages is now under active investigation (Wargovich 2000). Various bioassays that detect phenomena or cellular activities characteristic of each stage of cancer are used to examine the effects of specific foods and phytochemicals on cancer metabolism in vitro. Such assays provide one type of evidence of the effects that phytochemicals have on the pathogenesis and etiology of cancer. In vitro studies can provide information regarding mechanisms of actions for phytochemicals in cancer metabolism, but cannot address the issue of bioavailability in the body. Whole animal feeding studies can provide more significant results, since the bioavailability of a phytochemical agent is a prerequisite to its potential chemoprotective action. However, speciesdependent differences are known to occur and must be considered when interpreting results (Byers 2000). Epidemiological evidence is used to assess the association between specific dietary factors and cancer incidence.

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C. Cardiovascular Disease A major determinant in the development of cardiovascular disease is the formation of atherosclerotic plaques in blood vessels. Plaques narrow arteries; reduced blood flow can lead to heart attacks and strokes. In the late stages of development, plaques can be the site of thrombus (clot) formation, which can further obstruct blood flow, sometimes quite precipitously. The development of plaque is strongly influenced by the nature of circulating low density lipoprotein (LDL). Oxidation of native LDL modifies these molecules so that they are preferentially removed from the bloodstream and sequestered by macrophages adherent to, or localized in, the walls of coronary and arterial blood vessel walls. The accumulation of LDL by macrophages causes a substantial increase in macrophage size. Low density lipoprotein also stimulates growth factors and other chemical messengers that result in smooth muscle cell proliferation in the arterial wall plaque, and recruitment of more macrophages into the lesion. This results in the narrowing of blood vessels where the plaques occur (German et al. 1997; Chia 1998). Platelets, non-nucleated components that circulate in the blood, help in the blood clotting process by self-aggregating. In CVD there is a tendency for platelets to aggregate (clump) on the surfaces of atherosclerotic plaques, forming a clot that is incorporated into the plaque. Platelet aggregation that occurs rapidly on the surface of a plaque is frequently responsible for the blockage that precipitates a heart attack or stroke (Chia 1998; German et al. 1997). Fruit components, particularly flavonoids, have been extensively studied with respect to their effects on LDL oxidation and platelet aggregation (Frankel et al. 1993; German et al. 1997; Manach et al. 1996). As with cancer research, evidence for the cardio-protective properties of certain foods and phytochemicals comes from in vitro, in vivo, and epidemiological data. D. Fruit Phytochemicals In addition to essential nutrients, fruit phenolics, and to a lesser extent carotenoids, are the major phytochemical groups of interest with respect to the role of fruit in human health. 1. Phenolics. Phenolics are the most important group of secondary compounds in fruit, both in terms of abundance (Macheix et al. 1990) and potential health benefits. The phenolics are a large and diverse group of compounds that have an aromatic ring bearing one or more hydroxyl

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groups. Various subgroups of phenolics are distinguished by their number of carbons and the basic structure of the carbon skeleton. The hydroxycinnamic acid derivatives and the flavonoids are the most abundant subgroups of phenolic components in fruit (Fig. 7.2). Free hydroxycinnamic acids are relatively rare in fruit, but their esters are common. Chlorogenic acid, which is caffeic acid esterified at C-9 to quinic acid, is a hydroxycinnamic acid ester that is widely distributed in fruit (Macheix et al. 1990). The flavonoids are phenyl-2-benzopyrilium structures that are organized into various subgroups based on the oxidation state of their C ring (Fig. 7.2 and 7.3). These subgroups include anthocyanins, flavonols, flavan 3-ols (catechins), flavanones, and flavones. Anthocyanins and flavonols are widely distributed and occur almost exclusively as glycosides. Flavan 3-ols, also found in many fruit species, can occur in oligomeric forms (proanthocyanidins) and polymeric forms (condensed tannins). Flavanones and flavones are largely restricted to the Family Rutaceae, which includes the genus Citrus (Macheix et al. 1990).

Hydroxycinnamic acid

Flavonoid (Anthocyanidin) Fig. 7.2.

Representative structures of major fruit phenolic components.

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Fig. 7.3.

W. KALT

Chemical structure of major flavonoid subgroups.

Fruit species vary widely in regard to their total phenolic content and their predominant phenolic subgroup (Macheix et al. 1990; Table 7.2). Phenolic components change dramatically as fruit mature (Wang and Lin 2000) and in response to environmental factors, such as light and temperature (Price et al. 1995; Lancaster 1992; Kalt et al. 1999). Fruit phenolics serve various ecological functions; they provide resistance against the effects of mechanical stress (e.g., insect wounding) and biological stress (e.g., fungal infection), while also serving as visual attractants for

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the purpose of seed dispersal (Macheix et al. 1990). Phenolics influence the food quality of fruits by contributing to color, astringency, bitterness, and aroma (Macheix et al. 1990). Research on the healthful properties of the hydroxycinnamates and their ester derivatives is relatively new and has concentrated mainly on antioxidant characteristics (Meyer et al. 1998). In contrast, the effects of flavonoids on human health have been of interest for many years because of their various bioactivities (Cody et al. 1986; Harborne 1993; Manthey and Buslig 1998). Flavonoids, like hydroxycinnamates, are antioxidants; this characteristic is believed to be of great importance to the healthful properties of these compounds. The relationship between flavonoid structure and antioxidant capacity has been investigated (Cao et al. 1997; Rice-Evans et al. 1997). The antioxidant capacity of the flavonoids is influenced by O-dihydroxy structure on the B ring, the 2,3 double bond in conjunction with the 4-oxo function on the C ring, and the presence of both hydroxyl groups in position 3 (C ring) and 5 (A ring) (Fig. 7.3). These features influence the molecule’s ability to capture electrons while retaining stability, thereby preventing the propagation of oxy-free radical chain reactions. In addition to the antioxidant characteristics, other potential healthpromoting bioactivities of the flavonoids include anti-allergic, antiinflammatory, anti-viral, and anti-cancer properties (Cody et al. 1986; Harborne 1993; Manthey and Buslig 1998). Middleton and Kandaswami (1993, 1994) outline the various anti-carcinogenic activities reported for flavonoids, including: (1) induction of detoxification enzymes; (2) inhibition of adduct formation between xenophobic agents and DNA; (3) inhibition of tumor promotion; (4) inhibition of cancer cell proliferation; (5) reversible inhibition of proliferation of hormone-dependent cancer cell lines; (6) promotion of cell differentiation; and (7) inhibition of cancer cell metastasis by affecting cell-to-cell adhesion. Certain flavonoids are beneficial against CVD because they inhibit blood platelet aggregation and provide antioxidant protection to LDL (Frankel et al. 1993). This topic is discussed in detail with respect to grapes and wine (Section III). Although relatively little is known about the absorption and metabolism of phenolics, the appearance of anthocyanins (Miyazawa et al. 1999), catechin (and catechins derivatives) (Donovan et al. 1999; Bell et al. 2000), flavonols (Paganga and Rice-Evans 1997), and non-flavonoid phenolics (Caccetta et al. 2000; Paganga and Rice-Evans 1997) in plasma demonstrates digestive absorption and metabolism of phenolic phytonutrients in humans. Phenolic aglycones (e.g., catechins) are sufficiently hydrophobic that they could passively diffuse through biological membranes, while glycosides (e.g., anthocyanins) are hydrophilic.

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Transport of flavonoid glycosides into cells requires some type of transport mechanism, which may be mediated by interaction of their sugar moieties with sugar transporters. Quercitin and phloridzin glycosides in blood plasma have been reported (Paganga and Rice-Evans 1997). Deglycosylation of flavonoid glycosides may be an early step in metabolism; deglycosylation can occur in the lumen of the gut, or within the cells of the gut epithelium. Further metabolism of flavonoids includes possible hydroxylation and/or methylation on the B ring. Conjugation of flavonoids with either sulfate or glucuronide also occurs, and the final profile of flavonoid conjugates appears to be highly species dependent. De-esterification of hydroxycinnamate esters occurs most likely through the enzymatic action of the gut microflora (Williamson et al. 2000). 2. Carotenoids. The carotenoids belong to a larger group of secondary compounds called the terpenoids, which are compounds based on the five-carbon unit, isoprene. Carotenoids are hydrophobic molecules possessing a long conjugated hydrocarbon tail. Different classes of carotenoids include: (1) lycopenes, which have a long unconjugated hydrocarbon structure; (2) carotenes (e.g., β-carotene), which have a long unconjugated hydrocarbon structure with additional six-carbon ring(s); and (3) xanthophylls (e.g., zeathanthin) with six-carbon ring(s) containing one or more oxygen functional groups (Fig. 7.4). In photosynthesis the carotenoids serve as accessory pigments and also provide antioxidant protection by quenching activated oxygen species and pigment radicals resulting from photosynthetic light capture and electron transport (Horton 1996). Carotenoids have an ecological role as visual attractants, with colors ranging from yellow to red. Some carotenoids are used commercially as food colorants. Carotenoids are precursors to vitamin A, and for 50 years human health research has focused only on the carotenoid groups that possess this provitamin A activity (Krinsky 1998). Provitamin A carotenoids include α-carotene, β-carotene, and βcryptoxanthin; about 40 others have also been identified. Plant carotenoids provide the only source of vitamin A to the animal kingdom (Astorg 1997) and more than 80% of the world’s vitamin A is provided by horticultural crops (Simon 1997). Although much of the prior research focused on carotenoids with provitamin A activity, the antioxidant properties of dietary carotenoids are now being investigated. Unlike the phenolic antioxidants, which are water soluble, the carotenoids are lipophilic. Therefore the carotenoids may play a significant role in providing antioxidant protection to membrane lipids, which are a major target for oxidative damage. Carotenoids are susceptible to oxidation, and β-carotene, which has been extensively studied, can

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Fig. 7.4.

281

Chemical structures of representative carotenoids found in fruit.

become a pro-oxidant (i.e., promote oxidation) under conditions of high oxygen pressure (Astorg 1997). The research on the chemopreventive properties of the carotenoids has focused primarily on their anti-cancer activity, although their cardioprotective activity has been examined (Palace et al. 1999). Specific carotenoids (lutein and zeaxanthin) have also been reported to be protective against macular degeneration, a prevalent eye disorder of the elderly (Eye Disease Case-Control Study Group 1993). Research on the anti-cancer properties of carotenoids was reviewed by Astorg (1997) and is summarized below. Various carotenoids demonstrate activities against cancer initiation, including protection against DNA damage by UV radiation, oxidants, and carcinogens, as well as carcinogen detoxification by way of cytochrome P450 induction. Carotenoids have been shown to suppress growth of cell lines and inhibit enzyme induction and gene expression related to cell proliferation. Gap junctions, which contain arrays of tiny plasma membrane connections between adjacent cells, are suppressed in cancerous tumors; in their absence the autonomous growth of neoplastic cells is favored. Carotenoids have been reported to increase the gap junctions needed for cell-to-cell com-

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munication. Certain carotenoids have been shown to enhance immune response by increasing several elements of the immune response process in animal models. Due to the substantial epidemiologic and experimental evidence of the role of β-carotene in reducing cancer risk, human trials were initiated in the early 1980s. Results of these trials have been, for the most part, negative. In the Finnish Alpha-Tocopherol, BetaCarotene Trial, incidence of lung cancer among smokers actually increased in the group supplemented with the β-carotene alone or with α-tocopherol. In the American Beta Carotene and Retinol Efficiency Trial of smokers who were exposed to asbestos, lung cancer increased in the supplemented group. The pro-oxidant effect of β-carotene under the oxidative stress conditions induced by heavy smoking may have enhanced tumor promotion. High β-carotene supplementation may reduce the absorption of other carotenoids that may be effective antioxidants (Astorg 1997). These widely cited studies illustrate how a high dose of single dietary components can have adverse effects on cancer incidence and bring to mind another adage of Hippocrates: the difference between a food, drug, and poison is dose. A third study among exsmokers or non-smokers found no effect of β-carotene supplementation on cancer incidence (Astorg 1997). Several carotenoids, including lutein, β-crytoxanthin, lycopene, αcarotene, and β-carotene were detected in the plasma of subjects fed carotenoid-containing fruits and vegetables (Broekmans et al. 2000). Factors affecting the bioavailability of carotenoids from vegetables were recently reviewed by Clevidence et al. (2000).

II. CITRUS The genus Citrus belongs to the Rutaceae and the subfamily Aurantiodeae. Members of this subfamily are characterized by fruit with a juicy pulp made of vesicles within segments. Major commercial citrus species include orange (C. sinensis), grapefruit (C. paradisi), lemon (C. limon), lime (C. aurantifolia), and mandarin (C. reticulata). Citrus is made up of an outer peel, which includes epidermis, flavedo, albedo, and endocarp. The flavedo in the subepidemal region contains chromoplasts (which give the fruit its green, yellow, or orange coloring) and numerous oil glands filled with aromatic essential oils. The albedo (mesocarp) is rich in pectin, cellulose, and hemicellulose. The endocarp (inner flesh) consists of segments (carpels) that are filled with closely compacted juice vesicles and contain pigments, sugars, and organic acids; these are the primary source of citrus juice. Seeds may be found

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within the segments adjacent to the core, at the center of the fruit (Girard and Mazza 1998). The United States produces about 10% of the world’s citrus crop. Its annual citrus crop is in excess of 6.8 million metric tonnes, making it one of the largest U.S. fruit crops in terms of tonnage and value. Oranges constitute about 70% of the U.S. total citrus production, a large proportion of which is processed into either juice or juice concentrate. Smaller percentages of the grapefruit and lemon crops are used for juices, while tangerines and other minor citrus crops are primarily consumed fresh. The processing of citrus fruit into juice gives rise to a large volume of by-products that contain rind and pulp (Girard and Mazza 1998). A major collaborative research effort was undertaken in Florida, during the development of the citrus juice industry in the 1950s, to develop ways to use the huge volumes of citrus rinds generated by the juice industry. This included an investigation of the health properties of citrus products; various citrus components are currently being studied with respect to health, including phenolics, carotenoids, liminoids, pectin, and of course, vitamin C. A. Citrus Phenolics Health research on citrus phenolics has focused almost exclusively on their flavonoid components, although hydroxycinnamates do occur in these fruits (Table 7.2). 1. Composition and Localization. There are four classes of flavonoids in citrus: flavanones, flavones, flavonols, and anthocyanins (blood oranges only) (Fig. 7.3). The predominance of flavones and flavanones is a distinctive characteristic of citrus, while flavonols and anthocyanins are widely distributed among other fruit species (Macheix et al. 1990). More than 60 different flavonoids have been identified in citrus species (Benavente-García et al. 1997). During fruit maturation, flavonoid levels have been reported to increase, decrease, and fluctuate (Baldwin 1993). Peel flavonoids form part of the resistance mechanisms of citrus, possessing antimicrobial and antiviral activity, and protecting peel by absorbing UV radiation. The flavanones (including flavanone glycosides) are the most abundant flavonoid group in citrus, while their occurrence in other fruits is rare (Macheix et al. 1990). The flavanone profiles of citrus species are unique, and are therefore useful in taxonomic classification. Hesperidin (hesperitin rutinoside) is a major flavanone component in oranges, where it is

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localized mostly in the flavedo and albedo (i.e., peel). Naringin is the predominant flavanone in grapefruit (Ameer et al. 1996). It is found not only in the flavedo and the albedo of the fruit, but also in the core and segment membranes (Macheix et al. 1990; Girard and Mazza 1998). Although hesperidin is predominant in oranges, it is only a minor flavanone component in grapefruit. Lemon peel contains both the hesperitin and eriocitrin (eriodictyol 7-rutinoside) flavanones (Macheix et al. 1990). Flavanone content and profile of citrus products is of interest to food manufacturers because flavanones affect taste and bitterness. The principal compound responsible for the immediate bitterness of citrus is naringin, although neohesperidin and poncitrin also contribute. The liminoids, discussed in Section C, are responsible for a delayed development of bitterness (Goodwin and Goad 1970). Hesperidin is not soluble and thus contributes to the haze in orange and lemon juices. Although flavanones are the predominant class of flavonoids in citrus, they are probably not as important as other flavonoid classes in terms of human health (Benavente-García et al. 1997). Flavones (and their glycosides) are found in lower concentrations in citrus than flavanones. Flavones are localized in the essential oil of the flavedo. Examples of citrus flavones include luteolin, diosmin, and apigenin. The polymethoxylated flavones, such as nobiletin (hexamethoxyflavone), sinensetin, and tangeretin (pentamethyoxyflavones) are of particular interest since polymethoxylation is associated with more potent biological activity (Macheix et al. 1990; Benavente-García et al. 1997). The third major group of citrus flavonoids are the flavonols (and their glycosides), which include: quercetin, kaempferol, rutin, limocitrol, limocitrin, and isolimocitrol (Macheix et al. 1990). The first three of these flavonols are widely distributed among many fruit species. The last three are found in lemon, and contain three, two, and three methoxy groups, respectively. Citrus flavonoid extracts have been manufactured by citrus processors and utilized as food supplements for human and animal health use for several decades (Girard and Mazza 1998). The citro-flavonoids, which are extracted from the peel, contain all three major classes of the citrus flavonoids. “Hesperidin complex,” taken from de-oiled orange peel extract, contains several flavonoids, including flavonols, flavones, and flavanones (Macheix et al. 1990). 2. Anti-cancer Effects. Citrus flavonoids have been reported to have effects on all three stages of cancer development (i.e., initiation, promotion, and progression). UV radiation can affect the initiation of can-

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cer since it induces damage to DNA, a precursor to the disease. Flavonoids may directly protect DNA by absorbing UV radiation, and/or by acting as antioxidants in quenching UV-generated free radicals. The citrus flavanone, naringenin, and the flavonol, rutin, have been shown to protect template DNA from UV damage in vitro (Kooststra 1994). Flavonoids may also protect against cancer initiation by detoxifying chemical mutagens directly. Various mutagens and citrus flavonoids (naringin, hesperidin, nobiletin, and tangeretin) were surveyed in a bacterial bioassay. All flavonoids provided varying levels of protection against the mutagenicity of the compounds tested (Calomme et al. 1996). The induction stage (i.e., initiation and promotion) of cancer development has been shown to be slowed by the flavonol, quercetin, in a study examining tumor incidence and growth in rats treated with the carcinogen azoxymethanol (Benavente-García et al. 1997). Quercetin treatment also slowed the growth of human squamous carcinoma cells both in vitro and in vivo. In normal human lung tissue, however, higher levels of quercetin were required before growth inhibition was observed, suggesting differential effects between normal and cancerous cells (Attaway 1994). The polymethoxylated flavones, tangeretin and nobiletin, exhibited a marked inhibitory effect on the growth of cancerous squamous cells, as compared with quercetin and taxifolin. The increased biological activity of polymethoxylated flavonoids may be due to the decreased polarity of these compounds, facilitating greater membrane uptake (Kandaswami et al. 1991; Williamson et al. 2000). Quercetin-fed and luteolin-fed mice had a lower incidence of tumors than controls after treatment with the carcinogen 20-methylcolanthrene (Benavente-García et al. 1997). Citrus flavonoids inhibit non-specific in vitro markers of tumor promotion, including ornithine decarboxylase induction, incorporation of inorganic phosphate into membrane lipids, and protein kinase activation (Manach et al. 1996). The progression phase of cancer, which is characterized by tumor invasion into surrounding tissue and metastasis, is the most important determinant in disease prognosis. Chemotherapy and radiation therapy are often effective against localized disease, but are frequently minimally effective once cancer cells have extensively invaded tissues or metastasized. Some flavonoids may possess properties that are effective against this progression phase. The invasion of malignant mouse cells and malignant human cells into chick tissue fragments was inhibited by the polymethoxylated flavone, tangeretin (Bracke et al. 1989, 1994). Tangeretin may inhibit the spread of tumors into surrounding tissue by affecting the protein E-cadherin. E-cadherin separates normal epithethial cells from underlying connective tissue and facilitates homotypic cell-

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to-cell adhesion. Disruption, or down regulation, of E-cadherin is purported to allow cancer cells to invade tissue. Although tangeretin was effective in an assay demonstrating this effect (i.e., promotion of cell-tocell adhesion and inhibition of cell invasion), the other polymethoxylated flavone, nobiletin, and the flavanones, hesperidin and naringin, were not effective (Bracke et al. 1994), suggesting that specific properties of tangeretin are responsible. The structure-activity properties of 27 citrus flavonoids were recently examined in vitro, with respect to their effect on the rate of cell division of several cancerous and normal cell lines. It was determined that (1) an ortho-catechol moiety on the flavonoid B ring and (2) a C2-C3 double bond were important for activity. For the polymethoxylated flavones, the presence of a C-3 hydroxyl and C-8 methoxyl group was essential for high activity (Kawaii et al. 1999). 3. Cardiovascular Protection. Citrus flavonoids, and flavonoids in general, are protective against CVD due mainly to their effect on capillaries, erythrocytes, and LDL. Flavonoids decrease the permeability of capillaries; this reduces the seepage of plasma constituents into tissue that causes tissue edema (swelling) during inflammation. Drugs that are currently used to counteract vascular permeability are based on flavonoids, mainly citrus hesperidin and rutin. Flavonoids also dilate blood vessels to improve circulation. Patients with chronic venous insufficiency are treated with flavonoids or flavonoid derivatives to improve circulation (Benavente-García et al. 1997). Citrus flavonoids, like flavonoids from other species, affect the properties of platelets. Aggregation of platelets, the first step in the clotting process, is induced by various “agonists,” (activators of platelet aggregation), including collagen, ADP, and arachidonic acid. The polymethoxylated citrus flavones, tangeretin and nobiletin, are potent inhibitors when ADP or collagen are used to induce platelet aggregation in vitro, while naringin and hesperidin are not active (Beretz and Cazenave 1988). Flavonols, none of which were methoxylated, differed in their effectiveness when aggregation was induced by arachidonic acid (Benavente-García et al. 1997). Thus, some flavonoids may reduce the inappropriate aggregation of platelets and the associated deleterious effects. However, the effects of citrus flavonoids on platelet properties appear to depend on both the particular flavonoid and the aggregation agonist, suggesting that different mechanisms can affect platelet aggregation (Benavente-García et al. 1997). Contradictory in vivo evidence for an effect of citrus flavonoids on platelet aggregation has been recently reported (Keevil et al. 2000). In this study grape juice inhibited platelet

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aggregation in human subjects, while orange and grapefruit juice did not. The difference in response may be due to the higher total phenolic content of the grape juice and/or differences in the flavonoid profiles and absorption of citrus and non-citrus juices. Only the citrus juices would contain flavanones and flavones, while only the grape juice would contain anthocyanins. As previously mentioned, the oxidation of LDL by ROS plays an important part in atherogenesis because oxidized LDL is sequestered by macrophages in the walls of blood vessels, leading to plaque formation (German et al. 1997; Chia 1998). Flavonoids inhibit the oxidation of LDL in vitro and may also provide antioxidant protection to the α-tocopherol (vitamin E) present in LDL particles (Hertog et al. 1993). 4. Anti-inflammatory Effects. The anti-inflammatory effect of flavonoids is well documented (Gabor 1986; Middleton and Kandaswami 1993). Recent studies on citrus flavonoids, including hesperidin and diosmin, suggest that these compounds work by inhibiting the enzymes lipoxygenase, cyclooxygenase, and phospholipase A2, which are involved in inflammatory response (references in Benavente-García et al. 1997). Phospholipase A2 and other phospholipases release arachidonic acid from cell membrane phospholipids. Arachidonic acid is then used as a substrate by lipoxygenase to form the leukotrienes that enhance the inflammatory response, and by cyclooxygenase to form prostaglandins, some of which cause vascular dilatation, increased edema, and affect platelet aggregation. Polymethoxylated flavones may affect the inflammatory process via cytokines that modulate the actions of other cells involved in the inflammatory response; these flavonoids were reported to inhibit phosphodiesterase activity, thereby suppressing cytokine synthesis (Manthey et al. 1999). Various methoxylated flavones were active; the most active were highly methoxylated (3,5,6,7,8,3′,4′-heptamethoxyflavone). 5. Other Effects. Certain citrus beverages can influence the metabolism of some pharmacologic agents, specifically those that are normally catabolized by certain cytochrome P450 isozymes. When these agents are administered along with grapefruit juice, their plasma concentration is significantly increased due to their decreased metabolism by cytochrome P450, with potentially harmful side effects (e.g., arrhythmias) (Spence 1997). Since this effect is observed after oral, but not intravenous, administration, citrus components are most likely having an effect on the intestinal and not the hepatic cytochrome P450 isozyme. Several citrus components, including flavones, flavonols, as well as non-flavonoid

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components have been shown to inhibit cytochrome P450 isozymes in vitro (Chan et al. 1998). B. Carotenoids Carotenoids are the pigments responsible for the color of citrus peel, fruit, and juice, and are therefore important to the visual quality of this fruit. Lycopene is the primary pigment that colors red and pink grapefruit. While α-carotene is not generally abundant in Citrus, some varieties of red grapefruit are a particularly good source. Carotenoids that can be converted to vitamin A are desirable because of its metabolic role. Sixteen carotenoids with provitamin A activity have been identified in citrus, the major ones being α-carotene, β-carotene, and β-cryptoxanthin (Roussef and Nagy 1994). C. Other Components 1. Liminoids. Liminoids are a group of polycylic terpenoid derivatives that are present in orange, grapefruit, lemon, and lime (Fig. 7.5). The most widely distributed liminoid in citrus is limonin, which is found in the seeds, endocarp, flavedo, and albedo. Like flavonoids, liminoids contribute to the bitterness of citrus juices, but the bitterness from liminoid forms gradually after the juice stands for several hours or after heating (Goodwin and Goad 1970). This is due to the slow conversion of limonin monolactone, which is released from disrupted tissue, to the bitter limonin dilactone, which can occur in the acidic milieu of the juice. Liminoid bitterness is lessened when riper fruit are used in processing, since limonin monolactone declines as the fruit matures (Baldwin 1993). Another liminoid found in citrus is nomilin. Liminoids have recently been considered as chemo-preventive agents because they can induce the detoxification enzyme glutathione S-transferase. Glutathione S-transferase catalyzes the conjugation of the tripeptide glutathione (GSH) with xenobiotic electrophiles, including carcinogens. Thus, this enzyme plays a protective role against the initiation stage of cancer. The essential structural feature for GSH Stransferase induction in the liminoids is the furan ring. The increased water solubility of the GSH-conjugate facilitates its excretion from the body (Lam et al. 1994). Liminoid treatment has been reported to inhibit tumor initiation in vivo in the forestomach, lung, and skin of mice, with nomilin being generally more effective than limonin. In an in vivo hamster cheek model, testing against oral cancers, both limonin and limonin

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Chemical structure of major Citrus liminoids.

glycoside treatments produced lower tumor loads, but nomilin was ineffective (Miller et al. 1994). The differences among liminoids in their ability to inhibit tumor initiation in different tissues may be related to their relative induction of different GSH S-transferase isozymes (Lam et al. 1994). Although limonin appears to possess chemo-protective properties, its low concentration in fruit, its intense bitterness, and its limited water solubility may render it useless. However, limonin glycosides are more concentrated in citrus, are tasteless, and are soluble in water. The predominant limonin glycoside is limonin 17-α-D-glucopyranoside (Girard and Mazza 1998). In orange juice, the combined concentration of limomin and nomilin is 1–2 ppm, while limonin 17-α-D-glucopyranoside is 176–180 ppm. When consumed, limonin glycoside is probably converted

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to limonin by glycosidic enzymes in the digestive tract (Girard and Mazza 1998). 2. Vitamin C. Vitamin C is an essential nutrient (i.e., a dietary component required to prevent specific deficiency-related diseases). Although this review is focused primarily on non-essential fruit phytonutrients, vitamin C is worth mentioning since, like phenolics and carotenoids, it functions as an antioxidant. Vitamin C is present in a high concentration in citrus fruits. Even though only about one quarter of the vitamin C content of the whole fruit remains in the juice, a 227 ml (8-oz) serving of either grapefruit or orange juice will provide 100% of the RDA intake for vitamin C (Rouseff and Nagy 1994). After processing, vitamin C remains in the peel and pulp and especially in the flavedo (Baldwin 1993). Vitamin C is the least stable antioxidant in foods, and various processing factors have been shown to reduce its content. Neutral pH, high temperature, extended heating, and the presence of metal ions can all reduce the vitamin C content of foods. Vitamin C from citrus products is relatively stable because of the low pH of citrus products, and the high content of vitamin C in these fruit. In general, vitamin C is retained better in foods that are normally high in this antioxidant nutrient (Klein and Kurilich 2000). As an antioxidant, vitamin C is considered to be an important first line of defense against ROS and works with other antioxidant compounds and antioxidant enzymes, such as GSH reductase and ascorbate peroxidase. Vitamin C also helps to maintain enzyme-bound metal ions in correct reduction states for catalytic activity. It is implicated in the deactivation of carcinogens (i.e., anti-initiation activity) and may impede the reaction of nitrates with amines and amides, which can form potent carcinogenic nitrosamines within the digestive tract (anti-initiation activity) (Mirvish et al. 1975). 3. Fiber. Citrus fruits contain pectin, cellulose, and hemicellulose. All of these compounds are resistant to breakdown by digestive enzymes, and therefore are sources of dietary fiber. Pectin is the fiber source in greatest abundance in citrus, and is present in both the edible and inedible portions of the fruits. Greater amounts of pectin occur in the fruit solids, such as the albedo (peel), juice sacs, and membranes, as compared with the juice, but the bitterness and astringency of these high pectin components limits their use in citrus products. Pectin is beneficial to human health by regulating blood sugar levels and reducing serum cholesterol. It regulates blood sugar by both delaying gastric emptying and increasing the intestinal mucosa. This slows the uptake of glu-

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cose into the serum and the concomitant rise in insulin. Pectin may also be useful as a dietary aid in managing non-insulin dependent diabetes (Baker 1994). Pectin supplementation protects against CVD by reducing serum cholesterol. In both diet-induced (in rats) (Vorster 1984) and genetically predisposed (in minipigs) (Ahrens et al. 1986) hypercholesterolemia, symptoms were reduced with pectin supplementation. Pectin may also promote the excretion of fats, bile acids, and cholesterol (references in Baker 1994). Modified citrus pectin was reported to have beneficial effects in specific in vitro assays used to detect growth suppression of rat prostate cancer cells (Hsieh and Wu 1995). 4. Limonene. Limonene, a terpenoid, is the major component of the Citrus volatiles, which are made up almost entirely of terpene hydrocarbons. Limonene, which is obtained by distillation during citrus juice processing, has been shown to block and suppress carcinogenic events. The addition of limonene or limonene-rich citrus oils (orange, lemon, grapefruit, or tangerine) to a semi-purified diet induced GSH Stransferase activity in the liver and small bowel mucosa. Feeding resulted in inhibition of forestomach, lung, and mammary tumors (Hocman 1989; Wattenberg et al. 1986). Limonene has also been shown to inhibit the conversion of proto-oncogenes to their activated farnesylated forms, in both in vitro cell cultures and in vivo mammalian systems (Crowell et al. 1991; Wattenberg 1983; Maltzman et al. 1989). 5. Folic Acid. Citrus fruit are relatively good sources of folic acid and may contribute substantial amounts of folate to the diet, by virtue of the large volume of citrus products consumed. Folic acid acts as a coenzyme in numerous biological reactions, and is used to transport single carbon fragments during amino and nucleic acid synthesis. Decreased folic acid intake is linked to pathological disorders such as CVD and cancers. Severe deficiencies during pregnancy may lead to neural tube defects in offspring. Both animal and in vitro studies have shown that folic acid deficiency can cause chromosomal damage that precedes cancer development (Rouseff and Nagy 1994).

III. GRAPES AND WINE The European grape, Vitis vinifera, is native to Asia Minor and has a long history throughout the world. Egyptian hieroglyphics, dating from 2400 B.C. depict grape and wine production. The medicinal value of grapes

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and wine was recognized by the ancient Egyptians, and also by Hippocrates (5th–4th century B.C.). Grapes and wine form part of the Ayurvedic medicine of East India, as well as the medicine of South Africa, the Middle East, and China. V. vinifera is mentioned in several pharmacopoeias, including those of the United Kingdom and the United States (Bombardelli and Morazzoni 1995). V. vinifera is the species used almost exclusively for wine production in Europe and the rest of the world. Other North American grape species include V. labrusca (e.g., ‘Concord’) and the native V. rotundifolia (muscadine), as well as other native species that are used as root stocks for V. vinifera (Kanellis and Roubelakis-Angelakis 1993). Recent intensive research on the healthful properties of wine originated from the epidemiological evidence known as the “French paradox.” The television documentary program “60 Minutes” brought the paradoxical findings to the attention of the American public in 1991. They reported that although the consumption of saturated fats in France was about three times higher than in the United States, the incidence of CVD was only about one third. The possible cardio-protective effects of higher consumptions of garlic, cheese, and fruits and vegetables were considered, but it was the higher consumption of red wine that received the greatest attention. Although research initially focused on the possible cardio-protective effects of the alcohol in wines, most evidence now suggests that it is the phenolic components in grapes and wine that provide the protective effects against CVD (Constant 1997). A. Phenolics The phenolic phytochemicals of grape and wine have been, and continue to be, extensively studied because of their impact on the color (especially anthocyanins) and organoleptic properties of wine (e.g., tannins) (Van Buren 1970). There is also a substantial interest in investigating their health properties. Grapes, and the wines made from them, contain two major classes of phenolic components: the hydroxycinnamate derivatives and the flavonoids. The hydroxycinnamate ester chlorogenic acid (caffeic acid esterified with quinic acid), although widely distributed and often predominant in many fruit species, is not in Vitis. Instead the hydroxycinnamate ester, caffeoyl tartaric acid (caffeic acid esterified with tartaric acid), predominates in this genus (Macheix et al. 1990). Grape flavonoids include the flavan 3-ols (catechins), flavonols, and anthocyanins; the latter two are present as glycosides. The flavan 3-ols also occur in oligomeric forms (proanthocyanins) and polymeric forms (condensed

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tannins). Polymers of the simple phenolic acids, including gallic acid and ellagic acid (tannic acid), occur in muscadines but not in wine grapes (Waterhouse and Walzem 1998). Grape peels and seeds are the richest source of phenolics, especially the phenolic flavonoids. The peel contains anthocyanin pigments, which are not found in the seeds, although the seeds are a richer source of all other flavonoid classes, in particular proanthocyanidins. Hydroxycinnamates are found almost exclusively in grape juice/pulp (Waterhouse and Walzem 1998). The differential distribution of the phenolics throughout the fruit has implications in juice and wine making. For example, fermentation of red grape cultivars in the presence of peel creates red, anthocyanin-rich wine, which is also high in phenolic content. Red grape fermentation without the peel yields a white wine, which is substantially lower in total phenolics. When grape extraction time was extended and seeds crushed, more of the simple phenolics (C6) and flavan 3-ols were recovered in the wine (Meyer et al. 1997). Useful phenolic components are extracted from the by-products of wine and juice making (e.g., pomace, including peels and seeds) and are used for products such as natural colorants or dietary supplements (e.g., grape seed proanthocyanidins). Without the seeds, red grapes are consistently and substantially higher in their total phenolic content than white grapes (Waterhouse and Walzem 1998). The phenolic content and composition of grapes can be drastically affected by season and management practices, leading to year-to-year differences in wine qualities. Although the total phenolic content of grapes changes little during maturation, the content among some phenolic groups does change (Waterhouse and Walzem 1998). Price et al. (1995) report that the flavonoid content of Pinot Noir grapes was affected by sun exposure; the peel of fruit from clusters exposed to full sun had 10 times more of the flavonol glycoside quercetin than fruit from shaded clusters. Anthocyanin content was not affected. Clusters exposed to the sun had a lower concentration of the hydroxycinnamate derivative, caffeoyl tartaric acid. Flavonols may accumulate in response to UV radiation and protect the exposed tissue from potential damage. Changes in the phenolic profile of grapes can also occur when they are processed into wine. During vinification, caffeoyl tartaric acid may be converted to the GSH conjugate, S-glutathionylcaftaric acid (Girard and Mazza 1998). The chemistry of this conversion is well characterized because of its effects on wine quality. Although both the hydroxycinnamate derivative and glutathione are radical scavengers, it is not known whether the conjugation product also has scavenging activity (Meyer et al. 1997).

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A grape phenolic compound of particular note is resveratrol (3,5,4′trihydroxystilbene; Fig. 7.6). In grapes, resveratrol functions as a phytoalexin; the content of resveratrol increases with fungal infection (Langcake and Pryce 1976), and grapes with high levels of resveratrol appear to have greater resistance to Botrytis infection (Jeandet et al. 1995). Resveratrol content is associated with fungal infection pressure; grapes produced in damper, cooler regions have higher levels of resveratrol (Soleas et al. 1997). Resveratrol has been measured in V. vinifera, V. labrusca, and V. rotundifolia (Ector et al. 1996). Since the compound is localized primarily in the peel of the fruit, the length of time peel is present during fermentation strongly correlates with the resveratrol content of the finished wine (Okuda and Yokotsuka 1996). Soleas et al. (1997) summarize results of various wine surveys and conclude that Pinot Noir wines have a relatively high resveratrol content compared with other red wines, and especially when compared with white wine. Resveratrol occurs in trans or cis isomers, but most of the literature does not distinguish between these two forms.

trans-Resveratrol

cis-Resveratrol Fig. 7.6.

Chemical structure of cis- and trans-isomers of resveratrol.

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B. Evidence of Health Benefits 1. Anti-cancer Effects. The health properties of resveratrol have received a great deal of attention following the appearance of evidence that this compound is active as an anti-cancer agent (Jang et al. 1997). Resveratrol was active in vitro in bioassays designed to test biochemical markers for all three stages of cancer development (i.e., initiation, promotion, and progression). With respect to cancer initiation, resveratrol was shown to have antioxidant and anti-mutagenic activity and to induce quinone reductase, a drug-metabolizing enzyme, which can detoxify carcinogens. Resveratrol was shown to be a selective inhibitor of human cytochrome P450 1A1 (Chun et al. 1999). Cytochrome P450 1A1 plays a role in the activation of procarcinogens; its activity is considered one of the most important in the initiation of tumors. Resveratrol reduced the growth and induced differentiation of human leukemia cells in vitro. Resveratrol also reduced the incidence of tumor occurrence when tumor inducers and promoters were combined in an animal cell model for carcinogenesis (Jang et al. 1997). Resveratrol has been reported to stop the cell cycle at the S/G2 stage (Della Ragione et al. 1998); this allows for cell differentiation and counteracts the promotion phase of carcinogenesis. In cancer, the balance between cell proliferation and cell death is disrupted leading to tumor growth. Morphological and ultrastructural evidence from cancer cells suggests that resveratrol induces cell death (apoptosis) (Surh et al. 1999). The effect of resveratrol on cell division is due, at least in part, to the inhibitory effect of resveratrol on ribonucleotide reductase and DNA synthesis in mammalian cells (Fontecave et al. 1998). Like some other flavonoids (e.g., isoflavones), resveratrol appears to possess phytoestrogenic activity (Gehm et al. 1997). Resveratrol at a low concentration was reported to inhibit estradiol binding to estrogen receptors, activate the expression of estrogen-related genes, and stimulate the proliferation of estrogen-dependent human breast cancer cells. The various estrogenic effects of resveratrol were found to be specific to certain cell-types, and in this way were reminiscent of the tissue- and speciesspecific nature of the therapeutic agent tamoxifen. While resveratrol stimulated growth of estrogen-dependent human breast cancer cells, it inhibited the growth of estrogen-independent mouse mammary cell cultures. Clearly further research is required to determine the potential negative and positive effects of resveratrol in the human diet, and to assess its potential use as a therapeutic agent (Gehm et al. 1997).

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2. Cardiovascular Protection. An early and significant report by Frankel et al. (1993) shows that low concentrations of red wine phenolics inhibit the oxidation of low-density LDL in vitro. The direct measurement of the antioxidant effects of grape and wine phenolics on LDL is significant because LDL oxidation is associated with atherosclerotic plaque formation. Proanthocyanidin flavonoids, purified from V. vinifera, effectively inhibited the oxidation of vitamin E and lipids, and prevented the hemolysis of red blood cells (Facino et al. 1998). The activity of the wine grape proanthocyanidins against vitamin E oxidation may be of interest because vitamin E is a component of LDL particles. The vitamin E sparing activity of other grape phenolics are considered less potent than their proanthocyanidins (Facino et al. 1998). Grape and wine phenolics have been examined using other specific tests related to cardiovascular protection. Aggregation of whole blood platelets in humans was observed to be inhibited 2 h after ingesting red wine and grape juice, but no effect was observed after ingesting white wine (Folts 1998). Inhibiting platelet aggregation potentially reduces the risk of blood vessel occlusion that can occur when blood clots at the site of atherosclerotic plaque accumulation. Another aspect of cardiovascular protection, investigated with respect to grapes and wine, is vasodilation. Dilation of blood vessels, important for the free flow of blood, is restricted in areas of atheosclerotic plaque. Vasodilation occurs with the release of nitric oxide (NO) from the endothethial cells of blood vessels. Fitzpatrick et al. (1997) report that grapes and wine stimulate the release of NO from the endothelial cells in vitro. Grape skins from both red and white wines were effective, but the pulp had no effect. Red wine skins were more effective than white wine skins, but there were differences among the red varieties. Although several individual grape skin components were studied, no single component was associated with the vasodilatory effect. Resveratrol can protect LDL against oxidation, although other phenolic wine compounds appear to be more effective (Frankel et al. 1993; Frankel et al. 1995; Soleas et al. 1997). Like other grape phenolics, resveratrol inhibits the aggregation of platelets (Pace-Asciak et al. 1995) and thus protects against clotting. In addition, resveratrol is known to have lipid-lowering effects (Arichi et al. 1982; Soleas et al. 1997). Flavonoids generally possess anti-inflammatory activity. The antiinflammatory activity of resveratrol appears to be related to its effect on arachidonate metabolism, specifically the 5-lipoxygenase and 15-lipoxygenase pathways, which are powerful mediators of inflammatory reactions (Goldberg et al. 1997). The various biological effects of resveratrol

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have been reviewed by Soleas et al. (1997) and more recently by Fremont (2000). Although relatively little is known about the digestion and absorption of grape and wine phenolics, epidemiological and in vitro evidence suggests that their inclusion in the diet has a positive impact on health. Phenolic absorption has been inferred from measured changes in the antioxidant capacity of serum before and after its ingestion. The consumption of red wine significantly increased the antioxidant capacity of serum, while the consumption of white wine did not (Whitehead et al. 1995). Red wine anthocyanins were detected in urine, indicating that they had been absorbed. Two anthocyanins were unchanged by absorption and excretion, while other anthocyanins were modified (Lapidot et al. 1998). The flavan 3-ol, catechin, was detected mainly in modified forms (methylated, sulfated, and/or glucuronidated) in blood serum after ingestion of a single serving of wine (Bell et al. 2000). Both catechin and its derivatives reached their maximum plasma concentration between 1 and 2 h after ingestion (Donovan et al. 1999). 3. Antioxidant Activity. Several wine varieties have been tested for their ability to prevent LDL oxidation in vitro. For a selection of 20 California wines, inhibition ranged from about 50 to 100% for red wines, and 3 to 6% for the white wines. When red and white wines were tested at the same level of phenolic components, the inhibitory effect ranged from 37 to 65%, and 27 to 46%, respectively (Frankel et al. 1995). The phenolic profiles of these wines were determined and related to their antioxidant effects on LDL. Antioxidant activity was strongly correlated with the total phenolic content (r = 0.94), and correlations with nine individual phenolic components ranged between an r value of 0.92 for gallic acid to 0.38 for the anthocyanin malvidin 3-glucoside (Frankel et al. 1995). For grape extracts fermented with crushed seeds, the higher levels of simple phenolics and flavan 3-ols obtained were highly correlated with the inhibition of LDL oxidation in the resulting wines (Meyer et al. 1997). Clearly, many types of phenolics may contribute to protecting LDL against oxidation in vivo. LDL oxidation studies of different types of grape juices, with a standardized total phenolic content, indicate that anthocyanins are more highly correlated with the inhibition of LDL in Concord grape juice as compared with other Vitis species. In white grape juices, flavan 3-ols and hydroxycinnamates correlated higher with the inhibition of LDL. When juices were not standardized to the same total phenolic content, the Concord grape juice and red wines had a higher anti-LDL oxidation activity than the white grapes

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(Frankel et al. 1998). The value of in vivo studies was illustrated by Caccetta et al. (2000), who reported that although red wine ingestion increased the plasma phenolic acid concentration, the ex vivo oxidizability of LDL was not changed.

IV. VACCINIUM Major commercial North American Vaccinium fruit crops include highbush blueberries (V. corymbosum), lowbush blueberries (V. angustifolium), rabbiteye blueberries (V. ashei), and cranberries (V. macrocarpon). Minor commercial North American species include huckleberries (V. membranaceum and V. ovalifolium) and partridgeberries (V. vitis-idaea). In Europe, commercial Vaccinium species include bilberries (V. myrtillus) and lingonberries (V. vitis-idaea); in China, V. uliginosum; and in S. America, V. floribundum (C. Finn, pers. commun.). The European bilberry is probably the best known Vaccinium for human health use. The earliest record of the medicinal use of bilberry dates from the Middles Ages; it has consistently been used by herbalists and physicians from the 18th century to the present, with published reports dating back to the beginning of the 20th century. Since the 1960s, a standardized extract of bilberry has been widely used in research (Morazzoni and Bombardelli 1996). Today, extracts containing a standardized content of bilberry anthocyanins form the basis of a substantial health products industry (Kalt and Dufour 1997). The general health properties of bilberries and blueberries are reviewed in Morazzoni and Bombardelli (1996) and Kalt and Dufour (1997), and most recently by Camire (2000). Blueberries were used in the early days of North American settlement as a food in different forms (e.g., pemmican, cakes, stews) and as a medicine to treat coughs, diarrhea, and various female illnesses (Kalt and Dufour 1997). Cranberries were traditionally used by native North Americans in poultices to treat wounds and as a treatment for blood poisoning. Cranberries are widely known in folk medicine for being beneficial to urinary tract health (Eck 1990). As with grapes and wine, it is the phenolic, and in particular, the flavonoid components of the Vaccinium species that are beneficial to human health. A. Phenolic Components Compared with other commercial fresh fruits, members of the genus Vaccinium have a relatively high phenolic content; highbush and low-

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bush blueberries contain about three times the total phenolic content of strawberry or raspberry (Kalt et al. 1999). Like other fruits, blueberries contain several classes of phenolics of which the hydroxycinnamate ester, chlorogenic acid, and the anthocyanin flavonoids are major contributors (Table 7.2) (Schuster and Herrmann 1985; Machiex et al. 1990; Kalt and McDonald 1996). The anthocyanin pigments are important to the color quality of whole and processed fruit products, and are also considered to be the major active component in bilberry health products. As a result, anthocyanin research of Vaccinium species spans the fields of horticulture, food, and medicine. Members of the genus Vaccinium are classified in taxonomic Sections. The two main North American blueberry species, highbush blueberries and lowbush blueberries, are both members of the Section Cyanococcus, and contain anthocyanin pigments in only the peel. The European bilberry (V. myrtillus) belongs to the Section Myrtillus; some members of this Section, including bilberries, contain anthocyanins in both the peel and the flesh of the fruit. The cranberries belong to the Section Oxycoccus; their peel has a low concentration of anthocyanin and their fruit flesh has essentially no pigment. B. Health Properties Attributed to Bilberry Myrtocyan® is a commercial water/alcohol extract of bilberry, standardized to 36% anthocyanins, used to study the medicinal properties of bilberry. Myrtocyan® is not pure bilberry anthocyanin, but rather a mixture of phenolic compounds highly enriched in anthocyanins (Kalt and Dufour 1997). A cardiovascular protective property of Myrtocyan® is its ability to inhibit blood platelet aggregation induced by ADP, collagen, or arachidonic acid (Morazzoni and Magistretti 1990). Rats fed Myrtocyan® had prolonged bleeding times and blood coagulation pathways were not affected (Morazzoni and Magistretti 1990). Numerous other bioactivities have specifically investigated bilberry anthocyanins. For example, bilberry anthocyanins are reported to have beneficial effects on collagen, the major protein component of connective tissue. Anthocyanins are reported to strengthen connective tissue by increasing the cross linking of collagen fibrils. They may also inhibit enzymes, like elastase and collagenase, that can break down collagen (Morazzoni and Bombardelli 1996). Decreases in capillary fragility and permeability after administration of either bilberry extract or Myrtocyan® have been demonstrated in various in vivo animal studies with both oral administration and injection. Capillary permeability increases in the inflammatory process and bilberry anthocyanins have an antiinflammatory effect (Morazzoni and Bombardelli 1996).

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A notable pharmacological application for bilberry extracts is in ophthalmology. After treatment with bilberry extracts, alone or in combination with vitamin E and β-carotene, improvements in night vision, faster adaptation to darkness, and improved dark adaptation to dazzling with bright light have been reported (references in Morazzoni and Bombardelli 1996). A more recent study of the effect of anthocyanosides on night vision, however, found no significant difference in several indicators of night vision up to 24 h after oral doses of anthocyanosides were administered (Levy and Glovinsky 1998). The purity of the anthocyanoside preparation was not indicated, but it may be an important aspect, given that other flavonoid components contribute to the effects on night vision. Other eye-related studies have used Myrtocyan®. In a double-blind, placebo-controlled study of patients with diabetic or hypertensive retinopathy, indicators of improvements were reported in 77–90% of the patients (Morazzoni and Bombardelli 1996). Since various tissues of the eyes are subjected to severe oxidative stress (Trevithick and Mitton 1999), the positive effects of bilberry phenolics on certain ophthalmological conditions may be due, in part, to their antioxidant properties. C. Anti-cancer Activity Anti-cancer activity in vitro was evaluated for a selection of Vaccinium species. Fruits of bilberry, lowbush blueberry, cranberry, and lingonberry were fractionated according to their solubilities and chemical reactivities. A hexane/chloroform fraction was a potent inducer of quinone reductase activity in mouse hepatoma cells. Madhavi et al. (1998) later identified β-sitosterol, chlorophyll, lipid, and the carotenoids lutein and zeaxanthin as components of the hexane/chloroform fraction. Quinone reductase is a detoxifying enzyme that can neutralize electrophiles, some of which are known carcinogens. Other fractions containing Vaccinium flavonoids were active in an in vitro assay for ornithine decarboxylase (ODC). During tumor progression ODC is elevated and polyamine products of its reaction are prevalent in proliferating cells. The proanthocyanidin fraction of blueberry, cranberry, and lingonberry were all effective at inhibiting ODC activity (Bomser et al. 1996), demonstrating their in vitro activity against tumor progression. D. Neurological Function Lowbush blueberries are reported to affect age-related neurological declines in motor and memory abilities (Joseph et al. 1999). The declines

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typically associated with age were delayed in rats fed extracts of spinach, strawberry, and vitamin E (Joseph et al. 1998). In a later study, dietary supplements of aqueous blueberry, strawberry, and spinach extracts were reported to be effective in actually reversing certain declines (Joseph et al. 1999). Blueberry supplementation may be particularly effective in maintaining Ca++ homeostasis. The extrusion or sequestration of Ca++ is required for normal neuronal function; Ca++ homeostasis declines during aging as a result of oxidative stress. Reversals in motor behavioral deficits were more marked in the blueberry-fed treatment group than in the other treatment groups (Joseph et al. 1999). E. Urinary Tract Health A feature strongly associated with cranberries, but apparently common to all Vaccinium species, is their role in preventing and treating urinary tract infection (UTI) (Ofek et al. 1996). E. coli is the organism largely responsible for UTI. Two types of E. coli can adhere to the urinary tract (UT) tissue, each with their own mechanism of adherence. Type 1 E. coli adheres to tissue by a mechanism mediated by a specific sugar interaction between it and the UT tissue, but P-type E. coli possess proteinaceous fimbriae that can adhere to UT tissue. Adherence greatly favors colonization and increases in the E. coli population. Howell et al. (1998) have conducted bioactivity-directed fractionation of cranberry phenolics and found that proanthocyanidin flavonoids have an anti-UTI activity against P-fimbriated E. coli. Structural characterization of the active proanthocyanidins indicate that they consist mainly of oligomers of 4 to 5 epicatechin units (Foo et al. 2000). The anti-adherence activity of a wide selection of Vaccinium species were compared; although species differed, they all had effective anti-adherence activity compared with other fruits (A. B. Howell and W. Kalt, unpubl.). F. Antioxidant Activity The ability of fruit and vegetable antioxidants to reduce disease risk has been discussed. Ames et al. (1993) have suggested that oxidative damage is responsible for several cancers, CVD, certain neurological diseases, immune dysfunction, and cataracts. In a survey of 20 different fruits and vegetables, using the oxygen radical absorbing capacity (ORAC) assay, lowbush blueberries rated high for their antioxidant capacity (Cao et al. 1996; Wang et al. 1996; Prior et al. 1998). Similar ORAC values have been reported for highbush and rabbiteye blueberries and bilberries (Prior et al. 1998). Highbush and lowbush blueberries had ORAC levels about

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three times higher than strawberries and raspberries (Kalt et al. 1999). The antioxidant protection provided by phenolics of various fruit species, including blueberries, against the oxidation of both LDL and phosphotidyl choline-containing liposomes was examined by Heinonen et al. (1998; Table 7.3). Protection of LDL from oxidation was greater for cherries and blackberries than blueberries, while liposomes were best protected by the blueberry phenolic extract. The ORAC assay is one of a few aqueous assays used to measure total antioxidant capacity. An advantage of the ORAC assay is that it provides a kinetic profile of the antioxidant protection reaction (Cao et al. 1995), although it does not use a biological molecule (e.g., LDL) as a substrate for oxidation. The ORAC method is commonly used to measure the antioxidant capacity of water-soluble components in the presence of a peroxyl radical generator. The peroxyl radical ROO may be relatively abundant in vivo because it is formed extensively during autocatalytic lipid peroxidation, and it has a relatively long half-life. The ORAC of compounds has also been measured during transition metal-catalyzed radical formation (Fenton chemistry) (Cao et al. 1997). The difficulties and limitations in interpretation of in vitro antioxidant measurements are reviewed by Frankel and Meyer (2000). In blueberries, ORAC was positively correlated with the content of total phenolics and anthocyanins (Prior et al. 1998). Thus, studies have been conducted to assess horticultural factors that may influence the content of phenolics, anthoocyanins, and ORAC. Since lowbush blueberries grow wild, there are thousands of genotypes. A survey of wild genotypes indicated substantial variability in the content of phenolics, anthocyanins, and antioxidant capacity. Up to 5-fold differences in ORAC were observed among 135 genotypes, with 80% of the clones varying by about 2.5-fold (Duy 1999). This variation suggests that it may be possible to select genotypes with high antioxidant capacity. During blueberry ripening, anthocyanin content increases from nil to approximately 10 mg malvidin 3-glucoside equivalents per g dry weight, although total phenolic levels undergo little change during the same period. There is also little change in ORAC during anthocyanin synthesis and fruit ripening. In ripening blueberries it appears likely that the total phenolic pool contributes to the antioxidant capacity. Anthocyanins make a greater contribution to the phenolic pool after the fruit turns from white to their characteristic blue color when ripe (W. Kalt, unpubl.). Since blueberries are frequently used as a food ingredient, it is important to know how food preparation and processing changes their antiox-

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idant capacity. Heat and oxygen reduce the ORAC of blueberries, due in part to their adverse effects on anthocyanin integrity. Also, over a wide range of processed blueberry food products, ORAC was negatively correlated with the degree of polymerized anthocyanins (Kalt et al. 2000).

V. OTHER FRUITS A. Strawberries Strawberries are reported to have higher antioxidant capacity than melons, Citrus, and other fruits (Wang et al. 1996). Strawberries and raspberries have a higher concentration of vitamin C than blueberries (Kalt et al. 1999). In two recent studies in neurobiology, strawberries were found to affect traits associated with age-related declines, especially in neuronal signal transduction, memory, and motor skills. Age-related declines in neurological traits were delayed (Joseph et al. 1998) and some losses in neurological and behavioral function were actually reversed (Joseph et al. 1999) with dietary supplements. The rat diets in these studies were supplemented with a standardized level of antioxidant capacity (as ORAC) (Cao et al. 1996) from different food sources, including strawberries, spinach, vitamin E, and blueberries. Ellagic acid, a phenolic component that is reported to have anticancer activity, has been studied in strawberries and other tender fruits. Ellagic acid inhibits cancer initiation by: (1) stimulating the detoxification of carcinogens via enzymes (e.g., GSH S-transferase); (2) inhibiting the chemical activation of pro-carcinogens into the highly reactive forms that can cause DNA damage; (3) binding to activated forms of carcinogens; and (4) forming DNA adducts, thereby preventing the binding of carcinogens or their metabolites to DNA (Maas et al. 1991). Antimutagenic and anti-cancer activity of ellagic acid against various carcinogens has also been reported (references in Maas et al. 1991). Ellagitannins, which may give rise to ellagic acid, are readily soluble in water, while pure ellagic is not water soluble and is poorly absorbed. Ellagic acid occurs in certain subfamilies of the Family Rosaceae. In strawberries, it is found mostly in the leaves and achenes, but it is also present in the fruit pulp (Maas et al. 1991). Ellagic acid content varies among varieties, but is almost always higher in the flesh of greenharvested fruit than in red fruit. The genetic inheritance of ellagic acid is unknown (Maas et al. 1991). While strawberries, raspberries, and

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blackberries are reported to contain 630, 1500, and 1500 µg/g dry matter, respectively, the content in oranges, grapefruit, red and white grapes, blueberries, and cranberries was less than 100 µg/g dry matter (Daniel et al. 1989). B. Apples Research on the cardio-protective properties of flavonoids (Hertog et al. 1993) considers apples, tea, and onions to be major food sources of dietary flavonoids. Contributions of various components to the antioxidant capacity of long-life apple juice have been determined by compositional analysis and measured antioxidant capacity in pure compounds (Miller 1998). Hydroxycinnamates, particularly chlorogenic acid, made the greatest contribution to total antioxidant capacity (60%), while the chalcone flavonoids (phloridzin and phloretin glycoside) contributed about 15%, the flavan 3-ol, epicatechin, contributed 4% and vitamin C, 6%. The remainder was not accounted for. Although hydroxycinnamates are not as effective antioxidants as the flavonoids (TEAC assay; Rice-Evans et al. 1997), their contribution was significant based on their high concentration in apple juice. The antioxidant and in vitro anti-cancer activity of Red Delicious apples was reported recently. Almost all of the antioxidant capacity was due to fruit components other than vitamin C. The in vitro proliferation of colon cancer cells was reduced in a dose-dependent manner by the addition of apple extracts. The growth of human liver tumor cells was also reduced by apple extracts. Both antioxidant and anti-cancer activities were greater in extracts obtained from unpeeled, rather than peeled apples (Eberhardt et al. 2000); apple peel contains a higher level of phenolic components than the fruit flesh. Variation in the content of phenolic components of apples was comprehensively examined by Awad et al. (2000). Chlorogenic acid and several classes of flavonoids were differentially distributed between peel flesh, core, and seeds of apple fruit. Higher levels of certain anthocyanins and flavonol glycosides were reported in fruit from outer positions on the tree, with their greater exposure to the sun. The phenolic profile differed in fruit of the same cultivar, grown in different orchards. Differences among cultivars, as well as between mutants within a cultivar, were observed. C. Melons Thus far, coverage of bioactive components of fruit have emphasized the health functionality of phenolic components. However, the carotenoids,

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which have been intensively studied for their role in human health, occur at high levels in some fruits. The muskmelon or cantaloupe (Cucumis melo var. reticulatis) has a high content of β-carotene as well as vitamin C. The cantaloupe is the only commonly consumed fruit that can provide the U.S. RDA for vitamin A in a 1-cup serving (Lester 1997). Although honeydew melon (Cucumis melo var. inodorus) contains no vitamin A, it is a good source of vitamin C; cantaloupe and honeydew melon provide 130 and 90%, respectively, of the RDA for vitamin C per 1-cup serving (Lester 1997). Watermelon (Citrullus sp.) has almost no β-carotene, but contains a high level of lycopene. Indeed, watermelon contains more lycopene (4.1 mg/100 g FW) than raw tomatoes (3.1 mg/100 g FW) (USDA-NCI Carotenoid Data Base), although tomatoes have been more intensively studied for their content of this phytonutrient.

D. Other Fruits Prunes and prune juice have been examined for their phenolic components and their protective effects against LDL oxidation. Hydroxycinnamates, especially neochlorogenic and chlorogenic acid, constituted more than 95% of total phenolic content of prunes. Prune extracts have a high antioxidant capacity against LDL oxidation (Donovan et al. 1998; Table 7.3). The antioxidant capacity of wild and domesticated Rubus fruit of varying color was measured using two antioxidant assays, and was found to be most strongly correlated with total phenolic content, and less strongly with anthocyanin content. Vitamin C did not make a large contribution to antioxidant activity (Deighton et al. 2000). The antioxidant capacity of some other common and lesser-known fruit crops have been examined using different kinds of assays (Table 7.3). These results highlight differences in fruit crops, but also differences in the assays used to measure antioxidant activity. The ranking of blackberries, blueberries, raspberries, and strawberries was different when different oxidizable substrates were used. The apparent antioxidant activity of Sea buckthorn (Hippophae rhamnoides) was relatively low in the aqueous assay, in which phycoerythrin was oxidized, but it was very high in the lipophilic assay that used linoleic acid. In contrast, blueberries had a high antioxidant activity in both of these assays (Table 7.3). Much of the current research on fruit acknowledges phenolic components as major contributors to antioxidant capacity. However, fruits such as melons and Sea buckthorn, which are high in lipophilic antioxidants, may best be measured in non-aqueous assays.

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306 Table 7.3.

W. KALT Antioxidant capacity of various fruit crops. Oxidized Substrate

Fruit species Apple Banana Blackberry Blueberry Blueberry (albino) Buffaloberry Cherry (Bing) Cherry (Burlat) Chokeberry Cranberry Crowberry Elderberry Grape (red) Grape (white) Grapefruit (white) Viburnum Kiwi fruit Lingonberry Melon Orange Pear Plum Prune Raspberry Saskatoon berry Sea buckthorn Strawberry

β or R-phycoerythrin (µmol/g DW)

LDLy (µmol/mg GAE)z

Liposome PCx (inhibition % of hexanal)r

Linoleic acid (inhibition % of hexanal)r

(%)

13.2w 9.0w 343v 438u

14.14v 13.77u

83.9t 64.8t

67.8t 77.1t

92.1s

302v 110v

14.13v 3.65v 70.0t

68.8t

72.7t

63.6t

82.8t 78.8t

51.8t

t

t

v

574 345v 677v 469v 36.0w

v

13.01 13.43v 12.85v 17.44v

26.2w 48.3w 874v 36.5w 582v 12.9w 51.7w 9.6w 79.1w

13.65v 15.98v

142u

17.9u

345v

15.3v

95.0v 154w

9.48v 24.1u

93.6s 53.9

27.4

z GAE, gallic acid equivalents; yLDL, low density lipoprotein; xPC, phosphotidyl choline in lecithin liposomes; wWang et al. 1996; vKalt et al., unpublished; uKalt et al. 1999; t Heinonen et al. 1998; sVelioglu et al. 1998; rMeasured at 10 µM GAE.

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VI. CONCLUSIONS Hundreds of epidemiological studies have demonstrated a negative correlation between specific cancers and fruit and vegetable intake. Although fewer data exist on the relationship between plant food consumption and CVD, observational studies report a protective effect of plant food intake against various CVD endpoints, including cardiovascular mortality, myocardial infarction, and stroke (Gillman 1996). To date, information on the role of fruit and vegetable supplementation in neuroprotection has been limited to animal intervention studies (Joseph et al. 1998, 1999). Our understanding of the mechanistic basis for associations between plant food components and disease etiology is evolving through information obtained from in vitro studies. Animal and human studies also contribute to this understanding, and are essential in demonstrating in vivo efficacy. Ultimately, in vivo biomarkers of treatment-dependent physiological effects will be required to conclusively demonstrate a link between food components and disease outcome. In the meantime, the increased awareness of food phytonutrients has renewed consumer interest in the possibility of managing personal health and disease risk through lifestyle, and in particular, dietary choices. What does this mean to the horticulturist? Certainly many fruit crops warrant further health research by virtue of their phytonutrient content; these will become more apparent as modern methods are applied to fruit phytochemical analysis (Song et al. 2000). Both genetic transformation and conventional breeding aimed at enhancing nutritional quality open many possibilities for new cultivar development. The impacts of production, storage, and processing practices on fruit phytonutrient content are other areas of opportunity for the horticulturist and food scientist. A continuum of research and product development in horticulture and food science is unfolding against the backdrop of publicly funded initiatives to encourage fruit and vegetable consumption. The U.S. National Cancer Institute’s “5 a day” program encourages consumers to include at least five servings of fruits and vegetables per day in their diet. Ultimately, these programs are designed to decrease the social and economic impact of rising health care costs and the aging American population. Taken together, the science and industry of fruit phytonutrients will have a substantial and positive impact on the field of horticulture.

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Wang, H., G. Cao, and R. L. Prior. 1996. Total antioxidant capacity of fruits. J. Agr. Food Chem. 44:701–705. Wang, S. Y., and H.-S. Lin. 2000. Antioxidant activity in fruits and leaves of blackberry, raspberry and strawberry varies with cultivar and developmental stage. J. Agr. Food Chem. 48:140–146. Wargovich, M. J. 2000. Anticancer properties of fruits and vegetables. HortScience 35:573–575. Waterhouse, A. L., and R. L. Walzem. 1998. Nutrition of grape phenolics. p. 359–385. In: C. A. Rice-Evans and L. Packer (eds.), Flavonoids in health and disease. Marcel Dekker, New York. Wattenberg, L. W., A. B. Hanley, G. Baraney, V. L. Sparnins, L. K. T. Lam, and G. R. Fenwick. 1986. Inhibition of carcinogenesis by some minor dietary constituents. p. 109– 203. In: Y. Hayashi (ed.), Diet, nutrition and cancer. Sci. Soc. Press, Tokyo. Wattenberg, L. W. 1983. Inhibition of neoplasia by minor dietary constituents. Cancer Res. 43:2448s–2453s. Whitehead, T. P., D. Robinson, S. Allaway, J. Syms, and A. Hale. 1995. Effect of red wine ingestion on the antioxidant capacity of serum. Endoc. Met. 41:32–35. Williamson, G., A. J. Day, G. W. Plumb, and D. Couteau. 2000. Human metabolic pathways of dietary flavonoids and cinnamates. Biochem. Soc. Trans. 28:16–22.

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8 Producing Sods over Plastic in Soilless Media Henry F. Decker Buckeye Bluegrass Farms, Inc. Box 176, Ostrander, Ohio 43061

I. INTRODUCTION A. Conventional Sod Production B. Environmental Drawbacks II. PRODUCING SODS IN SOILLESS MEDIA A. The Concept B. Harvesting Soilless Sods C. Environmental Benefits III. DEVELOPMENT OF THE CONCEPT A. Seed Mats, Sheets, and Carriers B. Pregerminated Seed Mats C. Seedling Turfs D. Steps Toward Growing a Mature Sod IV. PRODUCING MATURE SODS OVER PLASTIC A. Defining a Mature Sod B. Early Experiments C. Materials Tested D. Nettings E. Machines to Insert Netting V. PRODUCING SODS FOR GOLF GREENS VI. SOLVING THE PROBLEM OF A STABLE CONTINUUM A. The Mulch/Medium/Matrix (MMM) System B. Using Vegetative Propagating Material VII. SUBSEQUENT PROPOSALS IN THE GENRE VIII. MANUFACTURING SODS IX. NEW MACHINERY A. The Liquid Mulch Sod Planting System B. Using Selected Composts in the LMSP System

Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 317

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X. FUTURE POTENTIAL A. Producing Vegetative Propagating Material B. Growing Non-Grass Sods C. Growing Forage Crops D. Filtering Waste Water XI. SUMMARY LITERATURE CITED

I. INTRODUCTION A. Conventional Sod Production In the conventional system of producing sods directly in soil, the soil is plowed, harrowed, graded, and seeded usually in the spring or fall. Almost continuous cultivation of the grass, involving mowing, irrigation, fertilizing, and application of pesticides, is required for 1 to 2 years until the sod has “knitted,” that is, will hold together in a sheet or roll while being harvested. Since the sod cutting machine used for harvesting cuts off the bulk of the root system, leaving it behind in the soil, the knitting or binding of the grass plants into a sod is due largely to the production of soil-level stems known as tillers, rhizomes, and stolons. The slowness of their development accounts for the relatively lengthy period of time necessary to grow a conventional sod to the point where it will remain intact when harvested. After a conventional sod is harvested and laid on a new site, it may take several weeks to regenerate a new root system and hence to bind to and root into the new soil surface. The sod must be kept moist during this period, often requiring large quantities of water. At the beginning of the new millennium, there is over 300,000 acres (122,000 ha) of farmland in the United States devoted to the production of turfgrass sods. The average sod farm has about 250 acres (101 ha) under cultivation and sells about one half of this production each year with gross incomes per acre of about $4,500 ($11,000/ha). Across the industry about $15,000,000 is spent each year on pesticides (Anon. 1998, 1999). To produce conventional premium bluegrass sod in the United States can require as much as 100 cubic yards/acre (189m3/ha) of topsoil, which amounts to a transfer of over 30 million cubic yards (23,000,000 m3) of topsoil per year. B. Environmental Drawbacks The conventional method of producing sods on soil has several difficult environmental drawbacks. Aside from the extensive use of pesticides and topsoil, conventional sods are often very heavy, difficult to handle,

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and expensive to ship. The production cycle is often lengthy and fragmented, requiring much mowing, watering, and fertilizing. To eliminate some of these drawbacks and to help find solutions to the nation’s growing problem with extensive quantities of waste materials, the National Science Foundation supported a study beginning in 1988, proposed by Buckeye Bluegrass Farms in Ostrander, Ohio, to determine the feasibility of growing grass sods in a unique manner in which topsoil was replaced in the production cycle by waste materials such as composted yard mulch, sewage sludge, flume sands, and other waste materials. The volume of waste materials that could be used in the proposed novel sod growing system is not small. If a suitable waste material could be substituted for topsoil at the rate of 100 cubic yards per acre, then just a modest sod farm of say 500 acres (1,235 ha), producing two crops per year, could utilize all the yearly dry sludge production of, for example, Philadelphia (which reports approximately 100,000 cubic yards or 76,511 m3 of dry sludge per year). Expressed another way, there is an estimated 300,000 acres (741,000 ha) of cultivated sod under production each year in the United States. If we assume that only 5%, or 15,000 acres (37,050 ha) of this production were devoted to the new sod growing system discussed below, using sewage sludge as the main ingredient, then this 15,000 acres would use the equivalent of the total dry sludge of New York, Chicago, Philadelphia, Boston, and Washington combined. Clearly there is an environmental possibility here of great magnitude for the U.S. sod industry. If the technology can be refined and adapted to cost-effective production on a large scale, then an entirely new avenue of resource recovery could be introduced into the handling and disposition of otherwise troublesome waste materials along with a better and more economical method of growing grass sods while conserving topsoil.

II. PRODUCING SODS IN SOILLESS MEDIA A. The Concept Compared to the conventional method of grass sod production on soil, producing sods in prepared growing media over plastic sheeting, or any root-impervious barrier, has several unusual and superior features that make the concept very attractive. By capitalizing on the tremendous capacity of grasses to quickly form a primary, fibrous root system (a

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single grass plant has been shown by Dittmer, 1976, to produce over 639 m2 of root surface in 17 weeks or less ), high-quality grass sods can be produced much more quickly than with the older method of growing sods directly in soil. By germinating the grass seed over plastic sheeting in suitable growing media, often derived from composted waste materials, the entire primary rooting system of the grasses remains intact during the growing period. Being unable to penetrate the plastic sheeting and being bathed in a moist, rich nutrient mix spread evenly across the surface of the plastic, the rapidly growing fibrous root system is “trapped” and quickly binds and knits the growing medium into a sod that can be harvested in just a few weeks rather than a year or more. A tall fescue sod, for example, which takes as much as two years to produce on soil, can form a high-quality, harvestable sod on plastic in as little as 7 to 10 weeks (Decker 1989). B. Harvesting Soilless Sods The new sod can be harvested by simply rolling it off the plastic sheeting in big rolls typically 4 feet (1.2 m) wide and 2 feet (0.6 m) or more in diameter (Fig. 8.1). It is similar in effect to the Big Roll System devel-

Fig. 8.1.

Harvesting a seven-week-old tall fescue sod in a big roll off of plastic sheeting.

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oped by Beck (1971) for the conventional sod industry. Novel machinery has been developed for sod production over plastic. It includes the rolling cutter invented by Holmes (1993) that cuts the sod without cutting the plastic sheeting and the prong utility harvester proposed by Decker (1995) that can roll the sod off the plastic, load it directly onto a truck bed, and install it at the laying site. In some cases it is possible to leave the plastic sheeting in the field to be used again for subsequent crops. Since it is harvested with its primary root system intact, rather than being cut off by a sod cutter, as in the conventional method, the new sod binds and roots rapidly to its new laying site. C. Environmental Benefits There are several impressive, often environmentally positive, benefits to this innovative production system: waste materials used as growing media are converted into a useful and marketable product (Decker 1991b); the waste materials are used outside the food chain in a closed, environmentally safe, manner; since the sod is grown in a shorter period, much less water, fertilizer, and mowing are required to produce it; topsoil is conserved; many valuable plant nutrients can be recovered and recycled rather than being lost; if grown solely in composted organic matter, the sod is often lighter, thinner, easier to handle and less costly to ship; and pesticide use is often reduced to a bare minimum or eliminated completely (Decker 1991c).

III. DEVELOPMENT OF THE CONCEPT A. Seed Mats, Sheets, and Carriers The first step on the way to the goal of producing mature sods on a field scale in large rolls over plastic was the development of the seed mat or seed sheet. The concept of adhering seed to a sheet or mat, which is then laid or unrolled on a prepared soil surface, ready for watering, goes back to a United Kingdom patent issued to James MacDonald in 1912. The various materials and their combinations used to construct seed mats or sheets are extensive but, typically, some type of biodegradable backing such as tissue paper, crepe paper, or papier-mâché and one or more layers of fibrous material such as burlap, cotton, flax, or pulp and other cellulose fibers, cotton fabrics, cotton wadding, geotextiles, nonwoven plastics and fabrics, Sphagnum peat moss, straw, wood chips, sawdust and various paper, cotton, and plastic webs, meshes, or nettings

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are combined in layers in which or to which seed, fertilizers, various pesticides, various adhesives, and sometimes gel particles are embedded, attached, glued, asphalted, or stitched together. As well as MacDonald (1912) other seed mat examples include those of Pratt (1934), Clapp (1934), Watson (1949), Kuestner (1952), Chohamin (1958), Tietz and Tietz (1959), Marshall (1959), Allen (1960), Merrill and Tinker (1963), Amar et al. (1964), Lippoldt and Woods (1970), Muldner (1975, 1980, 1982), Yoshimi (1978), Knolle and Grimm (1979), Ball (1982, 1983), Baron (1982), Gaughen (1986), Kaneko et al. (1992), Besing (1994), Sakate et al. (1995), Ecer (1995), Welch (1999), Marshall and Marshall (1999); and others. A more or less typical seed mat is described by Muldner (1982) as follows. A laminated mat for growing lawns or other vegetation on soil comprises a base sheet (tissue paper) of water-pervious, biodegradable web material. Joined to the base sheet by an adhesive binder (rubber cement) is a bed formed of dried gel particles (Terra-Sorb), seeds, and dried compressed peat particles. Secured to the bed of seeds, gel, and peat particles is an upper laminate (an unspecified fibrous, non-woven, synthetic web material claimed to biodegrade in 12 to 18 months) comprising a fibrous, porous veil that protects and retains the bed there below. The upper veil is water-pervious to pass water through to the bed of gel particles, peat particles, and seeds. The gel particles absorb up to 200 times their weight in water, facilitating wetting of the peat particles and seeds, and creating sufficient weight in the mat to resist high wind lift forces. An example of a less complex seed carrier is that of Watson et al. (1964) described as follows. The seed is secured with a water-soluble adhesive (polyvinyl alcohol) to a film of 1–6 mil green, translucent polyethylene. The film is then unrolled over the prepared site to be planted with the seed side of the film down against the soil. Water vapor collects beneath the film, dissolving the adhesive, which allows the seed to drop onto the prepared soil below the film and germinate. After the seeding has been protected long enough to become sufficiently established, the film is removed from the planting site. Hydrogels and other super absorbents in seed mats are also proposed by McFarland and O’Connor (1988) and Welch (1999). Kaneko et al. (1992) use an “adhesive agent containing powdery ferro-magnetic substances” to position the seed in their seed mats. Peat paper and peat matrix seed carriers are proposed by Schmidt (1981), Fjeldsa (1981), and Lopez (1993). Mats, films, or carriers comprising plastic foams have been proposed by Ohsol (1966), Swan (1972), Rubens and Clarke (1974),

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Wood et al. (1974), Wood and Frisch (1975), Gluck et al. (1977), and King and Vallans (1982). Slayter and Thomas (1940) use Owens Corning glass “wool” as the carrier for the seed and fertilizer. Knop and van Banning (1991) use rockwool in their “grow-mat.” Like rolls of sod, these early, ungerminated seed blankets were unrolled over a prepared soil surface and watered. Mats of polyolefin, polypropylene, polyester, rayon, and other nonwoven fibers and fabrics have been popular materials for seed mat production, as in Hansen and Oace (1961), Franklin and Gagle (1971), Clark (1993, 1998), McFarland and O’Connor (1988), Anton (1993), Robertson (1993), Taniguchi et al. (1993), Molnar and Molnar (1993, 1994a, 1994b, 1995a, 1995b, 1996), Molnar et al. (1994, 1996), and Molnar and Mitchell (1996). Seed mats have been reviewed by Ball (1982, 1983); and several authors such as Watson et al. (1964), Baker (1972), Muldner (1975), Ball (1982, 1983), Yoshimi (1978), Gaughen (1986), McFarland and O’Connor (1988) disclose devices for the commercial production of seed sheets and mats. B. Pregerminated Seed Mats Seed in fibrous, burlap blankets (Watson 1949; Tietz and Teitz 1959) or in nonwoven, largely flax cloth (Baron 1982) can be watered and pregerminated before being rolled up and transported to the installation site. Obholzer in 1962 described seed mats for use on soccer fields in Germany. Wire netting, burlap, or an unspecified material with holes is placed over a hard, smooth surface such as rolled soil. Spread evenly over the wire netting or burlap to a depth of 3 cm is a growing mixture comprising peat and seed. This is then watered for 2 to 3 weeks until the seed germinates and then the seed mat is rolled up, delivered, and installed on its new site. Four or five more weeks of growth in place are required before the seed mat is rooted sufficiently to its new site and mature enough for a playing field (several more weeks would be required on an American football field). Miyachi (1991) used vegetative propagating material of bermudagrass sandwiched between two layers of plastic netting. The layers of the sandwich are lightly threaded together, rolled up, and taken to the planting site, where the sprig mat is unrolled, covered with soil, and watered. In one embodiment, he “mulches” the double netting with perforated plastic film, which is removed after the sprig mat is planted, germinates, and begins to raise the film. All of the above seed mats, sheets, or blankets are taken to the installation site, sometimes pregerminated as in Watson (1949), Tietz and

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Tietz (1959), and Obholzer (1962), and laid or unrolled, sometimes covered with soil as in Miyachi (1991), and then watered. There is no mention in these processes of germinating the materials on a root-impermeable base such as plastic sheeting to trap the primary rooting of the grasses to form a mature sod before it is transported to the installation site. Tietz and Tietz (1959) take a step toward this concept in that they start with an “impervious material such as Saran Wrap” on which seed and plant food are deposited on the burlap, moistened, and covered and wrapped up in a second layer of the plastic and “hermetically sealed” into a “live grass package” that goes to market pregerminated. C. Seedling Turfs Preceding the concept of producing mature sods over plastic was the idea of pregrowing a “seedling turf” in a mixture of growing medium (consisting typically of peat and/or sand) and seed. The mixture is spread over an impervious base such as plastic sheeting. In the late 1960s and early 1970s three commercial seedling turfs were produced in England and Scotland. The first was “Tana Grass” developed by Goodall (1972a) in which a layer of very light growth medium was laid on a shallow lagoon of water and covered with a thin layer of polystyrene on which seed was broadcast. When the seed had germinated into seedlings, the young turf was rolled up and transported to the laying site. Goodall (1972b) changed his technique (Shildrick 1974) to placing “shoddy” (cheap woolen fabric) over a base of polyethylene sheeting over which was placed a layer of moistened sand or soil. After sufficient growth the planting was rolled up in the base of plastic sheeting and transported to its new site. Goodall also considered the use of unspecified nettings and of “vegetative cuttings” in place of seed. A second seedling turf, “Netlon Bravura Turf,” was developed by Loads (1975). In this young turf, the grass seed was mixed in a growing medium consisting of perlite and lignite and spread over plastic sheeting to which a fine plastic net had been lightly bonded. As in Goodall above, in two weeks or so, the grass had rooted sufficiently to roll the seeding up in the plastic sheeting and netting and to transport it to a prepared site where it was unrolled and the plastic sheeting slipped out from under the germinated seed mat. Later, Loads (1978) eliminates the netting, allows the grass to grow approximately 4 weeks, but still rolls it up and transports it in the base of plastic sheeting. For this process, like Dawson (1976) in Scotland,

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Loads grew the seed mats in containers comprised of rectangular frames of wooden boards (“kerbs”), which would make such an operation on a field scale economically prohibitive. A third turf mat, and perhaps the one closest to a true sod, was that developed by Dawson (1976) of the Scottish Agricultural Industries Ltd. and called the “S.A.I. Turf Process” or “Rolawn.” It consisted of germinating the seed outdoors in a 0.5 to 0.75 inch (12 to 19 mm) layer of peat containing fertilizer. For protection, a cover sheet of interwoven polypropylene was placed over the young seedlings; and, like Loads’ method, the grass was grown in rectangular wooden frames. In a study reported by Shildrick (1974), the three processes were compared in a seven-month field trial at the Institute’s facilities in Bingley. All three systems with attentive care eventually provided “satisfactory swards.” Despite the lack of netting, Dawson’s “S.A.I. Turf Mat was the most impressive in its strength at laying and in the vigor of grass growth.” Since most of these seed mats were not yet mature sods, Shildrick notes that the turf “becomes increasingly the reflection of the user’s management (mowing, feeding, top-dressing) to achieve satisfactory levels.” Two other processes from the United Kingdom about the same time as the three above (and possibly arriving too late for inclusion in the Shildrick trials) were those of Mercer (1974) and Blackburn (1975). Mercer placed a growing medium containing seed over a root-impervious film to which had been bonded “at a plurality of spaced points . . . a flexible reticulate sheet material.” After sufficient germination and growth of the new grass, the bottom layer is stripped off the reticulate sheet “to expose the grass roots prior to laying the grass lamina in situ.” Blackburn uses as many as three layers of plastic to effect his germinated seed mat: one is the base; the second is the wrapper; and the third is perforated with holes smaller than the seed. The seed is mixed with compost and spread in a thin layer over the perforated plastic. The germinated seed roots through the perforations and in 3 weeks is rolled up inside the second layer of black plastic off of the base. When rolled out on a prepared site, the wrapping layer of plastic, as in Goodall (1972), Mercer (1974), and Loads (1975), is removed from under the root surface. Under a grant from Frank Mercer, studies were initiated at Lancaster University in England from 1971 into 1973 that confirmed the net sod techniques of Loads and Mercer and refined them into the “Tuft 1 system.” Mercer also provided a grant to Michigan State University (MSU) to examine and test the net mesh produced by the Netlon Co. of Blackburn, Lancashire, England (Beard 1976). As with Loads and others, the

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studies at MSU confirmed that a netted seedling turf could be produced in 2–3 weeks under greenhouse conditions and in 4–6 weeks under field conditions using mixtures of fine fescue, Kentucky bluegrass, and perennial ryegrass. It was reported that Kentucky bluegrass grown alone took two to three times as long to produce a netted seedling turf. The MSU studies also noted one of the yet unsolved problems in sod production over plastic. Under field conditions the Tuft 1 system “was prone to soil erosion during intense rainfalls.” In planting grass lawns of the size that require a grading tractor (the typical U.S. home lawn), seed mats, seed sheets, and seedling turfs, while very appealing, have a major problem. They have to compete price-wise with the less expensive conventional seeding method in the hands of a skilled grader using a Brillion seeder and a straw blower. D. Steps Toward Growing a Mature Sod An important new concept in the evolution of producing mature sods over plastic occurred at this time, probably concurrently or at least independently, in the United States and in the United Kingdom. The thought was expressed by J. L. Dawson, developer of the S.A.I. Turf Mat, in 1975 (pers. commun.) in response to an article by Decker (1975) that netting was unnecessary and that the strength of the turf mat arose “solely from the knitting together of the mass of the primary rooting systems.” These were the first expressions of the concept that the trapping of the primary rooting system over a root-impervious surface could be sufficient to produce a mature sod without any nettings, meshes, or other reinforcements. None of the publications quoted so far had recognized this significant feature of the primary rooting in grasses. It had been assumed that producing a grass sod over plastic would require a net or mesh or fabric. J. L. Dawson grew his “turves” in long beds or containers with wooden sides, as did Loads (1975, 1978), covered the seeding outdoors with interwoven polypropylene, and grew it in fine peat . . . any one of which would be prohibitive in the United States. Nevertheless, he introduced three important features: nettings were eliminated; the “turves” during their growth were cut and watered; and the grass was grown for six to eight weeks before transporting. These features are most likely the reasons why Shildrick (1974) referred to Dawson’s “S.A.I. Turf Mat” as the “most impressive in its strength at laying and in the vigor of grass growth” when compared to the seedling turfs of Loads and Goodall. Dawson was close to producing a mature or true sod over plastic sheeting.

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IV. PRODUCING MATURE SODS OVER PLASTIC A. Defining a Mature Sod The works cited above involve seed carriers, grass mats, or “seedling turfs” sometimes comprising the germination of grass seeds for a few weeks, usually over a mesh or netting that are frequently rolled up in the plastic base and transported to the laying site. They are not yet true or mature sods, which are distinguished by several features: 1. Grasses in a true sod have begun to tiller, that is, produce additional plants from the same crown, or to produce stolons (above ground running stems) or rhizomes (below ground running stems). These secondary processes typically do not occur in abundance until the new planting has been mowed several times. Except for Dawson (1976) none of the processes above mention or indicate mowing. 2. In a true sod, the grasses will hold together, like a blanket when lifted off the plastic. They can withstand the rigor of being harvested, handled, loaded, and installed in big rolls 4 feet (1.2 m) or more wide and 2 feet (0.6 m) or more in diameter. 3. They do not require any artificial nettings or have to be wrapped in plastic for support when they are transported and laid. 4. A true sod can be played on in just a few days after it is installed. A germinated seed mat, on the other hand, may take several weeks or even months before it becomes “hardened off” and can withstand the severe stress of, for example, a football game. None of the seed mats or “seedling turfs” cited above meets all these criteria.

B. Early Experiments The first extensive experiments leading to the production of mature sods (rather than seed mats or seedling turfs) over plastic sheeting on a field scale began at Ohio Wesleyan University (OWU) in Delaware, Ohio in the late 1960s. The research was moved in 1972 to an operating sod farm (Buckeye Bluegrass Farms) near Ostrander, Ohio. An article about the research appeared on the front page of the Business Section of the April 20, 1975 Sunday edition of the Columbus (Ohio) Dispatch and was widely distributed by the Associated Press. The possibility that large quantities of topsoil could be replaced with composted waste materials to produce a valuable crop attracted the attention of the National Science Foundation (NSF), which eventually funded much of the research. The experiments in Ohio were completely independent and had an entirely different emphasis from those that were underway at the same

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time in England, Scotland, and elsewhere in Europe. At OWU the challenge was to develop sods on a scale commensurate with U.S. commercial and conventional grass sod production. This meant developing techniques that could be applied to large outdoor areas and that would be competitive with conventional sod production directly on soil. Any new growing system had to be stable in severe thunderstorms. The final product also had to be a mature sod, unlike the seed mats produced at this same time in the United Kingdom and Germany. The production process had to allow for mowing the growing turf several times before harvest to encourage tillers, stolons, and rhizomes; and the turf had to hold together well enough to withstand harvesting, handling, shipping, and installing in big rolls. It was also obvious that the new system would lend itself to the production and harvesting of sods in big rolls 4 feet (1.2 m) wide and 2 feet (0.6 m) or more in diameter. This concept of producing mature, finished sods in big rolls over plastic sheeting without any netting seems to have arisen independently in the United States. While Dawson also concludes that netting was not as important as increasing the primary rooting of the grasses, the seed mats developed in the United Kingdom in the late 1960s and early 1970s were not mature enough sods to be of interest in the United States. Severe storms, wooden curbs, lack of mowing to increase tillering before harvest, having to use covers in open areas, too weak to harvest in big rolls . . . made the previously developed systems uncompetitive with the U.S. conventional sod industry. C. Materials Tested In the early work at OWU many different materials were tested for use as bases, growing media, and binders. This work was assembled into a comprehensive 1973 U.S. patent application (# 371,462), and a summary of some of the materials and experimental combinations in this work appear in Table 8.1. Of the materials tested for a base, it quickly became obvious that 0.5to 4-mil plastic films worked best. Several of the growing media that were tested alone or in various combinations worked, but a few—such as various composts and manures and peat/sand combinations that were already standards in the golf industry—were obviously superior. In later experiments for the National Science Foundation, flume sands (incinerated sewage sludge from Columbus) and spent mushroom soils from Campbell Soup’s mushroom producing facility in Jackson were tried alone or worked into combinations. Composted yard mulch was also

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Materials tested as bases, growing media, and binders.

BASES Packed clay, cement, various plastic foams, compressed cellulose, fiber boards (American Can Corporation), various cardboards, mold proof papers (Union Bag Camp), brown Kraft papers, perforated and unperforated 0.5-, 1-, 2-, 4- and 6-mil clear, white, and black plastic films, and plastic films with cellulose wadding attached (Kimberly Clark’s “Kim-Pac”). GROWING MEDIA Vermiculite, shredded paper, spun plastic, pulp fiber mulch, cotton batting, wood chips and bark, sawdust, ground corn cobs, seed hulls, calcined clay, various sands, peats, and manures, composted leaves, garbage, and sewage sludges, ground aspen bark, and various combinations. BINDERS Burlap or jute netting, woven cellulose netting, sisal netting, cheesecloth, various plastic foams, polyester fiber netting, polypropylene and other plastic nettings.

beginning to appear in abundance, and this material alone or in combinations with composted sewage sludge, sand, or flume sand proved to make superior growing media (Roberts et al. 1995). D. Nettings Various nettings, textiles, webs, and meshes have been used to stabilize seed mats and seedling turfs. Those that were considered in the early years of the OWU experiments are listed in Table 8.1. At about the same time, Loads in 1970 in England, as mentioned earlier, had begun experimenting with a net mesh (“Netlon”) to produce seedling turfs; and Michigan State University tested the same mesh in the production of sods over plastic as well as sods grown in the conventional manner on soil (Beard 1976; Beard et al. 1980). DuPont’s Vexar netting was tested in 1972 by Cockerham (1984) at the Rancho Verde Turf Farm of Cal Turf in California. Burns (1980, 1981, 1982) at the University of Georgia proved that sods of tall fescue, bermudagrass, and centipedegrass could be produced much more quickly if plastic netting was placed on the surface or slightly below the soil in new seedings. As a result of the above works, plastic netting became popular in the conventional sod on soil industry to help speed sod binding and to increase its integrity during harvest.

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E. Machines to Insert Netting Machines have been invented (Grether 1975; Huggett 1976; and Rehbein 1979) for conventional sodding on soil that skim off a thin layer of soil, unroll netting on the skimmed surface, and redeposit the skimmed soil back over the netting. Parish et al. (1991) developed a machine to lay reinforcing material in sod fields of centipedegrass. In the early OWU experiments, as with Dawson (1976) in Scotland, it was felt that grasses grown on plastic formed sods so quickly that adding artificial nettings, meshes, or fabrics seemed an unnecessary cost. There was also some uneasiness about difficulties that might arise in the subsequent care of a turf, especially on golf greens, and also about the remote potential for injury caused by nettings that were not biodegradable.

V. PRODUCING SODS FOR GOLF GREENS One of the early directions in the production of sods over plastic was for golf greens. The first grower to produce conventional sods exclusively for golf greens was Alec Rohozo of Sewickley, Pennsylvania (Decker 1999a). Starting in the 1950s he began covering his sod farm with sands and soils recommended by the United States Golf Association (USGA). He became the first grower to custom match the growing medium of the sod with the soil profile of the green on which the sod would be installed. Over the following decades his custom-grown sods were installed on many of the country’s most prestigious golf courses, including six times for The Masters at the Augusta National in Augusta, Georgia. The first actual attempt to produce greens-quality sods over plastic in peat/sand was that of a Sun Oil Executive named Voorhees Anderson in Wilmington, North Carolina, in 1973. He and his cousin, F. B. Anderson, experimented with growing greens-quality sods over plastic for the Myrtle Beach golf course area using the technology that had been developed to that time at Ohio Wesleyan University (pers. commun.). Later in an article in Grounds Maintenance, Beard (1976) mentions that beside an “extremely rapid production time” another advantage of growing sods over plastic is “the flexibility to produce sod with prescribed rootzone mixes, which can be of particular importance on sport fields and greens where root zone modification is utilized.” The work in Wilmington came to an end abruptly in part because of the premature death of Voorhees Anderson and because the technology at the time lacked provisions for stabilizing young sod plantings in thunderstorms. A thin 0.5 to 0.75 inch (1.3 to 1.9 cm) layer of a sand/peat

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growing medium placed on plastic sheeting and sprigged with bermudagrass was very hard to stabilize in strong, drying winds often followed by a heavy rain. The exposure of a young turf in its first few weeks to sheet erosion or gapping from even a modest thunderstorm ended in too many complete crop failures. Many subsequent methods proposed for growing sods over plastic have been essentially inoperable because the critical problem of thunderstorms and gapping have been left unaddressed. In 1993 these same problems clouded the future of a young sod company, RapidTurf Inc., of Rincon, Georgia. Despite newly issued patents (Egan 1994, 1997) for growing golf greens-quality sods over plastic in sand/peat, RapidTurf was struggling with the same difficulties of stability that had plagued the 1973 attempt in Wilmington, North Carolina. Aware of the earlier NSF supported research that had been conducted in Ohio, RapidTurf approached Buckeye Bluegrass Farms. An association was formed whereby the research efforts in Ohio were moved in the fall of 1993 to Georgia (Decker 1998a). One of the primary goals was to solve the problem of stability in the production of greens-quality sods grown in sand/peat over plastic. Reaching this goal in a cost-effective manner required some intense research over the next two and a half years.

VI. SOLVING THE PROBLEM OF A STABLE CONTINUUM It became obvious that several key problems had to be solved before the production of mature sods over plastic sheeting could literally get off the ground. First, the young sod had to be stabilized in thunderstorms. Second, the growing material had to be stable on the plastic and spread with extreme evenness and thickness. Gaps over plastic produced holes in the sod. Third, to be cost-effective, the amount of growing material placed on the plastic had to be thin enough to produce sods that were one-half inch (1.3 cm) thick or less. Fourth, the costs of any additional materials such as covers, nettings, meshes, and nonwoven fabrics had to be cost competitive with conventional sod production. If all growing conditions are maximized, grasses will root and grow quickly over plastic sheeting. A new sod planting will have formed enough root mass in 3–4 weeks to bind the growing medium/mulch into a fairly stable structure that will withstand at least a modest thunderstorm. Turf covers are uneconomical on a field scale, but pregerminating the seed or sprigs and treating them with growth hormones can be helpful in closing the 3–4 week window of danger from severe thunderstorms. Various glues, tacks, adhesives, and gels can help. But the

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most effective system that protects seedings and spriggings from devastating thunderstorms, starting day one of the planting, and which concomitantly produces a cost effective continuum over the plastic, is by the use of vegetative mulches. A. The Mulch/Medium/Matrix (MMM) System A new concept began to emerge in the 1978 summer experiments at Buckeye Bluegrass Farms. In the conventional sequence of doing a lawn seeding into soil (the growing medium), the new site is finish graded, fertilized, and seeded. It is then typically covered with a mulch such as wheat or pine straw followed by careful watering. In the new concept, the conventional seeding sequence is reversed. First, a vegetative mulch such as wheat straw is placed over the plastic film; secondly, a carefully determined and exact quantity of a growing medium (topsoil now being replaced by waste organic composts or sand) is applied; and then seeding takes place (or the seed is mixed with the growing medium when it is applied). The growing medium and seed filter down, or are irrigated down, into the interstices of the mulch layer, where it is protected and thus stabilized in heavy rains. There is another important feature of the MMM sequence. Since the mulch protects the young seedlings, it is possible to subsequently run tractors and other equipment over the new planting without disturbing development. The results from this simple resequencing were dramatic. In the right combinations this MMM system was surprisingly resistant to laminar flow and hence to sheet erosion (Decker 1991a, 1993, 1996a). At the first irrigation, the mulch is soaked with water so that it expands and becomes essentially immovable even in a 4-inch (10 cm) rain. In effect, and what proved to be a key in the evolution of this technology, was that the biodegradable mulch combined with a suitable growing medium formed a relatively inexpensive stable continuum that was highly resistant to gapping in storms and was essential and central to the successful production of sods over plastic sheeting. If the MMM system is carefully constructed, even a sod as thin as 0.25-inch ( 0.6 cm) can be stabilized in severe thunderstorms. The MMM system brought the growing of sods over plastic down to the basics: the propagating material, the growing medium, and the mulch. Covers and nettings were obviated, and growing sods over plastic became economically viable. It was found that using a mulch alone, such as straw or wood fibers, resulted in too much material with a very low cation exchange capacity (cec); and comminuted straw had a tendency to blow off or quickly

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wash off the plastic, making it difficult to form a stable continuum. A carefully selected or contrived growing medium (preferably with a high cec) that would fill the interstices of the mulch was an essential ingredient to form and stabilize the continuum and ultimately to produce a high-quality sod. B. Using Vegetative Propagating Material The mulch/medium/matrix system was established using several types of seeded cool-season turfgrasses (Decker 1991a, 1993; Roberts et al. 1995); and it was also tried with warm-season vegetative propagating material, particularly bermudagrass sprigs. There was an immediate and readily apparent advantage in using vegetative propagating material instead of seed. Vegetative propagating material such as sprigs have much more bulk than seed and hence act like and add to the mass of mulch necessary to form the continuum over the plastic. Instead, then, of planting the sprigs into the sand growing medium, tests were established in which the sequence was reversed and the sprigs were first placed like a mulch on the plastic and then covered with sand. The sprig planting rate was increased to produce more of a mulch effect that included more meristems, and these two simple steps alone made a significant improvement in the chances of the young sod surviving to maturity. In a third step, the chances of obtaining a fine turf for a golf green were even more improved by replacing coarse-fiber mulches such as wheat and pine straw with finer mulches such as paper pulp and processed wood fibers (Decker 1996a). Georgia is a good place to experiment with waste paper pulps, and several experiments demonstrated that using them in the matrix system with sand and the vegetative propagating material was very effective and often responsible for the difference between total failure and total success. A fourth improvement arose from experiments with various gels, glues, tacks, and adhesives that essentially cemented the matrix into systems that were resistant to thunderstorms yet still maintained a high degree of viability.

VII. SUBSEQUENT PROPOSALS IN THE GENRE Practically all of the early work on producing sods over plastic, especially in the United Kingdom, is reported in the U.S. Patent and Trademark Office literature. Other than the experiments at OWU from 1969 to 1972 only a few research projects concerning soilless sod production

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were undertaken in the United States in the 1970s. Beard (1976), in greenhouse and small plots, studied the experiments of Loads (1975) in using a mesh or netting (manufactured by the Netlon Co. of Blackburn, Lancashire, England) for use in the production of seedling turfs over plastic and also as a reinforcement of conventional sod on soil production. More extensive studies by Beard et al. (1980) proved that growing conventional sods with netting “could reduce production time substantially.” Busey and Burt (1977) at the University of Florida, in testing bahiagrass for roadside plantings, grew sods “in waste-product media” over plastic “in order to harvest the entire root system. In contrast to commercially prepared sod, this system permitted rapid penetration by roots into dry sand, and successful establishment on a difficult site.” Also at the University of Florida, Neel et al. (1978) planted sprigs of Tifgreen bermudagrass and seed of bahiagrass in composts of sewage sludge, composted wood chips, and composted sugar cane by-product. The growing media were spread 10 cm deep on 3 m × 3 m sheets of 0.1mil black polyethylene. Such a depth would be economically prohibitive on a field scale (the LMSP planter mentioned later grows sods in 0.25 to 0.50 inches (0.6 to 1.3 cm) of growing medium), but the results show that excellent quality sods of both grasses were produced in media that contained composted heat-treated sewage sludge in 51 to 65 days. The sods rooted to their laying sites more quickly than comparable commercial sod. It was indicated that this effect was the result of the experimental sods having intact root apical meristems that were not cut off during the harvesting process, as is the case with conventional sods grown on soil. Research activity began to increase in the 1980s and 1990s. Chamoulaud (1980) in France produced mature sods over plastic in a compost layer made from crushed wood bark. Chamoulaud’s system was tried in the United States but was apparently not successful. Rogers and Goodrich (1988) successfully produced high-quality sods over plastic in growing media comprising “prescribed proportions of water-absorbent wood fiber and nonwater absorptive cellulose particles (preferably ricehulls).” The rice hulls helped to aerate the growing medium and when dry the sod was light and easy to handle and less expensive to transport. Airhart et al. (1983) germinated several wildflower species in peat mixes in 5 cm deep plastic half flats in greenhouse mist beds. Various commercial peat mixes were used as the growing media and cheesecloth netting was used to strengthen the rooting. Later, the wildflower sods were transplanted outside into sandy loam field plots. It was reported that several species made satisfactory sods: black-eyed susan, evening

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primrose, and yarrow at a recommended (1×) field seeding rate, while coneflowers required a 5× seeding rate and dame’s rocket a 10× seeding rate. Subsequent work (Barker 1993; Barker and O’Brien, 1994, 1998; O’Brien and Barker 1995, 1997) evaluated several types of composts produced from municipal solid wastes, biosolids and wood chips, “agricultural wastes” (cranberry fruit pomace and chicken manure), and deciduous tree leaves. Turfgrass and wildflower mixes were seeded into plastic greenhouse trays or outdoors in small, plastic-lined plots filled 5 cm deep with the selected composts. Best results were obtained with mature biosolids compost and with the agricultural waste compost. Aging of each compost improved its sod production capacities. It was claimed that weed-free, marketable, wildflower sods could be produced in as little as 8 weeks. Kabbaz and Petrovic (1987, 1988) of Cornell conducted greenhouse flat and small plot field studies on “accelerated sod production” over black plastic using a bluegrass/perennial ryegrass mixture in 1 inch (2.54 cm) of three distinct media: composted pharmaceutical residue, composted stable waste/horse manure, and aerobically digested sewage sludge. Typically, seed germination and growth was slowest in the sewage sludge due to its high soluble salt content. After it was leached, no other problems with this growing medium were noted. In the greenhouse, harvestable sods (tearing at greater than 30 lbs. of force) were produced in the pharmaceutical compost in 7 weeks, in composted horse manure in 8 weeks; and in the sewage sludge in 12 weeks. In a special presentation by Luft (1988), he states that in growing sods over plastic: “No additional supporting fabric (i.e., netting) is required. The root system of the grass forms a natural fabric of immense strength and flexibility.” These results support the 1975 observations by Decker and Dawson mentioned earlier. Lin Wu (1988) produced “immature” sods of perennial ryegrass, tall fescue, and Kentucky bluegrass in 5 to 6 weeks on a greenhouse bench in 1-foot-square (0.33 m2) plastic pans filled with a peatmoss growing medium adjusted to pH 7.0 with dolomitic limestone. Walton (1990) produced mature sods in 60 days by placing over plastic sheeting a 2-inch layer of spent mushroom mulch followed by Conwed plastic netting, then seeding, followed by another 1.0- to 1.5-inch layer of mushroom compost. Milstein (1998) also used spent mushroom soil to produce various types of plant sod mats. Milstein (1990) placed polyester fabric such as Dupont’s Reemay in perforated plastic flats covered with suitable growing media and seeded in the production of wildflower sod mats. Later Milstein (1998) proposed

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plant sod mats using spent mushroom soil comprising two layers of the growth medium separated by a sheet of polyester, polypropylene, or polyethylene fabric over a base of perforated polyethylene. Cisar and Synder (1992) studied the feasibility of sod production over plastic on a solid waste compost. The compost was spread 5 cm deep on six separate 1 m × 18 m sheets of black polyethylene and seeded or sprigged with bahiagrass, bermudagrass, and St. Augustinegrass. At first the grasses showed a temporary poor growth and discoloration typical of growth on unwashed or unirrigated composts with high salinities. Nevertheless, at 5 months it is reported that the sods were harvestable: “whereas for conventional field production 9 to 24 months generally is required to produce a harvestable product.” Also the compost grown sods had similar or higher tear resistance than conventionally produced sods; and also when transplanted onto a sandy soil, they had longer and heavier roots than commercially grown sod. Clark (1993) placed a nonwoven polypropylene mat (Typar) over a black film of polyvinyl and covered it with a growing medium and seed to produce light sod mats. Many other nets and webs are used for reinforcing sports playing surfaces (Robey 1977; Halliday and Martin 1990) or for soil erosion (Sakate et al. 1991). Molnar and Molnar (1993, 1994a,b, 1995a,b, 1996), Molnar et al. (1994, 1996), Molnar and Mitchell (1996), and Mitchell et al. (1994) have done extensive work using nylon, polyolefin, coherent sheets of stable fibers, and other nonwoven plastic fabrics and plastic nettings to reinforce several kinds of horticultural sod mats. Weber and Delucia (1993) employed a biodegradable latex web composed of natural softwood Kraft fibers and synthetic polyester fibers as a base for a seed mat. The cellulose fiber decomposes, while the polyester easily disperses as mulch. Solomou (1993) describes a more or less “seedling turf” reminiscent of the early works of Loads, Goodall, and others in the United Kingdom mentioned earlier. Coarse sand is placed over a perforated plastic sheeting, then a layer of spun polyester followed by nettings or meshes of various kinds, including hessian or cotton mesh or woven or welded polyolefin, followed by seed mixed in pulp or sawdust. The grass is never mowed and, when harvested, the rooting of the seed mats is extracted from the coarse sand and the layer of spun polyester below the mesh. The seedling roots are shaken or washed free of excess sand, suggestive of Bouchard (1994). Egan (1994, 1997) proposed producing sods over plastic in sand/peat mixes for golf greens similar to the methods of Rohoza and Anderson

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mentioned earlier. Krick (1995) notes that a Kentucky bluegrass/perennial ryegrass sod mix grown on plastic provided a “superior athletic surface when established on a sand based rootzone.” Farrow et al. (1995) proposed producing sods by placing a mixture of carpet pieces in a layer of compost over a root-impervious barrier, followed by a second layer of compost containing seeds, rooted cuttings, and other propagating materials. In an extension of the earlier seed mat proposals in which burlap was commonly used (Watson 1949; Tietz and Tietz 1959) or flax fibers (Baron 1982) or seed was inlaid in a “carpet” of jute and hemp fibers (Anon. 1988), Hensler et al. (1996) grew seeds and sprigs of several warmseason turfgrasses (bermudagrass, centipedegrass, St. Augustinegrass, and zoysiagrass) in two to three layers of kenaf fiber mats placed over 6-mil black plastic sheeting. The seed was embedded in a kenaf fiber mat carrier that was placed over one or two additional kenaf fiber mats. Vegetative planting material was placed directly over two or three fiber mats and covered with rayon netting. “By 15 weeks all plots of sprigged species were nearly 100% covered, and of the seeded plots bermudagrass and centipedegrass were at 100% coverage.” Similar to the jute, hemp, flax, and kenaf fiber seed mats mentioned above are the recycled wood fiber mats (“Ecomats”) reported by Rogers et al. (1996) and Sorochan and Rogers (1998). Several differences were studied in the growth of four turfgrasses on the wood fiber mats over plastic: “After three months, perennial ryegrass sod had greater turf density than tall fescue, Kentucky bluegrass, and supina bluegrass sods. High seeding rates resulted in greater turf density than low seeding rates. Use of organic rather than mineral nitrogen sources, and higher rates of nitrogen fertilization, also resulted in greater turf density. Potassium and phosphorus rates had no effect on turf density.” Another interesting proposal is that of Bergiven (1996), who interplants natural grass into a perforated plastic web base or backing, which is comprised of synthetic grass blades. The natural grass roots grow through the perforated backing and the natural grass blades then combine with the synthetic grass blades to form a more durable playing turf. Bergiven’s invention is also described in Popular Mechanics (Wheeler 1996). A more recent idea is that of Strombom (1999), who proposes killing with herbicides the grass and grass root system of a conventional grass sod, then harvesting the killed sod and placing it on plastic sheeting where it is implanted with seeds or other propagating material of annual or perennial ornamental plants. After the new planting has matured, it

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is then harvested as a new sod having used for support the original, nonliving, conventional grass sod. Roberts et al. (2000a) have demonstrated that it is feasible to produce wildflower sods in flume sand-dominant growing media. Wildflower sods could be produced in 6 to 12 weeks when grown over 4-mil plastic sheeting. “During this time the primary rooting system knitted together into a usable sod which was easily removed from its container and readily transplanted into the field. The addition of 0.5 inch (1.3 cm) plastic netting had no apparent effect on wildflower sod establishment.”

VIII. MANUFACTURING SODS In the early 1987 experiments supported by the NSF at the research farm in central Ohio, sods were grown in contiguous beds 32 feet (9.8 m) wide by 200 feet (61 m) long. These test beds were graded and crowned to a 2% to 3% “road grade” with the plastic sloping to 4 inches (10 cm) wide drainage trenches containing perforated plastic drain lines or 12 inches (30 cm) high verti-drains. This graded bed system was much like the “Farming Method” of Houston (1971) and the later proposal of Gramckow (1996). The irrigation lines with sprinklers were placed in the same trench just above the perforated plastic drain lines, which were also underlain with plastic. The trenches were backfilled with gravel (Decker 1996a). Both the drain and the irrigation lines grade to manifolds at the end of the beds, which in turn grade to a holding pond; all of which can also be underlain with plastic to provide a closed system. As mentioned later, such a closed system can be used to form an effective wastewater “biofilter,” an idea that was submitted in a 1991 research grant proposal to the U.S. Environmental Protection Agency. The bed size was about the average size (700 square yards, 585m2) of a residential lawn in Columbus. At harvest one bed yielded a truckload of sod and the bed could be replanted the same day to be ready to harvest again in 7 to 10 or more weeks depending on the weather and the type of sod. In the graded bed system, which has been used by some of our earlier growers, the 4–6 mil plastic, with a few cultural adjustments, can be used over again (some of the plots going 10 years plus). The plastic on the bed is exposed to UV light only very briefly while the plot is being replanted as soon after harvesting as possible, usually the same day. Also only a small part of the total production is at risk each day from thunderstorms. Holmes (1993) invented a sod cutter that would cut sod with-

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Fig. 8.2. Prototype of sod cutter that cuts sod without cutting the plastic, invented by Professor Robert Holmes of Ohio State University.

out cutting the plastic (Fig. 8.2); and a simple device was invented (Decker 1995) for rolling the sod off the plastic and for loading it in big rolls on a truck. The same device can be used at the installation site to quickly unload and unroll the big sod rolls. Instead of having one large field to plant and then later having to harvest it all at once, this “BioSod” system shifts each element of sod production—planting, growing, mowing, harvesting—onto a daily basis, which is more like a manufacturing than a farming process. The above system, of course, is a necessary adaption to Midwest type heavy clay soils with sluggish drainage, in which case unperforated plastic is required since it is imperative that the soil under the plastic remain as dry as possible. However, on the Coastal Plain and other areas of the country where soils drain rapidly and the grading is less critical, the graded bed system is obviated by draining excess water through perforated plastic into the sandy soil below. The sod can be grown in much wider, larger, and more economical flat beds. The plastic used is on the order of 1–2 mils and is considered expendable. Another feature that has eliminated the need for the narrow, graded bed system has been the recent development of the liquid mulch sod planter (LMSP), which is described later. This machine is effective on

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wider beds, which are much more economical. On upland soils that are more sluggishly drained, gently sloping land is preferred. This is interrupted at wide intervals by grassy swales as in the “Farming Method” of Houston (1971). Care is taken to shingle or seal the unrolling plastic sheeting as it is dispensed from beneath the LMSP.

IX. NEW MACHINERY Machines for producing sods over plastic were patented as early as 1975 by Brem and in 1988 by Heard. Brem’s machine unrolls a plastic base, places on it a dry growing medium or a mulch and distributes seed, all in one operation. Heard (1988) indicates a rough sketch of an apparatus “which has elements for laying an impervious plastic sheeting and for dispensing pellitized straw on the plastic sheeting and for dispensing grass seed on the straw, all in one pass of the apparatus.” Neither of these machines introduces an essential growing medium into a mulch layer to form a stable mulch/medium/matrix, stabilizes young sods in thunderstorms, or provides a stable continuum across the plastic. Brem indicates growing media but not the concept of a mulch layer, while Heard has a straw mulch but no growing media. In earlier experiments in the 1970s, we found it very difficult to grow sods on plastic with just a growing medium. It quickly washed off the plastic or gapped in even modest thunderstorms. With just a mulch such as straw, there was also a tendency to wash off the plastic as well as an insufficient cec to produce a high-quality sod quickly. A nutrient rich suitable growing medium with a high cec infiltrated into a more coarse and distinctive mulch gave the best results. In addition a mulch such as wheat straw is quite coarse and not suitable for growing greens-quality sods over plastic for golf courses, which has become one of the major markets for sods grown over plastic. A. The Liquid Mulch Sod Planting System A more effective process (Fig. 8.3) is the liquid mulch sod planting (LMSP) system (Decker 1998b). A specially designed hydroplanter directs a functionally sterile, complex slurry of mulch, various growing media and/or amendments, and seed and/or vegetative propagating material, onto the splashboard of a trailing planter assembly that drops the slurry in an even patina onto plastic sheeting that is unrolling under the splashboard. The uniformity and evenness with which the slurry is spread eliminates gapping and forms a continuum over the plastic sheet-

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Newly developed liquid mulch sod planter (LMSP).

ing. In keeping with the MMM concept, once slightly dried, the mulch slurry can be tracked and covered by a separate application of a suitable growing medium such as compost, sand, or other growing media or selected treatments. Sods grown using this innovative growing system are typically less than 0.5 inches (1.3 cm) thick and are harvested in less

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than 12 weeks. A second roller is specified in the LMSP to dispense additional plastic film, nettings, meshes, various fabrics, or seeded fiber mats to effect a sod quickly or to produce a specialty crop economically. Another significant feature of the LMSP system is the ability to pregerminate the seed and/or sprigs for specific lengths of time before planting. This helps to close the window of risk from severe thunderstorms. Sod produced in this manner can be essentially “manufactured” rather than harvested. That is, all the features of sod production—planting, growing, and harvesting—are carried out each day rather than on a fragmented, seasonal basis. B. Using Selected Composts in the LMSP System Since sods grown over plastic using the liquid mulch sod planting system require less than 0.5-inch (1.3 cm) of growing medium, sods produced in this manner for playing surfaces should include in their growing media selected composts to benefit from the significant advantages of these materials to suppress diseases, reduce pesticide usage, increase the capacity of soils to absorb and retain chemicals, and to greatly increase microbial activity. Composts apparently harbor pathogens that are antagonistic to various turf fungal diseases (Wilkinson 1994). Nelson (1999) indicates that while specific composts are being used to topdress playing surfaces, they should be incorporated into the rootzone as well, which “will provide a higher and longer-lasting disease suppression than the topdressing amendment.” Research by Roberts et al. (2000b) indicates that the growth of bentgrass (cv. ProCup) in a mix of 80% USGA spec sand to 10% composted sewage sludge (“Com-Til” from Columbus, Ohio) to 10% MetroMix over 4-mil polyethylene was clearly superior to the standard golf green mix of 80% sand to 20% sphagnum peat. Articles on the subject of disease suppression in turfgrasses using composts can be retrieved by searching the databases (using the key words “disease” and “compost”) at the Turfgrass Information Center at Michigan State University (www.lib.msu.edu./tgif).

X. FUTURE POTENTIAL The technique of producing sods over plastic sheeting has wide adaptability. It can be used to grow seeded or vegetatively propagated grasses, including all the common turfgrasses: bluegrasses, bentgrasses,

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bermudagrasses, buffalograsses, zoysiagrasses, and all of the fescues. It is particularly effective in growing tall fescue sods. Because of their weak formation of rhizomes, tall fescues often require two or more years to form conventional sods; however, they quickly produce very extensive primary rooting and hence have an advantage when grown over plastic sheeting. Other adaptations of the novel system include the following. A. Producing Vegetative Propagating Material (VPM) Sods grown in sterile growing media over plastic sheeting, which protects the sod from soil contamination, make it possible to separate the harvested sod into vegetative pieces that will maintain the purity of the original planting stock (Decker 1999b). Using this system, it may be possible to increase valuable cool-season cultivars, such as the bentgrasses, vegetatively, which would circumvent the outcrossing and dilution of genetic purity that occurs in the production of valuable seeded cultivars. This feature may be especially important in the production of transgenic cultivars that will shortly work their way into the marketplace. Buckeye Bluegrass Farms, with the help of a research grant from the U.S. Dept. of Agriculture, is exploring the possibilities of developing a machine that would harvest the sod in any one of three ways: directly off the plastic; directly off soil in the conventional manner if soil contamination is not important; or, in a combination of the two, by growing the sod over plastic sheeting a little thicker and then harvesting it with conventional sod harvesting equipment with the cutters set slightly higher so that the plastic sheeting is left behind undamaged. The plastic in this third alternative remains in place with a thin layer of root matter on top of it over which new growing media is placed and the cropping system repeated. In any of the three harvesting alternatives, if the intention is to produce VPM, the sod after cutting or lifting is brought up a chain conveyor to one or more pin rollers that effectively separate the VPM from its growing medium. The VPM is screened off and the growing medium is dropped back on the plastic to be used again in the growing of a subsequent crop. B. Growing Non-Grass Sods In the early 1970s, along with our experiments on grasses, we tried growing selected ground covers such as Pachysandra, ivy, Ajuga, strawberries, Euonymous, crownvetch, and others over plastic sheeting as

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sods (Decker 1975). One of our students at the time used the idea for the basis of his master’s thesis (Sterrett 1976; Sterrett and Sydnor 1977). A hill-row plastic base system that includes the use of plastic netting was worked out by Decker (1982) for the production of selected ground covers and other spreading type perennials as sods. Milstein (1990, 1998) reports growing wildflowers in a sod mat configuration. Extensive work has been done by Molnar and Molnar (1993, 1994a, 1994b, 1995a, 1995b, 1996), Molnar et al. (1994), Molnar and Mitchell (1996), and Mitchell et al. (1994), who report growing wildflowers, grasses, ground covers, and many other vegetable and ornamental plants in sod mats. Roberts et al. (2000a) report growing wildflower sods over plastic in flume-sand dominant growing media. J. Stageman (pers. commun.) of Sunset Specialty Turf in Florida has perfected growing techniques over plastic for producing sods of the perennial peanut, which is a potentially useful and attractive warm-season ground cover. C. Growing Forage Crops Luebben (1904) proposed crushing and embedding ungerminated grains of various kinds into mats or webs of compressed hay that would act as the carrier for his “Stock Food Package.” Frontczak and Dlugolecki (1982) proposed a system in which oats, rye, field peas, and others are seeded into layers of peat placed several inches thick on a rootimpervious surface. The peat is moistened and the seeds germinated for about 30 days, after which the peat is dried and then used as fodder. Buckeye Bluegrass Farms has been experimenting with growing various forage grasses, legumes, and grains in edible waste materials spread evenly over beds of plastic sheeting that can be irrigated. As with grass sods, the trapped primary rooting of the forages quickly bind the growing medium into a coherent mass that can be harvested off the plastic in large rolls of forage. The crop system can be repeated several more times per season than a conventional hay crop can; and the forages are grown to a point that corresponds to the level of their peak protein content, thus obtaining maximum nutrient efficiency while limiting production costs. D. Filtering Waste Waters Grasses cultured over plastic sheeting trap a vast amount of primary rooting and hence provide a gigantic absorptive surface that can be used to construct the ultimate biofilter (Decker 1996a). The entire system can be

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underlain with plastic (the sod plots, drain and irrigation lines, manifolds, and including the holding pond) so that the growing process is essentially a closed system. Wastewater effluents can be irrigated over the sod and impurities extracted by the extensive root surfaces growing on the plastic sheeting. If necessary, irrigations can be repeated until the effluent reaches a desired degree of purity, at which time it is released from the closed system into the environment. When the grass tissue begins to reach untenable concentrations of undesirable elements, the sod is harvested and transported to an appropriate laying site. Of potentially great importance to the turf industry would be the feature of this system to use wastewater in the production of sods. Turf is a nonfood crop and a turf facility such as that of RapidTurf Inc., for example, which is situated right next to a wastewater treatment plant outside Savannah, Georgia can be used to divert much of the recovery effluent from the plant to the irrigation of its turf fields. As the irrigation of lawns, turf farms, and golf courses becomes a more critical environmental issue, a system of this nature may become very attractive to the turf industry.

XI. SUMMARY The history of growing mature grass sods over plastic sheeting was preceded by a profusion of works involving seed mats, seed sheets, and seedling turfs. The latter were widely developed in the United Kingdom in the early 1970s by Goodall, Loads, Dawson, Mercer, and Blackburn. All of these ideas found their way to the U.S. Patent and Trademark Office along with a plethora of proposals for seed mats and seed sheets. Research was also underway in the United States at this time at Ohio Wesleyan University directed toward the production of mature sods over plastic rather than seedling turfs that were fragile and difficult to handle, required netting or a plastic base to hold them together, and were unplayable for several weeks after being installed. An important observation was made concurrently in the United States and in Scotland that the primary rooting of grasses grown over plastic (rather than the secondary rooting that is required to knit a conventional sod grown on soil) was sufficient to knit selected growing media into sods without having to use any reinforcements such as nettings, meshes, or fabrics. This discovery made it possible to quickly move beyond the seedling turf stage. All of the seedling turf patents and many of the subsequent mature sod patents that followed were unworkable on a field scale because they

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made no provisions for neutralizing the damaging effects of severe thunderstorms. This problem was not solved until the late 1970s by the development of the MMM growing system, in which suitable growing media were used to fill the interstices of selected mulches to form matrix continuums across the plastic that were highly stable and resistant to sheet erosion and gapping. Research support by the National Science Foundation was made to Buckeye Bluegrass Farms to study, in the production of sods, the feasibility of replacing topsoil with a varied assortment of growing media contrived from composted waste materials. This led to the development of the liquid mulch sod planting (LMSP) system, which has become an effective means of producing sods over plastic in a manufacturing sequence in which planting and harvesting and other facets of production are practiced daily on the land rather than at seasonal intervals. LITERATURE CITED Airhart, D. L., K. M. Falls, and T. Hosmer. 1983. Developing wildflower sods. HortScience 18(1):89–91. Allen, K. V. 1960. Seed planting mat. U.S. Patent 2,923,093. Amar, S. S. et al. 1964. Device for facilitating sowing and protecting plants reared from the seeds. U.S. Patent 3,154,884. Anonymous. 1988. Carpet produces lightweight sod. Landscape Management 27(9):22. Anonymous. 1998. Turf News. Jul/Aug p. 17–20. Anonymous. 1999. Turf News. Sept/Oct p. 14–15. Anton, A. 1993. Fibrous mat for growing plants. U.S. Patent 5,224,292. Baker, E. W. 1972. Seed sheets. U.S. Patent 3,659,396. Ball, H. J. 1982. Fibrous web for planting seeds, method of using same, apparatus for producing same. U.S. Patent 4,357,780. Ball, H. J. 1983. Fibrous web for planting seeds, method of using same, apparatus for producing same. U.S. Patent 4,414,776. Barker, A. V. 1993. Municipal solid waste compost as a medium for sod grown crops. HortScience 28:256. Barker, A. V., and T. A. O’Brien. 1994. Evaluation of composts for production of sod-grown crops. HortScience 29:443. Barker, A. V., and T. A. O’Brien. 1998. Sod production in composts. HortScience 33:203. Baron, G. 1982. Pre-grown turf and manufacturing of pre-grown turf. U.S. Patent 4,364,197. Beard, J. B. 1976. A new approach in growing turf: Net sod production. Grounds Maint. Sept.: 22–27, 44. Beard, J. B., D. P. Martin, and F. B. Mercer. 1980. Investigation of net-sod production as a new technique. p. 353–360. In: Proc. Third Int. Turfgrass Research Conference, Am. Soc. Agron. Madison, WI. Beck, M. 1971. Why we developed the big roll system. Weeds, Trees, Turf. Harvest Publ. Co., Oct. 1971. Bergevin, J. G. 1996. Surface for sports and other uses. U.S. Patent 5,489,317. Besing, D. J. 1994. “Grass Waffle” or “Seed Waffle”. U.S. Patent 5,274,951.

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Blackburn, J. 1975. Methods of producing grass carpets. U.S. Patent 3,890,739. Bouchard, B. L., and D. H. Bouchard. 1994. Method and apparatus for cleaning sod. U.S. Patent 5,293,714. Brem, J. W. 1975. Method and means of producing grass sod. U.S. Patent 3,903,816. Burns, R. E. 1980. Techniques for rapid sod production. p. 361–366. In: Proc. Third Int. Turfgrass Research Conference, Am. Soc. Agron. Madison, WI. Burns, R. E. 1981. Something new: Growing tall fescue sod with plastic netting. Georgia Agr. Res. 22(3):4–5. Burns, R. E. 1982. Rapid production of centipede sod using plastic net. Agron. Abstr., Nov. 1982. p. 140. Busey, P., and E. O. Burt. 1977. Genetics and management of bahiagrass for better highways. Agron. Abstr. 69, Nov. 1977. p. 109. Chamoulaud, M. C. 1980. Carpet of vegetable matter. U.S. Patent 4,232,481. Chohamin, J. M. 1958. Seed carrier unit. U.S. Patent 2,826,865. Cisar, J. L., and G. H. Snyder. 1992. Sod production on a solid waste compost over plastic. HortScience 27:219–222. Clapp, A. L. 1934. Composition for germinating seeds and its preparation. U.S. Patent 1,978,102. Clark, E. H. 1993. Turf-growing process. U.S. Patent 5,189,833. Clark, E. H. 1998. Turf-growing process. U.S. Patent 5,765,304. Cockerham, S. 1984. The use of netting in sod production. ASPA Special Rep. Turf News Mag. July/August 1984. Dawson, J. L. 1976. Improvements in and relating to turf mat production. U.K. Patent 1,455,133. Decker, H. F. 1975. Sewage sod system saves time. Weeds, Trees, Turf. 14 (6):40–41. Decker, H. F. 1982. Method for producing ground cover sods. U.S. Patent 4,336,668. Decker, H. F. 1989. Growing sod over plastic: turf in five weeks. Landscape Manag. 28:68–70. Decker, H. F. 1991a. Alternative method for producing tall fescue sod. U.S. Patent 4,986,026. Decker, H. F. 1991b. Compost use in sod production. BioCycle, March. p. 64–65. Decker, H. F. 1991c. Producing sod on plastic: pros and cons. ASPA TurfNews Mag. May/June. p. 14–18. Decker, H. F. 1993. Method for producing sod. U.S. Patent 5,177,898. Decker, H. F. 1995. Device for harvesting and loading or unloading and installing large rolls of sod. U.S. Patent 5,437,528. Decker, H. F. 1996a. Method for manufacturing sod. U.S. Patent 5,481,827. Decker, H. F. 1996b. Method for producing sod. Canada Patent 2,030,250. Decker, H. F. 1998a. Company profile: RapidTurf, Inc.: Natural grass grown on plastic. TPI Turf News Mag. Jan/Feb. p. 71–74. Decker, H. F. 1998b. Liquid mulch apparatus for manufacturing sods. U.S. Patent 5,806,445. Decker, H. F. 1999a. Alex Rohoza’s extensive research with bentgrass. TPI Turf News Mag. March/April. p. 50–51. Decker, H. F. 1999b. Method for the vegetative propagation of grasses. U.S. Patent 5,899,020. Dittmer, H. J. 1976. p. 419. Cited in P. H. Raven, R. F. Evert, and H. Curtis. Biology of plants, 2nd ed. Worth Publ. Inc., New York. Ecer, G. M. 1995. Light weight seeding sheet. U.S. Patent 5,417,010. Egan, M. A. 1994. Production of sod using a soil-less sand based root zone medium. U.S. Patent 5,301,466.

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Egan, M. A. 1997. Production of sod using a soilless sand based root medium. U.S. Patent 5,651,213. Farrow, W. H., V. Kumar, and W. H. Mitchell. 1995. Sod. U.S. Patent 5,404,671. Fjeldsa, O. 1981. Peat paper and a method for its manufacture. U.S. Patent 4,283,880. Franklin, M. L., and D. W. Gagle. 1971. Seed germination structure. U.S. Patent 3,557,491. Frontczak, S., and J. Dlugolecki. 1982. Methods of producing fodder. U.S. Patent 4,322,443. Gaughen, T. P. 1986. Seed mat and method and apparatus for manufacturing it. U.S. Patent 4,584,790. Gluck, M., W. Kriegner, and B. Elder. 1977. Foam body and process for the production thereof. U.S. Patent 4,007,556. Goodall, S. 1972a. New process grows turf without soil. Seedmen’s Dig. March. p.18. Goodall, S. 1972b. Transportable stand of plants. U.K. Patent 1,290,338. Gramckow, M. 1996. Crowned row mound sod production process. U.S. Patent 5,538,524. Grether, T. H. A. 1975. Seeding machine. U.S. Patent 3,905,313. Halliday, J., and K. F. Martin. 1990. Reinforcing a grassed surface. U.S. Patent 4,916,855. Hansen, P. E., and R. J. Oace. 1961. Grass-growing fabric. U.S. Patent 2,976,646. Heard, R. A. 1988. Pre-grown lawn turf product and method of growing. U.S. Patent 4,716,679. Hensler, K. L., B. S. Baldwin, and J. M. Goatley. 1996. Soilless sod establishment using an organic fiber medium. Agron. Abstr. 88:143. Holmes, R. 1993. Device for cutting sod grown over plastic sheeting. U.S. Patent 5,272,949. Houston, R. K. 1971. Farming method. U.S. Patent 3,556,026. Huggett, W. A. 1976. Grass growing. U.S. Patent 3,980,029. Kabbaz, M., and A. M. Petrovic. 1987. Accelerated sod production utilizing the root impermeable layer system. Agron. Abstr. Nov. p. 136. Kabbaz, M., and A. M. Petrovic. 1988. Accelerated sod production. TurfNews Mag. May/ June. p. 21–22. Kaneko, T., S. Kamijo, N. Kamezawa, and T. Kobayashi. 1992. Seeding and seedlinggrowing sheet and seedling-growing method. U.S. Patent 5,097,625. King, D. A., and S. J. Vallans. 1982. Production of artificial growing mediums. U.S. Patent 4,309,844. Knolle, J. C., and H. Grimm. 1979. Laminar seed carriers and method of preparing same. U.S. Patent 4,173,844. Knop, A. W., and R. J. H. M. van Banning. 1991. Grow-mat for cultivating plants and a method for manufacturing same. U.S. Patent 5,009,031. Krick, T. M. 1995. Establishment and fertility comparisons of trafficked athletic turf with sand based rootzones. M.S. thesis. Michigan State Univ., East Lansing. Kuestner, A. E. E. 1952. Sod unit. U.S. Patent 2,605,589. Lippoldt, R. F., and W. W. Woods. 1970. Mulch sheets and seed mats and method of making same. U.S. Patent 3,516,196. Loads, F. W. 1975. Growing of grasses. U.S. Patent 3,863,388. Loads, F. W. 1978. Production of turf. U.S. Patent 4,099,345. Lopez, M. J. 1993. Vegetation mat apparatus. U.S. Patent 5,199,215. Luebben, M. L. 1904. Stock food package. U.S. Patent 776,139. Luft, R. 1988. Light-tech sod introduces a new accelerated method. ASPA TurfNews Mag. May/June. p. 22, 24, 26. MacDonald, J. 1912. An improved process for the culture and laying of turf for lawns, golf putting greens, and the like. U.K. Patent 13,067. Marshall, J. C., and J. C. Marshall II. 1999. Seed mat and process for formation thereof. U.S. Patent 5,887,382. Marshall, P. F. 1959. Grass seed mat and process for making same. U.S. Patent 2,909,003.

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McFarland, T. M., and J. J. O’Connor. 1988. Meltblown and coform materials having application as seed beds. U.S. Patent 4,786,550. Mercer, F. B. 1974. Growing of grasses. U.S. Patent 3,845,584. Merrill, R. D., and M. L. Tinker. 1963. Seed planter. U.S. Patent 3,080,681. Milstein, G. 1990. Wildflower sod mat and method of propagation. U.S. Patent 4,941,282. Milstein, G. 1998. Spent mushroom growth media as a growing media for plant sod mats. U.S. Patents 5,802,763. Mitchell, W. H., C. J. Molnar, and S. S. Barton. 1994. Using composts to grow wildflower sod. Biocycle 35(2):62–63. Miyachi, Y. 1991. Mat-like lawngrass for transplantation and methods of manufacture and transplantation of the same. U.S. Patent 4,982,526. Molnar, C. J., and W. H. Mitchell. 1996. Plant sod mats. U.S. Patent 5,507,845. Molnar, C. J., and J. R. Molnar. 1993. Versatile herb, vegetable, flower and groundcover sod mat and method for propagation. U.S. Patent 5,224,290. Molnar, C. J., and J. R. Molnar. 1994a. Low cost, versatile sod mat and method for propagation. U.S. Patent 5,345,713. Molnar, C. J., and J. R. Molnar. 1994b. Versatile plant sod mat and method for propagation. U.S. Patent 5,346,514. Molnar, C. J., and J. R. Molnar. 1995a. Specialty sod mats constructed of nonwoven fabric. U.S. Patent 5,397,368. Molnar, C. J., and J. R. Molnar. 1995b. Specialty sod mat constructed of nonwoven fabric with apertures. U.S. Patent 5,464,455. Molnar, C. J., and J. R. Molnar. 1996. Low cost sod mat and method of propagation. U.S. Patent 5,490,351. Molnar, C. J., J. R. Molnar, and W. H. Mitchell. 1994. Sod mats constructed of stable fibers and a degradable matrix material. U.S. Patent 5,344,470. Molnar, C. J., J. R. Molnar, and W. H. Mitchell. 1996. Sod mats constructed of stable fibers and degradable matrix material and method of propagation. U.S. Patent 5,555,674. Muldner, L. C. 1975. Mat for growing lawns or other vegetation and process for producing same. U.S. Patent 3,914,901. Muldner, L. C. 1980. Mat for growing lawns or other vegetation. U.S. Patent 4,190,981. Muldner, L. C. 1982. Mats for growing lawns and other vegetation. U.S. Patent 4,318,248. Neel, P. L., E. O. Burt, and P. Busey. 1978. Sod production in shallow beds of waste material. J. Am. Soc. Hort. Sci. 103:549–553. Nelson, E. 1999. Using biological control strategies for turf. Part II. Diseases. Grounds Maint. 34(3):22, 24, 26. Obholzer, P. 1962. Method for producing a grass sod. German Patent 1,126,663. O’Brien, T. A., and A. V. Barker. 1995. Evaluation of fresh and year-old solid waste composts for production of wildflower and grass sods on plastic. Compost Science Util. 3(4):69–77. O’Brien, T. A., and A. V. Barker. 1997. Evaluating composts to produce wildflower sods on plastic. J. Am. Soc. Hort. Sci. 122:445–451. Ohsol, E. O. 1966. Planting seeds in a skin foam sheet. U.S. Patent 3,257,754. Parish, R. L., D. W. Wells, and P. E. Bergeron. 1991. Evaluation of turfgrass sod reinforcement methods. Louisiana Agr. 34(3):20–22. Pratt, V. E. 1934. Seed product and process of making same. U.S. Patent 1,971,504. Rehbein, G. 1979. Earth working implement. U.S. Patent 4,175,496. Roberts, B. R., H. F. Decker, K. J. Bagstad, and K. A. Peterson. 2000a. Biosolid residues as soilless media for growing wildflower sod. HortTech 11(2):33–37. Roberts, B. R., H. F. Decker, L. M. Ganahl, and E. Yarmark. 2000b. Biosolid residues as soilless media for growing creeping bentgrass sod. HortTech 11(3):194–199.

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Roberts, B. R., S. D. Kohorst, H. F. Decker, and D. Yaussy. 1995. Shoot biomass of turfgrass cultivars grown on composted waste. Environ. Manag. 19:735–739. Robertson, D. W. 1993. Plant mat and method. U.S. Patent 5,226,255. Robey, M. J. 1977. System and process for providing durability enhanced area. U.S. Patent 4,023,506. Rogers, J. N., J. C. Sorochan, and James R. Crum. 1996. Effect of mulch type for turf establishment. Proc. 66th Annual Michigan Turfgrass Conference 25:1–4. Rogers, R. B., and R. D. Goodrich. 1988. Sod-growing composition and method of using it. U.S. Patent 4,720,935. Rubens, L. C., and D. H. Clarke. 1974. A hydroponic bed for grow-plants. U.S. Patent 3,798,836. Sakate, M., T. Matsumoto, J. Katayama, N. Mitunaga, S. Tada, K. Hori, H. Kambe, and K. Izuka. 1995. Vegetation mat. U.S. Patent 5,421,123. Sakate, M., M. Shibata, K. Tamura, and M. Tsuyama. 1991. Net for grassing. U.S. Patent 5,033,231. Schmidt, E. G. 1981. Seed carrier and method of producing same. U.S. Patent 4,272,919. Shildrick, J. P. 1974. A comparison of three seedling turf products. J. Sports Turf Res. Inst. 50:95–107. Slayter, G., and J. H. Thomas. 1940. Agricultural application of glass wool. U.S. Patent 2,192,939. Solomou, C. J. 1993. Method for cultivation of turf. U.S. Patent 5,205,068. Sorochan, J. C., and J. N. Rogers III. 1998. Turfgrass research for high trafficked areas: I. Development of establishment techniques for sod production utilizing a refined wood fiber mat (Ecomat) as the growth media over an impermeable plastic barrier. Proc. 68th Annual Michigan Turfgrass Conference 27:9–13. Sterrett, R. B. 1976. The production of ground covers as a sod. M.S. thesis. Ohio State Univ., Columbus. Sterrett, R. B., and T. D. Sydnor. 1977. The production of ground covers in a sod-like manner. HortScience 12:492–494. Strombom, D. B. 1999. Sod mat for establishing plants. U.S. Patent 5,860,246. Swan, D. M. 1972. Grass seed mat. U.S. Patent 3,703,786. Taniguchi, M., Y. Fuzishima, M. Hanamaki, and M. Shibata. 1993. Mat with seed and method of producing same. U.S. Patent 5,245,785. Tietz, C. M., and P. Tietz. 1959. Method of producing a live grass package. U.S. Patent 2,876,588. Walton, W. E. 1990. Method of growing sod and sod product thereby formed. U.S. Patent 4,934,094. Watson, J. R., D. M. Lilly, and E. S. Conover. 1964. Seed planting method and apparatus for its practice. U.S. Patent 3,160,986. Watson, J. S. 1949. Method of making plant sod and product therefrom. Canada Patent 461,018. Weber, R. E., and M. L. Delucia. 1993. Biodegradable latex web material. U.S. Patent 5,191,734. Welch, R. L. 1999. Vegetable growing mat. U.S. Patent 5,860,245. Wheeler, M. 1996. Grass Tech: Is it plastic, or is it real ? It’s both. Popular Mechanics. May 1996. p. 66–67. Wilkinson, J. F. 1994. Applying compost to the golf course. Golf Course Manag. March 1994. p. 80–88. Wood, L. L., and K. C. Frisch. 1975. Method for preparing horticultural foam structures. U.S. Patent 3,889,417.

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Wood, L. L., C. L. Kehr, and K. C. Frisch. 1974. Seed-foam-fabric composite. U.S. Patent 3,812,618. Wu, Lin. 1988. Potential for juvenile sod production. HortScience 23:162–164. Yoshimi, M. 1978. Method for producing a lawn nursery strip. U.S. Patent 4,066,490.

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Subject Index Volume 27 A

G

Apple: functional phytonutrients, 304 scald, 227–267 Arabidopsis: Molecular biology of flowering, 1–39, 41–77

Genetics and Breeding: flowering, 1–39, 41–77 lingonberry, 108–111 Grape: functional phytochemicals, 291–298 irrigation, 189–225

C

H

Caparis, see Caper bush Caper bush, 125–188 Citrus functional phytochemicals, 282–291

Health phytochemicals, fruit, 269–315

D

I Irrigation: grape, 189–225

Dedication, Possingham, J.V., xi–xiii L F Flower and flowering: Arabidopsis, 1–39, 41–77 homeotic gene regulation, 41–77 Fruit: apple scald, 227–267 functional phytochemicals, 269–315 pear scald, 227–267 Fruit crops: grape irrigation, 189–225 lingonberry, 79–123 Functional phytochemicals, fruit, 269–315

Lingonberry, 79–123 M Molecular biology: floral induction, 3–20 flowering, 1–39, 41–77 N Nutrition (human), functional phytochemicals in fruit, 269–315

Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 353

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SUBJECT INDEX

P

V

Pear scald, 227–267 Plastic covers, sod production, 317–351 Postharvest physiology, scald, 227–267

Vaccinium, see Lingonberry: functional phytonutrients, 303 Vegetable crops: caper bush, 125–188

S Scald, apple and pear, 227–267 Sod production, 317–351 Strawberry, functional phytonutrients, 303–304

W Water relations, grape, 189–225

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Cumulative Subject Index (Volumes 1–27)

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, 163–166 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 Agapanthus, 25:56–57 Agaricus, 6:85–118 Agrobacterium tumefaciens, 3:34 Air pollution, 8:1–42 Alkaloids, steroidal, 25:171–196 Almond: bloom delay, 15:100–101 in vitro culture, 9:313 postharvest technology and utilization, 20:267–311

Alocasia, 8:46, 57. See also Aroids Alternate bearing: chemical thinning, 1:285–289 fruit crops, 4:128–173 pistachio, 3:387–388 Aluminum: deficiency and toxicity symptoms in fruits and nuts, 2:154 Ericaceae, 10:195–196 Amarcrinum, 25: 57 Amaryllidaceae, growth, development, flowering, 25:1–70 Amaryllis, 25:4–15 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, 15:147–156 embryogenesis, 1:4–21, 35–40 fig, 12:420–424 fruit abscission, 1:172–203 fruit storage, 1:314 ginseng, 9:198–201 grape flower, 13:315–337 grape seedlessness, 11:160–164 heliconia, 14:5–13 kiwifruit, 6:13–50 magnetic resonance imaging, 20:78–86, 225–266 navel orange, 8:132–133

Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 355

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356 Anatomy and morphology (cont.) orchid, 5:281–283 pecan flower, 8:217–255 petal senescence, 1:212–216 pollution injury, 8:15 waxes, 23:1–68 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, 6:357–372 Apple: alternate bearing, 4:136–137 anatomy and morphology of flower and fruit, 10:273–309 bioregulation, 10:309–401 bitter pit, 11:289–355 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 functional phytonutrients, 27:304 in vitro, 5:241–243; 9:319–321 light, 2:240–248 maturity indices, 13:407–432 mealiness, 20:200 nitrogen metabolism, 4:204–246 replant disease, 2:3 root distribution, 2:453–456 scald, 27:227–267 stock-scion relationships, 3:315–375 summer pruning, 9:351–375 tree morphology and anatomy, 12:265–305 vegetative growth, 11:229–287 watercore, 6:189–251 weight loss, 25:197–234 yield, 1:397–424 Apricot: bloom delay, 15:101–102 CA storage, 1:309 origin and dissemination, 22:225–266

CUMULATIVE SUBJECT INDEX Arabidopsis: molecular biology of flowering, 27:1–39, 41–77 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, 1:349–350 Asexual embryogenesis, 1:1–78; 2:268–310; 3:214–314; 7:163–168, 171–173, 176–177, 184–189; 10:153–181; 14:258–259, 337–339; 24:6–7; 26:105–110 Asparagus: CA storage, 1:350–351 fluid drilling of seed, 3:21 postharvest biology, 12:69–155 Auxin: abscission, citrus, 15:161, 168–176 bloom delay, 15:114–115 citrus abscission, 15:161, 168–176 dormancy, 7:273–274 flowering, 15:290–291, 315 genetic regulation, 16:5–6, 14, 21–22 geotropism, 15:246–267 mechanical stress, 17:18–19 petal senescence, 11:31 Avocado: CA and MA, 22:135–141 flowering, 8:257–289 fruit development, 10:230–238 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

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CUMULATIVE SUBJECT INDEX Banana: CA and MA, 22:141–146 CA storage, 1:311–312 fertilization, 1:105 in vitro culture, 7:178–180 Banksia, 22:1–25 Bean: CA storage, 1:352–353 fluid drilling of seed, 3:21 resistance to bacterial pathogens, 3:28–58 Bedding plants, fertilization, 1:99–100; 5:337–341 Beet: CA storage, 1:353 fluid drilling of seed, 3:18–19 Begonia (Rieger), fertilization, 1:104 Biennial bearing, see Alternate bearing Biochemistry, petal senescence, 11:15–43 Bioreactor technology, 24:1–30 Bioregulation, see Growth substances apple and pear, 10:309–401 Bird damage, 6:277–278 Bitter pit in apple, 11:289–355 Blackberry harvesting, 16:282–298 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 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 Botanic gardens, 15:1–62 Bramble, harvesting, 16:282–298 Branching, lateral: apple, 10:328–330 pear, 10:328–330 Brassicaceae, in vitro, 5:232–235 Breeding, see Genetics and breeding Broccoli, CA storage, 1:354–355 Brussels sprouts, CA storage, 1:355 Bulb crops, see Tulip development, 25:1–70 flowering, 25:1–70 genetics and breeding, 18:119–123 growth, 25:1–70

357 in vitro, 18:87–169 micropropagation, 18:89–113 root physiology, 14:57–88 virus elimination, 18:113–123 C CA storage, see Controlled-atmosphere storage Cabbage: CA storage, 1:355–359 fertilization, 1:117–118 Cactus: crops, 18:291–320 reproductive biology, 18:321–346 Caladium, see Aroids, ornamental Calcifuge, nutrition, 10:183–227 Calciole, 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, 10:196–197 foliar application, 6:328–329 fruit softening, 10:107–152 nutrition, 5:322–323 pine bark media, 9:116–117 tipburn, disorder, 4:50–57 Calmodulin, 10:132–134, 137–138 Caparis, see Caper bush Caper bush, 27:125–188 Carbohydrate: fig, 12:436–437 kiwifruit partitioning, 12:318–324 metabolism, 7:69–108 partitioning, 7:69–108 petal senescence, 11:19–20 reserves in deciduous fruit trees, 10:403–430 Carbon dioxide, enrichment, 7:345–398, 544–545 Carnation, fertilization, 1:100; 5:341–345 Carrot: CA storage, 1:362–366 fluid drilling of seed, 3:13–14 Caryophyllaceae, in vitro, 5:237–239 Cassava, 12:158–166; 13:105–129; 26:85–159 Cauliflower, CA storage, 1:359–362

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358 Celeriac, CA storage, 1:366–367 Celery: CA storage, 1:366–367 fluid drilling of seed, 3:14 Cell culture, 3:214–314 woody legumes, 14:265–332 Cell membrane: calcium, 10:126–140 petal senescence, 11:20–26 Cell wall: calcium, 10:109–122 hydrolases, 5:169–219 ice spread, 13:245–246 tomato, 13:70–71 Cellular mechanisms, salt tolerance, 16:33–69 Chelates, 9:169–171 Cherimoya, CA and MA, 22:146–147 Cherry: bloom delay, 15:105 CA storage, 1:308 origin, 19:263–317 Chestnut: blight, 8:281–336 in vitro culture, 9:311–312 Chicory, CA storage, 1:379 Chilling: injury, 4:260–261; 15:63–95 injury, chlorophyll fluorescence, 23:79–84 pistachio, 3:388–389 Chlorine: deficiency and toxicity symptoms in fruits and nuts, 2:153 nutrition, 5:239 Chlorophyll fluorescence, 23:69–107 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 functional phytochemicals, fruit, 27:269–315

CUMULATIVE SUBJECT INDEX honey bee pollination, 9:247–248 in vitro culture, 7:161–170 juice loss, 20:200–201 navel orange, 8:129–179 nitrogen metabolism, 8:181 practices for young trees, 24:319–372 rootstock, 1:237–269 viroid dwarfing, 24:277–317 Clivia, 25:57 Cloche (tunnel), 7:356–357 Coconut palm: asexual embryogenesis, 7:184 in vitro culture, 7:183–185 Cold hardiness, 2:33–34 apple and pear bioregulation, 10:374–375 citrus, 7:201–238 factors affecting, 11:55–56 herbaceous plants, 6:373–417 injury, 2:26–27 nutrition, 3:144–171 pruning, 8:356–357 Colocasia, 8:45, 55–56. See also Aroids Common blight of bean, 3:45–46 Compositae, in vitro, 5:235–237 Container production, nursery crops, 9:75–101 Controlled-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 tropical fruit, 22:123–183 tulip, 5:105 vegetable quality, 8:101–127 vegetables, 1:337–394; 4:259–260 Controlled environment agriculture, 7:534–545. See also Greenhouse and greenhouse crops; hydroponic culture; protected culture 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

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CUMULATIVE SUBJECT INDEX Corynebacterium flaccumfaciens, 3:33, 46 Cowpea: genetics, 2:317–348 U.S. production, 12:197–222 Cranberry: botany and horticulture, 21:215–249 fertilization, 1:106 harvesting, 16:298–311 Crinum, 25:58 Cryopreservation: apical meristems, 6:357–372 cold hardiness, 11:65–66 Cryphonectria parasitica, see Endothia parasitica Crytosperma, 8:47, 58. See also Aroids Cucumber, CA storage, 1:367–368 Cucurbita pepo, cultivar groups history, 25:71–170 Currant, harvesting, 16:311–327 Custard apple, CA and MA, 22:164 Cyrtanthus, 25:15–19 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., 1:v–viii Bukovac, M.J., 6:x–xii Campbell, C.W., 19:xiii Cummins, J.N., 15:xii–xv Dennis, F.G., 22:xi–xii De Hertogh, A.A., 26:xi–xii 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

359 Kamemoto, H., 24:x–xiii Looney, N.E., 18:xiii Magness, J.R., 2:vi–viii Moore, J.N., 14:xii–xv Possingham, J.V., 27:xi–xiii Pratt, C., 20:ix–xi Proebsting, Jr., E.L., 9:x–xiv Rick, Jr., C.M., 4:vi–ix Ryugo, K., 25:x–xii Sansavini, S., 17:xii–xiv Sherman, W.B., 21:xi–xiii Smock, R.M., 7:x–xiii Weiser, C.J., 11:x–xiii Whitaker, T.W., 3:vi–x Wittwer, S.H., 10:x–xiii Yang, S.F., 23:xi Deficit irrigation, 21:105–131 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, 18:171–213 Dieffenbachia, see Aroids, ornamental Dioscorea, see Yam Disease: and air pollution, 8:25 aroids, 8:67–69; 10:18; 12:168–169 bacterial, of bean, 3:28–58 cassava, 12:163–164 control by virus, 3:399–403 controlled-atmosphere storage, 3:412–461 cowpea, 12:210–213 fig, 12:447–479 flooding, 13:288–299 hydroponic crops, 7:530–534 lettuce, 2:187–197 mycorrhizal fungi, 3:182–185 ornamental aroids, 10:18 resistance, acquired, 18:247–289 root, 5:29–31 stress, 4:261–262 sweet potato, 12:173–175 tulip, 5:63, 92 turnip moasic virus, 14:199–238 waxes, 23:1–68 yam (Dioscorea), 12:181–183 Disorder, see Postharvest physiology bitterpit, 11:289–355 fig, 12:477–479

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360 Disorder (cont.) pear fruit, 11:357–411 watercore, 6:189–251; 11:385–387 Dormancy, 2:27–30 blueberry, 13:362–370 release in fruit trees, 7:239–300 tulip, 5:93 Drip irrigation, 4:1–48 Drought resistance, 4:250–251 cassava, 13:114–115 Durian, CA and MA, 22:147–148 Dwarfing: apple, 3:315–375 apple mutants, 12:297–298 by virus, 3:404–405

CUMULATIVE SUBJECT INDEX bloom delay, 15:107–111 CA storage, 1:317–319, 348 chilling injury, 15:80 citrus abscission, 15:158–161, 168–176 cut flower storage, 10:44–46 dormancy, 7:277–279 flower longevity, 3:66–75 flowering, 15:295–296, 319 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 Eucharis, 25:19–22 Eucrosia, 25:58

E Easter lily, fertilization, 5:352–355 Embryogenesis, see Asexual embryogenesis Endothia parasitica, 8:291–336 Energy efficiency, in greenhouses, 1:141–171; 9:1–52 Environment: air pollution, 8:20–22 controlled for agriculture, 7:534–545 controlled for energy efficiency, 1:141–171; 9:1–52 embryogenesis, 1:22, 43–44 fruit set, 1:411–412 ginseng, 9:211–226 greenhouse management, 9:32–38 navel orange, 8:138–140 nutrient film technique, 5:13–26 Epipremnum, see Aroids, ornamental Eriobotrya japonica, see Loquat 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, 15:158–161, 168–176 apple bioregulation, 10:366–369 avocado, 10:239–241

F Feed crops, cactus, 18:298–300 Feijoa, CA and MA, 22:148 Fertilization and fertilizer: anthurium, 5:334–335 azalea, 5:335–337 bedding plants, 5:337–341 blueberry, 10:183–227 carnation, 5:341–345 chrysanthemum, 5:345–352 controlled release, 1:79–139; 5:347–348 Easter lily, 5:352–355 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 zinc nutrition, 23:109–128 Fig: industry, 12:409–490 ripening, 4:258–259

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CUMULATIVE SUBJECT INDEX Filbert, in vitro culture, 9:313–314 Fire blight, 1:423–474 Flooding, fruit crops, 13:257–313 Floral scents, 24:31–53 Floricultural crops, see individual crops Amaryllidaceae, 25:1–70 Banksia, 22:1–25 fertilization, 1:98–104 growth regulation, 7:399–481 heliconia, 14:1–55 Leucospermum, 22:27–90 postharvest physiology and senescence, 1:204–236; 3:59–143; 10:35–62; 11:15–43 Protea, 26:1–48 Florigen, 4:94–98 Flower and flowering: Amaryllidaceae, 25:1–70 apple anatomy and morphology, 10:277–283 apple bioregulation, 10:344–348 Arabidopsis, 27:1–39, 41–77 aroids, ornamental, 10:19–24 avocado, 8:257–289 Banksia, 22:1–25 blueberry development, 13:354–378 cactus, 18:325–335 citrus, 12:349–408 control, 4:159–160; 15:279–334 development (postpollination), 19:1–58 fig, 12:424–429 grape anatomy and morphology, 13:354–378 homeotic gene regulation, 27:41–77 honey bee pollination, 9:239–243 induction, 4:174–203, 254–256 initiation, 4:152–153 in vitro, 4:106–127 kiwifruit, 6:21–35; 12:316–318 Leucospermum, 22:27–90 orchid, 5:297–300 pear bioregulation, 10:344–348 pecan, 8:217–255 perennial fruit crops, 12:223–264 phase change, 7:109–155 photoperiod, 4:66–105 pistachio, 3:378–387 postharvest physiology, 1:204–236; 3:59–143; 10:35–62; 11:15–43

361 postpollination development, 19:1–58 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 scents, 24:31–53 senescence, 1:204–236; 3:59–143; 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 abscission, citrus, 15:145–182 apple anatomy and morphology, 10:283–297 apple bioregulation, 10:348–374 apple bitter pit, 11:289–355 apple flavor, 16:197–234 apple maturity indices, 13:407–432 apple ripening and quality, 10:361–374 apple scald, 27:227–267 apple weight loss, 25:197–234 avocado development and ripening, 10:229–271 bloom delay, 15:97–144 blueberry development, 13:378–390 cactus physiology, 18:335–341 CA storage and quality, 8:101–127 chilling injury, 15:63–95 coating physiology, 26:161–238 cracking, 19:217–262 diseases in CA storage, 3:412–461 drop, apple and pear, 10:359–361 functional phytochemicals, 27:269–315 growth measurement, 24:373–431

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362 Fruit (cont.) kiwifruit, 6:35–48; 12:316–318 loquat, 23:233–276 maturity indices, 13:407–432 navel orange, 8:129–179 nectarine, postharvest, 11:413–452 nondestructive postharvest quality evaluation, 20:1–119 olive processing, 25:235–260 peach, postharvest, 11:413–452 pear, bioregulation, 10:348–374 pear, fruit disorders, 11:357–411 pear maturity indices, 13:407–432 pear ripening and quality, 10:361–374 pear scald, 27:227–267 pistachio, 3:382–391 plum, 23:179–231 quality and pruning, 8:365–367 ripening, 5:190–205 set, 1:397–424; 4:153–154 set in navel oranges, 8:140–142 size and thinning, 1:293–294; 4:161 softening, 5:109–219; 10:107–152 splitting, 19:217–262 strawberry growth and ripening, 17:267–297 texture, 20:121–224 thinning, apple and pear, 10:353–359 tomato parthenocarpy, 6:65–84 tomato ripening, 13:67–103 Fruit crops: alternate bearing, 4:128–173 apple bitter pit, 11:289–355 apple flavor, 16:197–234 apple fruit splitting and cracking, 19:217–262 apple growth, 11:229–287 apple maturity indices, 13:407–432 apple scald, 27:227–267 apricot, origin and dissemination, 22:225–266 avocado flowering, 8:257–289 avocado rootstocks, 17:381–429 berry crop harvesting, 16:255–382 bloom delay, 15:97–144 blueberry developmental physiology, 13:339–405 blueberry harvesting, 16:257–282 blueberry nutrition, 10:183–227 bramble harvesting, 16:282–298 cactus, 18:302–309

CUMULATIVE SUBJECT INDEX carbohydrate reserves, 10:403–430 CA and MA for tropicals, 22:123–183 CA storage, 1:301–336 CA storage diseases, 3:412–461 cherry origin, 19:263–317 chilling injury, 15:145–182 chlorosis, 9:161–165 citrus abscission, 15:145–182 citrus cold hardiness, 7:201–238 citrus, culture of young trees, 24:319–372 citrus dwarfing by viroids, 24:277–317 citrus flowering, 12:349–408 cranberry, 21:215–249 cranberry harvesting, 16:298–311 currant harvesting, 16:311–327 deficit irrigation, 21:105–131 dormancy release, 7:239–300 Ericaceae nutrition, 10:183–227 fertilization, 1:104–106 fig, industry, 12:409–490 fireblight, 11:423–474 flowering, 12:223–264 foliar nutrition, 6:287–355 frost control, 11:45–109 grape flower anatomy and morphology, 13:315–337 grape harvesting, 16:327–348 grape irrigation, 27:189–225 grape nitrogen metabolism, 14:407–452 grape pruning, 16:235–254, 336–340 grape root, 5:127–168 grape seedlessness, 11:164–176 grapevine pruning, 16:235–254, 336–340 honey bee pollination, 9:244–250, 254–256 jojoba, 17:233–266 in vitro culture, 7:157–200; 9:273–349 irrigation, deficit, 21:105–131 kiwifruit, 6:1–64; 12:307–347 lingonberry, 27:79–123 longan, 16:143–196 loquat, 23:233–276 lychee, 16:143–196 muscadine grape breeding, 14:357–405 navel orange, 8:129–179 nectarine postharvest, 11:413–452 nondestructive postharvest quality evaluation, 20:1–119 nutritional ranges, 2:143–164

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CUMULATIVE SUBJECT INDEX olive salinity tolerance, 21:177–214 orange, navel, 8:129–179 orchard floor management, 9:377–430 peach origin, 17:331–379 peach postharvest, 11:413–452 pear fruit disorders, 11:357–411; 27:227–267 pear maturity indices, 13:407–432 pear scald, 27:227–267 pecan flowering, 8:217–255 photosynthesis, 11:111–157 Phytophthora control, 17:299–330 plum origin, 23:179–231 pruning, 8:339–380 rambutan, 16:143–196 raspberry, 11:185–228 roots, 2:453–457 sapindaceous fruits, 16:143–196 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, 10:183–227 water status, 7:301–344 Functional phytochemicals, fruit, 27:269–315 Fungi: fig, 12:451–474 mushroom, 6:85–118 mycorrhiza, 3:172–213; 10:211–212 pathogens in postharvest storage, 3:412–461 truffle cultivation, 16:71–107 Fungicide, and apple fruit set, 1:416 G Galanthus, 25:22–25 Garlic, CA storage, 1:375 Genetic variation: alternate bearing, 4:146–150 photoperiodic response, 4:82 pollution injury, 8:16–19 temperature-photoperiod interaction, 17:73–123 Genetics and breeding: aroids (edible), 8:72–75; 12:169 aroids (ornamental), 10:18–25 bean, bacterial resistance, 3:28–58 bloom delay in fruits, 15:98–107 bulbs, flowering, 18:119–123

363 cassava, 12:164 chestnut blight resistance, 8:313–321 citrus cold hardiness, 7:221–223 cranberry, 21:236–239 embryogenesis, 1:23 fig, 12:432–433 fire blight resistance, 1:435–436 flower longevity, 1:208–209 flowering, 15:287–290, 303–309, 314–315; 27:1–39, 41–77 ginseng, 9:197–198 in vitro techniques, 9:318–324; 18:119–123 lettuce, 2:185–187 lingonberry, 27:108–111 loquat, 23:252–257 muscadine grapes, 14:357–405 mushroom, 6:100–111 navel orange, 8:150–156 nitrogen nutrition, 2:410–411 pineapple, 21:138–164 plant regeneration, 3:278–283 pollution insensitivity, 8:18–19 potato tuberization, 14:121–124 rhododendron, 12:54–59 sweet potato, 12:175 sweet sorghum, 21:87–90 tomato parthenocarpy, 6:69–70 tomato ripening, 13:77–98 tree short life, 2:66–70 Vigna, 2:311–394 waxes, 23:50–53 woody legume tissue and cell culture, 14:311–314 yam (Dioscorea), 12:183 Geophyte, see Bulb, tuber Geranium, fertilization, 5:355–357 Germination, seed, 2:117–141, 173–174; 24:229–275 Germplasm preservation: cryopreservation, 6:357–372 in vitro, 5:261–264; 9:324–325 pineapple, 21:164–168 Germplasm resources: pineapple, 21:133–175 Gibberellin: abscission, citrus, 15:166–167 bloom delay, 15:111–114 citrus, abscission, 15:166–167 cold hardiness, 11:63 dormancy, 7:270–271

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364 Gibberellin (cont.) floral promoter, 4:114 flowering, 15:219–293, 315–318 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 Gourd, history, 25:71–171 Graft and grafting: incompatibility, 15:183–232 phase change, 7:136–137, 141–142 rose, 9:56–57 Grape: CA storage, 1:308 chlorosis, 9:165–166 flower anatomy and morphology, 13:315–337 functional phytochemicals, 27:291–298 irrigation, 27:189–225 harvesting, 16:327–348 muscadine breeding, 14:357–405 nitrogen metabolism, 14:407–452 pollen morphology, 13:331–332 pruning, 16:235–254, 336–340 root, 5:127–168 seedlessness, 11:159–187 sex determination, 13:329–331 Gravitropism, 15:233–278 Greenhouse and greenhouse crops: carbon dioxide, 7:357–360, 544–545 energy efficiency, 1:141–171; 9:1–52 growth substances, 7:399–481 nutrition and fertilization, 5:317–403 pest management, 13:1–66 vegetables, 21:1–39 Growth regulators, see Growth substances Growth substances, 2:60–66; 24:55–138. See also Abscisic acid, Auxin, Cytokinins, Ethylene, Gibberellins abscission, citrus, 15:157–176 apple bioregulation, 10:309–401 apple dwarfing, 3:315–375 apple fruit set, 1:417 apple thinning, 1:270–300 aroids, ornamental, 10:14–18 avocado fruit development, 10:229–243

CUMULATIVE SUBJECT INDEX bloom delay, 15:107–119 CA storage in vegetables, 1:346–348 cell cultures, 3:214–314 chilling injury, 15:77–83 citrus abscission, 15:157–176 cold hardiness, 7:223–225; 11:58–66 dormancy, 7:270–279 embryogenesis, 1:41–43; 2:277–281 floriculture, 7:399–481 flower induction, 4:190–195 flower storage, 10:46–51 flowering, 15:290–296 genetic regulation, 16:1–32 ginseng, 9:226 grape seedlessness, 11:177–180 hormone reception, 26:49–84 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, 10:309–401 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 H Haemanthus, 25:25–28 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 Health phytochemicals, fruit, 27:269–315 Heat treatment (postharvest), 22:91–121 Heliconia, 14:1–55 Herbaceous plants, subzero stress, 6:373–417 Hippeastrum, 25:29–34 Histochemistry: flower induction, 4:177–179 fruit abscission, 1:172–203

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CUMULATIVE SUBJECT INDEX Histology, flower induction, 4:179–184. See also Anatomy and morphology Honey bee, 9:237–272 Horseradish, CA storage, 1:368 Hydrolases, 5:169–219 Hydroponic culture, 5:1–44; 7:483–558 Hymenocallis, 25:59 Hypovirulence, in Endothia parasitica, 8:299–310 I Ismene, 25:59 Ice, formation and spread in tissues, 13:215–255 Ice-nucleating bacteria, 7:210–212; 13:230–235 Industrial crops, cactus, 18:309–312 Insects and mites: aroids, 8:65–66 avocado pollination, 8:275–277 fig, 12:442–447 hydroponic crops, 7:530–534 integrated pest management, 13:1–66 lettuce, 2:197–198 ornamental aroids, 10:18 tree short life, 2:52 tulip, 5:63, 92 waxes, 23:1–68 Integrated pest management: greenhouse crops, 13:1–66 In vitro: abscission, 15:156–157 apple propagation, 10:325–326 aroids, ornamental, 10:13–14 artemisia, 19:342–345 bioreactor technology, 24:1–30 bulbs, flowering, 18:87–169 cassava propagation, 13:121–123; 26:99–119 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

365 propagation, 3:214–314; 5:221–277; 7:157–200; 9:57–58, 273–349; 17:125–172 thin cell layer morphogenesis, 14:239–264 woody legume culture, 14:265–332 Iron: deficiency and toxicity symptoms in fruits and nuts, 2:150 deficiency chlorosis, 9:133–186 Ericaceae nutrition, 10:193–195 foliar application, 6:330 nutrition, 5:324–325 pine bark media, 9:123 Irrigation: deficit, deciduous orchards, 21:105–131 drip or trickle, 4:1–48 frost control, 11:76–82 fruit trees, 7:331–332 grape, 27:189–225 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, 2:514–531 Lanzon, CA and MA, 22:149 Leaves: apple morphology, 12:283–288 flower induction, 4:188–189 Leek: CA storage, 1:375 fertilization, 1:118

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366 Leguminosae, in vitro, 5:227–229; 14:265–332 Lemon, rootstock, 1:244–246. See also Citrus Lettuce: CA storage, 1:369–371 fertilization, 1:118 fluid drilling of seed, 3:14–17 industry, 2:164–207 seed germination, 24:229–275 tipburn, 4:49–65 Leucojum, 25:34–39 Leucospermum, 22:27–90 Light: fertilization, greenhouse crops, 5:330–331 flowering, 15:282–287, 310–312 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 Lingonberry, 27:79–123 Longan, see Sapindaceous fruits CA and MA, 22:150 Loquat: botany and horticulture, 23:233–276 CA and MA, 22:149–150 Lychee, see Sapindaceous fruits CA and MA, 22:150 Lycoris, 25:39–43 M Magnesium: container growing, 9:84–85 deficiency and toxicity symptoms in fruits and nuts, 2:148 Ericaceae nutrition, 10:196–198 foliar application, 6:331 nutrition, 5:323 pine bark media, 9:117–119 Magnetic resonance imaging, 20:78–86, 225–266 Male sterility, temperature-photoperiod induction, 17:103–106

CUMULATIVE SUBJECT INDEX Mandarin, rootstock, 1:250–252 Manganese: deficiency and toxicity symptoms in fruits and nuts, 2:150–151 Ericaceae nutrition, 10:189–193 foliar application, 6:331 nutrition, 5:235–326 pine bark media, 9:123–124 Mango: alternate bearing, 4:145–146 asexual embryogenesis, 7:171–173 CA and MA, 22:151–157 CA storage, 1:313 in vitro culture, 7:171–173 Mangosteen, CA and MA, 22:157 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, 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 Modified atmosphere (MA) for tropical fruits, 22:123–183 Moisture, and seed storage, 2:125–132 Molecular biology: cassava, 26:85–159 floral induction, 27:3–20 flowering, 27:1–39, 41–77 hormone reception, 26:49–84 Molybdenum nutrition, 5:328–329

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CUMULATIVE SUBJECT INDEX 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 Narcissus, 25:43–48 Navel orange, 8:129–179 Nectarine: bloom delay, 15:105–106 CA storage, 1:309–310 postharvest physiology, 11:413–452 Nematodes: aroids, 8:66 fig, 12:475–477 lettuce, 2:197–198 tree short life, 2:49–50 Nerine, 25:48–56 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 Ericaceae nutrition, 10:198–202 fixation in woody legumes, 14:322–323 foliar application, 6:332 in embryogenesis, 2:273–275 metabolism in apple, 4:204–246 metabolism in citrus, 8:181–215 metabolism in grapevine, 14:407–452 nutrition, 2:395, 423; 5:319–320 pine bark media, 9:108–112

367 trickle irrigation, 4:29–30 vegetable crops, 22:185–223 Nondestructive quality evaluation of fruits and vegetables, 20:1–119 Nursery crops: fertilization, 1:106–112 nutrition, 9:75–101 Nut crops: almond postharvest technology and utilization, 20:267–311 chestnut blight, 8:291–336 fertilization, 1:106 honey bee pollination, 9:250–251 in vitro culture, 9:273–349 nutritional ranges, 2:143–164 pistachio culture, 3:376–396 Nutrient: concentration in fruit and nut crops, 2:154–162 film technique, 5:1–44 foliar-applied, 6:287–355 media, for asexual embryogenesis, 2:273–281 media, for organogenesis, 3:214–314 plant and tissue analysis, 7:30–56 solutions, 7:524–530 uptake, in trickle irrigation, 4:30–31 Nutrition (human): aroids, 8:79–84 CA storage, 8:101–127 functional phytochemicals in fruit, 27:269–315 steroidal alkaloids, 25:171–196 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 cranberry, 21:234–235 ecologically based, 24:156–172 embryogenesis, 1:40–41 Ericaceae, 10:183–227 fire blight, 1:438–441 foliar, 6:287–355 fruit and nut crops, 2:143–164 ginseng, 9:209–211 greenhouse crops, 5:317–403 kiwifruit, 12:325–332 mycorrhizal fungi, 3:185–191

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368 Nutrition (plant) (cont.) navel orange, 8:162–166 nitrogen in apple, 4:204–246 nitrogen in vegetable crops, 22:185–223 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, 1:79–139 O Oil palm: asexual embryogenesis, 7:187–188 in vitro culture, 7:187–188 Okra: botany and horticulture, 21:41–72 CA storage, 1:372–373 Olive: alternate bearing, 4:140–141 salinity tolerance, 21:177–214 processing technology, 25:235–260 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, 1:247–250 Orchard and orchard systems: floor management, 9:377–430 light, 2:208–267 root growth, 2:469–470 water, 7:301–344 Orchid: fertilization, 5:357–358 pollination regulation of flower development, 19:28–38 physiology, 5:279–315 Organogenesis, 3:214–314. See also In vitro; tissue culture Ornamental plants: Amaryllidaceae Banksia, 22:1–25 chlorosis, 9:168–169 fertilization, 1:98–104, 106–116 flowering bulb roots, 14:57–88

CUMULATIVE SUBJECT INDEX flowering bulbs in vitro, 18:87–169 foliage acclimatization, 6:119–154 heliconia, 14:1–55 Leucospermum, 22:27–90 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 and MA, 22:157–160 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 CA and MA, 22:160–161 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 wooliness, 20:198–199 Peach palm (Pejibaye): in vitro culture, 7:187–188 Pear: bioregulation, 10:309–401 bloom delay, 15:106–107 CA storage, 1:306–308 decline, 2:11 fire blight control, 1:423–474 fruit disorders, 11:357–411; 27:227–267 in vitro, 9:321 maturity indices, 13:407–432 root distribution, 2:456 scald, 27:227–267 short life, 2:6 Pecan: alternate bearing, 4:139–140

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CUMULATIVE SUBJECT INDEX 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 ecologically based, 24:172–201 fig, 12:442–477 fire blight, 1:423–474 ginseng, 9:227–229 greenhouse management, 13:1–66 hydroponics, 7:530–534 sweet potato, 12:173–175 vertebrate, 6:253–285 yam (Dioscorea), 12:181–183 Petal senescence, 11:15–43 pH: container growing, 9:87–88 fertilization greenhouse crops, 5:332–333 pine bark media, 9:114–117 soil testing, 7:8–12, 19–23 Phase change, 7:109–155 Phenology: apple, 11:231–237 raspberry, 11:186–190 Philodendron, see Aroids, ornamental 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, 17:125–172 Photoperiod, 4:66–105, 116–117; 17:73–123 flowering, 15:282–284, 310–312

369 Photosynthesis: cassava, 13:112–114 efficiency, 7:71–72; 10:378 fruit crops, 11:111–157 ginseng, 9:223–226 light, 2:237–238 Physiology, see 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, 7:201–238 conditioning 13:131–181 cut flower, 1:204–236; 3:59–143; 10:35–62 desiccation tolerance, 18:171–213 disease resistance, 18:247–289 dormancy, 7:239–300 embryogenesis, 1:21–23; 2:268–310 floral scents, 24:31–53 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 hormone reception, 26:49–84 juvenility, 7:109–155 lettuce seed germination, 24:229–275 light tolerance, 18:215–246 loquat, 23:242–252 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 olive salinity tolerance, 21:177–214 orchid, 5:279–315 petal senescence, 11:15–43 photoperiodism, 17:73–123 pollution injury, 8:12–16 polyamines, 14:333–356 potato tuberization, 14:89–188 pruning, 8:339–380 raspberry, 11:190–199 regulation, 11:1–14

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370 Physiology (cont.) root pruning, 6:158–171 roots of flowering bulbs, 14:57–88 rose, 9:3–53 salinity hormone action, 16:1–32 salinity tolerance, 16:33–69 seed, 2:117–141 seed priming, 16:109–141 subzero stress, 6:373–417 summer pruning, 9:351–375 sweet potato, 23:277–338 thin cell layer morphogenesis, 14:239–264 tomato fruit ripening, 13:67–103 tomato parthenocarpy, 6:71–74 triazoles, 10:63–105; 24:55–138 tulip, 5:45–125 vernalization, 17:73–123 volatiles, 17:43–72 watercore, 6:189–251 water relations cut flowers, 18:1–85 waxes, 23:1–68 Phytohormones, see Growth substances Phytophthora control, 17:299–330 Phytotoxins, 2:53–56 Pigmentation: flower, 1:216–219 rose, 9:64–65 Pinching, by chemicals, 7:453–461 Pineapple: CA and MA, 22:161–162 CA storage, 1:314 genetic resources, 21:138–141 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: CA and MA, 22:141–146 in vitro culture, 7:178–180 Plant protection, short life, 2:79–84 Plastic cover, sod production, 27:317–351 Plum: CA storage, 1:309 origin, 23:179–231 Poinsettia, fertilization, 1:103–104; 5:358–360 Pollen, desiccation tolerance, 18:195

CUMULATIVE SUBJECT INDEX Pollination: apple, 1:402–404 avocado, 8:272–283 cactus, 18:331–335 embryogenesis, 1:21–22 fig, 12:426–429 floral scents, 24:31–53 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: almond, 20:267–311 apple bitter pit, 11:289–355 apple maturity indices, 13:407–432 apple scald, 27:227–257 apple weight loss, 25:197–234 aroids, 8:84–86 asparagus, 12:69–155 CA for tropical fruit, 22:123–183 CA storage and quality, 8:101–127 chlorophyll fluorescence, 23:69–107 coated fruits & vegetables, 26:161–238 cut flower, 1:204–236; 3:59–143; 10:35–62 foliage plants, 6:119–154 fruit, 1:301–336 fruit softening, 10:107–152 heat treatment, 22:91–121 lettuce, 2:181–185 low-temperature sweetening, 17:203–231 MA for tropical fruit, 22:123–183 navel orange, 8:166–172 nectarine, 11:413–452 nondestructive quality evaluation, 20:1–119

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CUMULATIVE SUBJECT INDEX pathogens, 3:412–461 peach, 11:413–452 pear disorders, 11:357–411; 27:227–267 pear maturity indices, 13:407–432 pear scald, 27:227–257 petal senescence, 11:15–43 protea leaf blackening, 17:173–201 quality evaluation, 20:1–119 scald, 27:227–267 seed, 2:117–141 texture in fresh fruit, 20:121–244 tomato fruit ripening, 13:67–103 vegetables, 1:337–394 watercore, 6:189–251; 11:385–387 Potassium: container growing, 9:84 deficiency and toxicity symptoms in fruits and nuts, 2:147–148 foliar application, 6:331–332 nutrition, 5:321–322 pine bark media, 9:113–114 trickle irrigation, 4:29 Potato: CA storage, 1:376–378 fertilization, 1:120–121 low temperature sweetening, 17:203–231 tuberization, 14:89–198 Processing, table olives, 25:235–260 Propagation, see In vitro apple, 10:324–326; 12:288–295 aroids, ornamental, 10:12–13 bioreactor technology, 24:1–30 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 7:157–200 woody legumes in vitro, 14:265–332 Protaceous flower crop, see Protea Banksia, 22:1–25 Leucospermum, 22:27–90 Protea, leaf blackening, 17:173–201 Protected crops, carbon dioxide, 7:345–398 Protoplast culture, woody species, 10:173–201

371 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 Pumpkin, history, 25:71–170 Q Quality evaluation: fruits and vegetables, 20:1–119, 121–224 nondestructive, 20:1–119 texture in fresh fruit, 20:121–224 R Rabbit, 6:275–276 Radish, fertilization, 1:121 Rambutan, see Sapindaceous fruits Rambutan, CA and MA, 22:163 Raspberry: harvesting, 16:282–298 productivity, 11:185–228 Rejuvenation: rose, 9:59–60 woody plants, 7:109–155 Replant problem, deciduous fruit trees, 2:1–116 Respiration: asparagus postharvest, 12:72–77 fruit in CA storage, 1:315–316 kiwifruit, 6:47–48 vegetables in CA storage, 1:341–346 Rhizobium, 3:34, 41 Rhododendron, 12:1–67

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372 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: Amaryllidaceae, 25:1–79 aroids, 8:43–99; 12:166–170 cassava, 12:158–166; 26:85–159 low-temperature sweetening, 17:203–231 minor crops, 12:184–188 potato tuberization, 14:89–188 sweet potato, 12:170–176 sweet potato physiology, 23:277–338 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; 5:361–363 growth substances, 9:3–53 in vitro, 5:244–248 S Salinity: air pollution, 8:25–26 olive, 21:177–214 soils, 4:22–27

CUMULATIVE SUBJECT INDEX tolerance, 16:33–69 Sapindaceous fruits, 16:143–196 Sapodilla, CA and MA, 22:164 Scadoxus, 25:25–28 Scald, apple and pear, 27:227–265 Scoring, and fruit set, 1:416–417 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, 11:159–184 kiwifruit, 6:48–50 lettuce, 2:166–174 lettuce germination, 24:229–275 priming, 16:109–141 rose propagation, 9:54–55 vegetable, 3:1–58 viability and storage, 2:117–141 Secondary metabolites, woody legumes, 14:314–322 Senescence: chlorophyll senescence, 23:88–93 cut flower, 1:204–236; 3:59–143; 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 Signal transduction, 26:49–84 Small fruit, CA storage, 1:308 Snapdragon fertilization, 5:363–364 Sod production, 27:317–351 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

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CUMULATIVE SUBJECT INDEX orchard floor management, 9:377–430 plant relations, trickle irrigation, 4:18–21 stress, 4:151–152 testing, 7:1–68; 9:88–90 zinc, 23:109–178 Soilless culture, 5:1–44 Solanaceae: in vitro, 5:229–232 steroidal alkaloids, 25:171–196 Somatic embryogenesis, see Asexual embryogenesis Sorghum, sweet, 21:73–104 Spathiphyllum, see Aroids, ornamental Squash, history, 25:71–170 Stem, apple morphology, 12:272–283 Sternbergia, 25:59 Steroidal alkaloids, solanaceous, 25:171–196 Storage, see Postharvest physiology, Controlled-atmosphere (CA) storage cut flower, 3:96–100; 10:35–62 rose plants, 9:58–59 seed, 2:117–141 Strawberry: fertilization, 1:106 fruit growth and ripening, 17:267–297 functional phytonutrients, 27: 303–304 harvesting, 16:348–365 in vitro, 5:239–241 Stress: benefits of, 4:247–271 chlorophyll fluorescence, 23:69–107 climatic, 4:150–151 flooding, 13:257–313 mechanical, 17:1–42 petal, 11:32–33 plant, 2:34–37 protectants (triazoles), 24:55–138 protection, 7:463–466 salinity tolerance in olive, 21:177–214 subzero temperature, 6:373–417 waxes, 23:1–68 Sugar, see Carbohydrate allocation, 7:74–94 flowering, 4:114 Sugar apple, CA and MA, 22:164 Sugar beet, fluid drilling of seed, 3:18–19

373 Sulfur: deficiency and toxicity symptoms in fruits and nuts, 2:154 nutrition, 5:323–324 Sweet potato: culture, 12:170–176 fertilization, 1:121 physiology, 23:277–338 Sweet sop, CA and MA, 22:164 Symptoms, deficiency and toxicity symptoms in fruits and nuts, 2:145–154 Syngonium, see Aroids, ornamental T Taro, see Aroids, edible Tea, botany and horticulture, 22:267–295 Temperature: apple fruit set, 1:408–411 bloom delay, 15:119–128 CA storage of vegetables, 1:340–341 chilling injury, 15:67–74 cryopreservation, 6:357–372 cut flower storage, 10:40–43 fertilization, greenhouse crops, 5:331–332 fire blight forecasting, 1:456–459 flowering, 15:284–287, 312–313 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 Texture in fresh fruit, 20:121–224 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; 9:273–349; 10:153–181, 24:1–30 cassava, 26:85–159 dwarfing, 3:347–348 nutrient analysis, 7:52–56; 9:90

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374 Tomato: CA storage, 1:380–386 chilling injury, 20:199–200 fertilization, 1:121–123 fluid drilling of seed, 3:19–20 fruit ripening, 13:67–103 galacturonase, 13:67–103 greenhouse quality, 26:239 parthenocarpy, 6:65–84 Toxicity symptoms in fruit and nut crops, 2:145–154 Transport, cut flowers, 3:100–104 Tree decline, 2:1–116 Triazoles, 10:63–105; 24:55–138 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 fertilization, 5:364–366 in vitro, 18:144–145 physiology, 5:45–125 Tunnel (cloche), 7:356–357 Turfgrass, fertilization, 1:112–117 Turnip, fertilization, 1:123–124 Turnip Mosaic Virus, 14:199–238 U Urd bean, genetics, 2:364–373 Urea, foliar application, 6:332 V Vaccinium, 10:185–187. See also Blueberry; Cranberry; Lingonberry functional phytonutrients, 27:303 Vase solutions, 3:82–95; 10:46–51 Vegetable crops: aroids, 8:43–99; 12:166–170 asparagus postharvest, 12:69–155 cactus, 18:300–302 cassava, 12:158–166; 13:105–129; 26:85–159 CA storage, 1:337–394 CA storage and quality, 8:101–127 CA storage diseases, 3:412–461 Caper bush, 27:125–188

CUMULATIVE SUBJECT INDEX chilling injury, 15:63–95 coating physiology, 26:161–238 ecologically based, 24:139–228 fertilization, 1:117–124 fluid drilling of seeds, 3:1–58 gourd history, 25:71–170 greenhouse management, 21:1–39 greenhouse pest management, 13:1–66 honey bee pollination, 9:251–254 hydroponics, 7:483–558 lettuce seed germination, 24:229–275 low-temperature sweetening, 17:203–231 minor root and tubers, 12:184–188 mushroom cultivation, 19:59–97 mushroom spawn, 6:85–118 N nutrition, 22:185–223 nondestructive postharvest quality evaluation, 20:1–119 okra, 21:41–72 potato tuberization, 14:89–188 pumpkin history, 25:71–170 seed conditioing, 13:131–181 seed priming, 16:109–141 squash history, 25:71–170 steroidal alkaloids, Solanaceae, 25:171–196 sweet potato, 12:170–176 sweet potato physiology, 23:277–338 tomato fruit ripening, 13:67–103 tomato (greenhouse) quality: 26:239–319 tomato parthenocarpy, 6:65–84 tropical production, 24:139–228 truffle cultivation, 16:71–107 yam (Dioscorea), 12:177–184 Vegetative tissue, desiccation tolerance, 18:176–195 Vernalization, 4:117; 15:284–287; 17:73–123 Vertebrate pests, 6:253–285 Vigna, see Cowpea genetics, 2:311–394 U.S. production, 12:197–222 Viroid, dwarfing for citrus, 24:277–317 Virus: benefits in horticulture, 3:394–411 dwarfing for citrus, 24:277–317 elimination, 7:157–200; 9:318; 18:113–123

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CUMULATIVE SUBJECT INDEX fig, 12:474–475 tree short life, 2:50–51 turnip mosaic, 14:199–238 Volatiles, 17:43–72; 24:31–53 Vole, 6:254–274 W Walnut, in vitro culture, 9:312 Water relations: cut flower, 3:61–66; 18:1–85 deciduous orchards, 21:105–131 desiccation tolerance, 18:171–213 fertilization, grape and grapevine, 27:189–225 kiwifruit, 12:332–339 light in orchards, 2:248–249 photosynthesis, 11:124–131 trickle irrigation, 4:1–48 Watercore, 6:189–251 apple, 6:189–251 pear, 11:385–387 Watermelon, fertilization, 1:124 Wax apple, CA and MA, 22:164 Waxes, 23:1–68 Weed control, ginseng, 9:228–229 Weeds: lettuce research, 2:198 virus, 3:403

375 Woodchuck, 6:276–277 Woody species, somatic embryogenesis, 10:153–181 X Xanthomonas phaseoli, 3:29–32, 41, 45–46 Xanthophyll cycle, 18:226–239 Xanthosoma, 8:45–46, 56–57. See also Aroids Y Yam (Dioscorea), 12:177–184 Yield: determinants, 7:70–74; 97–99 limiting factors, 15:413–452 Z Zantedeschia, see Aroids, ornamental Zephyranthes, 25:60–61 Zinc: deficiency and toxicity symptoms in fruits and nuts, 2:151 foliar application, 6:332, 336 nutrition, 5:326; 23:109–178 pine bark media, 9:124

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Page 376

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Page 377

Cumulative Contributor Index (Volumes 1–27)

Abbott, J. A., 20:1 Adams III, W. W., 18:215 Aldwinckle, H. S., 1:423; 15:xiii Amarante, C., 28:161 Anderson, I. C., 21:73 Anderson, J. L., 15:97 Anderson, P. C., 13:257 Andrews, P. K., 15:183 Ashworth, E. N., 13:215; 23:1 Asokan, M. P., 8:43 Atkinson, D., 2:424 Aung, L. H., 5:45

Bower, J. P., 10:229 Bradley, G. A., 14:xiii Brennan, R., 16:255 Broadbent, P., 24:277 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

Bailey, W. G., 9:187 Baird, L. A. M., 1:172 Banks, N. H., 19:217; 25:197; 26:161 Barden, J. A., 9:351 Barker, A. V., 2:411 Bass, L. N., 2:117 Bassett, C. L., 26:49 Becker, J. S., 18:247 Beer, S. V., 1:423 Behboudian, M. H., 21:105; 27:189 Bennett, A. B., 13:67 Benschop, M., 5:45 Ben-Ya’acov, A., 17:381 Benzioni, A., 17:233 Bevington, K. B., 24:277 Bewley, J. D., 18:171 Binzel, M. L., 16:33 Blanpied, G. D., 7:xi Bliss, F. A., 16:xiii Boardman, K. 27 xi Borochov, A., 11:15

Caldas, L. S., 2:568 Campbell, L. E., 2:524 Cantliffe, D. J., 16:109; 17:43; 24:229 Carter, G., 20:121 Carter, J. V., 3:144 Cathey, H. M., 2:524 Chambers, R. J., 13:1 Charron, C. S., 17:43 Chen, Z., 25:171 Chin, C. K., 5:221 Clarke, N. D., 21:1 Coetzee, J. H., 26:1 Cohen, M., 3:394 Collier, G. F., 4:49 Collins, G., 25:235 Collins, W. L., 7:483 Colmagro, S., 25:235 Compton, M. E., 14:239 Conover, C. A., 5:317; 6:119 Coppens d’Eeckenbrugge, G., 21:133 Coyne, D. P., 3:28

Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 377

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Page 378

378

CUMULATIVE CONTRIBUTOR INDEX

Crane, J. C., 3:376 Criley, R. A., 14:1; 22:27; 24:x Crowly, W., 15:1 Cutting, J. G., 10:229

Flore, J. A., 11:111 Forshey, C. G., 11:229 Franks, R. G., 27:41 Fujiwara, K., 17:125

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; 24:319 Davies, P. J., 15:335 Davis, T. D., 10:63; 24:55 Decker, H. F., 27:317 DeEll, J. R., 23:69 DeGrandi-Hoffman, G., 9:237 De Hertogh, A. A., 5:45; 14:57; 18:87; 25:1 Deikman, J., 16:1 DellaPenna, D., 13:67 Demmig-Adams, B., 18:215 Dennis, F. G., Jr., 1:395 Dorais, M., 26:239 Doud, S. L., 2:1 Dudareva, N., 24:31 Duke, S. O., 15:371 Dunavent, M. G., 9:103 Duval, M.-F., 21:133 Düzyaman, E., 21:41 Dyer, W. E., 15:371

Geisler, D., 6:155 Geneve, R. L., 14:265 George, W. L., Jr., 6:25 Gerrath, J. M., 13:315 Gilley, A., 24:55 Giovannetti, G., 16:71 Giovannoni, J.J., 13:67 Glenn, G. M., 10:107 Goffinet, M. C., 20:ix Goldschmidt, E. E., 4:128 Goldy, R. G., 14:357 Goren, R., 15:145 Gosselin, A., 26:239 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 Gucci, R., 21:177 Guest, D. I., 17:299 Guiltinan, M. J., 16:1

Early, J. D., 13:339 Elfving, D. C., 4:1; 11:229 El-Goorani, M. A., 3:412 Esan, E. B., 1:1 Evans, D. A., 3:214 Ewing, E. E., 14:89

Hackett, W. P., 7:109 Halevy, A. H., 1:204; 3:59 Hallett, I. C., 20:121 Hammerschmidt, R., 18:247 Hanson, E. J., 16:255 Harker, F. R., 20:121 Heaney, R. K., 19:99 Heath, R. R., 17:43 Helzer, N. L., 13:1 Hendrix, J. W., 3:172 Henny, R. J., 10:1 Hergert, G. B., 16:255 Hess, F. D., 15:371 Heywood, V., 15:1 Hjalmarsson, I., 27:79–123 Hogue, E. J., 9:377 Holt, J. S., 15:371 Huber, D. J., 5:169 Hunter, E. L., 21:73 Hutchinson, J. F., 9:273 Hutton, R. J., 24:277

Faust, M., 2:vii, 142; 4:174; 6:287; 14:333; 17:331; 19:263; 22:225; 23:179 Fenner, M., 13:183 Fenwick, G. R., 19:99 Ferguson, A. R., 6:1 Ferguson, I. B., 11:289 Ferguson, J. J., 24:277 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 Fletcher, R. A., 24:53 Flick, C. E., 3:214

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Page 379

CUMULATIVE CONTRIBUTOR INDEX

Indira, P., 23:277 Ingle, M. 27:227 Isenberg, F. M. R., 1:337 Iwakiri, B. T., 3:376 Jackson, J. E., 2:208 Janick, J., 1:ix; 8:xi; 17:xiii; 19:319; 21:xi; 23:233 Jarvis, W. R., 21:1 Jenks, M. A., 23:1 Jensen, M. H., 7:483 Jeong, B. R., 17:125 Jewett, T. J., 21:1 Joiner, J. N., 5:317 Jones, H. G., 7:301 Jones, J. B., Jr., 7:1 Jones, R. B., 17:173 Kagan-Zur, V., 16:71 Kalt, W. 27:269 Kang, S. -M., 4:204 Kato, T., 8:181 Kawa, L., 14:57 Kawada, K., 4:247 Kelly, J. F., 10:ix; 22:xi Kester, D. E., 25:xii 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 Leal, F., 21:133 Ledbetter, C. A., 11:159 Li, P. H., 6:373 Lill, R. E., 11:413

379 Lin, S., 23:233 Lipton, W. J., 12:69 Littlejohn, G. M., 26:1 Litz, R. E., 7:157 Liu, Z., 27:41 Lockard, R. G., 3:315 Loescher, W. H., 6:198 Lorenz, O. A., 1:79 Lu, R., 20:1 Lurie, S., 22:91–121 Lyrene, P., 21:xi Maguire, K. M., 25:197 Manivel, L., 22:267 Maraffa, S. B., 2:268 Marangoni, A. G., 17:203 Marini, R. P., 9:351 Marlow, G. C., 6:189 Maronek, D. M., 3:172 Martin, G. G., 13:339 Mayak, S., 1:204; 3:59 Maynard, D. N., 1:79 McConchie, R., 17:173 McNicol, R. J., 16:255 Merkle, S. A., 14:265 Michailides, T. J., 12:409 Michelson, E., 17:381 Mika, A., 8:339 Miller, A. R., 25:171 Miller, S. S., 10:309 Mills, H. A., 2:411; 9:103 Mills, T. M., 21:105 Mitchell, C. A., 17:1 Mizrahi, Y., 18:291, 321 Molnar, J. M., 9:1 Monk, G. J., 9:1 Monselise, S. P., 4:128 Moore, G. A., 7:157 Mor, Y., 9:53 Morris, J. R., 16:255 Murashige, T., 1:1 Murr, D. P., 23:69 Murray, S. H., 20:121 Myers, P. N., 17:1 Nadeau, J. A., 19:1 Nascimento, W. M., 24:229 Neilsen, G. H., 9:377 Nelson, P. V., 26:xi Nerd, A., 18:291, 321

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380 Niemiera, A. X., 9:75 Nobel, P. S., 18:291 Nyujtò, F., 22:225 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; 24:373; 25:197 Ormrod, D. P., 8:1 Ortiz, R., 27:79 Palser, B. F., 12:1 Papadopoulos, A. P., 21:1; 26:239 Pararajasingham, S., 21:1 Parera, C. A., 16:109 Paris, H. S., 25:71 Pegg, K. G., 17:299 Pellett, H. M., 3:144 Perkins-Veazil, P., 17:267 Pichersky, E., 24:31 Piechulla, B., 24:31 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 Prange, R. K., 23:69 Pratt, C., 10:273; 12:265 Preece, J. E., 14:265 Priestley, C. A., 10:403 Proctor, J. T. A., 9:187 Puonti-Kaerlas, J., 26:85 Quamme, H., 18:xiii Raese, J. T., 11:357 Ramming, D. W., 11:159 Ravi, V., 23:277 Reddy, A. S. N., 10:107 Redgwell, R. J., 20:121 Reid, M., 12:xiii; 17:123 Reuveni, M., 16:33 Richards, D., 5:127 Rieger, M., 11:45 Roper, T. R., 21:215

Page 380

CUMULATIVE CONTRIBUTOR INDEX 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 Saltveit, M. E., 23:x San Antonio, J. P., 6:85 Sankhla, N., 10:63; 24:55 Saure, M. C., 7:239 Schaffer, B., 13:257 Schenk, M. K., 22:185 Schneider, G. W., 3:315 Schuster, M. L., 3:28 Scorza, R., 4:106 Scott, J. W., 6:25 Sedgley, M., 12:223; 22:1; 25:235 Seeley, S. S., 15:97 Serrano Marquez, C., 15:183 Sharp, W. R., 2:268; 3:214 Sharpe, R. H., 23:233 Shattuck, V. I., 14:199 Shear, C. B., 2:142 Sheehan, T. J., 5:279 Shipp, J. L., 21:1 Shirra, M., 20:267 Shorey, H. H., 12:409 Simon, J. E., 19:319 Singh, Z. 27:189 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. I., 13:1 Soule, J., 4:247 Sozzi, G. O., 27:125 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 Stroshine, R. L., 20:1 Struik, P. C., 14:89

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Page 381

CUMULATIVE CONTRIBUTOR INDEX Studman, C. J., 19:217 Stutte, G. W., 13:339 Styer, D. J., 5:221 Sunderland, K. D., 13:1 Sung, Y., 24:229 Surányi, D., 19:263; 22:225; 23:179 Swanson, B., 12:xiii Swietlik, D., 6:287; 23:109 Syvertsen, J. P., 7:301 Tattini, M., 21:177 Tétényi, P., 19:373 Theron, K. I., 25:1 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 Upchurch, B. L., 20:1 Valenzuela, H. R., 24:139 van Doorn, W. G., 17:173; 18:1 van Kooten, O., 23:69 van Nocker, S. 27:1 Veilleux, R. E., 14:239 Vorsa, N., 21:215 Wallace, A., 15:413 Wallace, D. H., 17:73

381 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 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, U. L., 2:1 Yahia, E. M., 16:197; 22:123 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 Ziv, M., 24:1 Zucconi, F., 11:1

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Plate 3.1A.

Lingonberry in Scandinavia: Pine heath with lingonberry and bilberry.

Plate 3.1B.

Lingonberry cultivar ‘Sanna’.

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Plate 3.1C.

Open flower of lingonberry.

Plate 3.1D. flower.

Anther from a lingonberry

Page 2